CA2506127C

CA2506127C - Methods of generating high-production of antibodies from hybridomas created by in vitro immunization

Info

Publication number: CA2506127C
Application number: CA2506127A
Authority: CA
Inventors: Luigi Grasso; Shaohong Liang; Nicholas E. Nicolaides; Philip M. Sass
Original assignee: Morphotek Inc
Current assignee: Eisai Inc
Priority date: 2002-11-15
Filing date: 2003-11-14
Publication date: 2013-07-09
Anticipated expiration: 2023-11-14
Also published as: AU2003295576A1; JP2006526983A; US20040214288A1; US20100311169A1; ATE444359T1; US8445229B2; EP1572971A4; US7754450B2; EP1572971A2; EP1572971B1; JP4555089B2; WO2004046330A2; AU2003295576B2; WO2004046330A3; CA2506127A1; DE60329526D1

Abstract

The invention provides methods for generating high titers of high-affinity antibodies from hybridoma cells produced by fusing myeloma cells with in vitro immunized donor cells. The hybridoma cells or mammalian expression cells with cloned antibody genes from the hybridomas producing the high-affinity antibodies may be mismatch repair defective due to defects of endogenous mismatch repair subunits of through expression of a dominant negative allele of a mismatch repair gene which allows the hybridoma cell to be hypermutable, may be rendered hypermutable by chemical means, or may be naturally mismatch repair deficient. High-affinity antibodies and high titer producer cells producing antibodies may be prepared by the methods of the invention.

Description

METHODS OF GENERATING HIGH-PRODUCTION OF ANTIBODIES FROM
HYBRIDOMAS CREATED BY IN VITRO IMMUNIZATION
FIELD OF THE INVENTION

[0002] The invention relates to the generation of hybridoma cells that produce high-affinity antibodies in high titers. More specifically, the invention relates to the use of an in vitro immunization method in conjunction with hybridoma technology using dominant negative mismatch repair genes or chemical inhibitors of mismatch repair to produce high titers of antigen specific antibodies of the IgG subclass, that bind to the antigen with high affinity.
BACKGROUND OF THE RELATED ART

[0003] The use of antibodies to block the activity of foreign and/or endogenous polypeptides provides an effective and selective strategy for treating the underlying cause of disease. In particular is the use of monoclonal antibodies (MAb) as effective therapeutics such as the FDA
approved ReoPro (Glaser, (1996) Nat. Biotechnol. 14:1216-1217), an anti-platelet MAb from Centocor; Herceptin (Weiner, (1999) Semin. Oncol. 26:43-51), an anti-Her2/neu MAb from Genentech; and Synagis (SaezLlorens, et al. (1998) Pediat. Infect. Dis. J.
17:787-791), an anti-respiratory syncytial virus MAb produced by Medimmune.

[0004] Standard methods for generating MAbs against candidate protein targets are known by those skilled in the art. Briefly, rodents such as mice or rats are injected with a purified antigen in the presence of adjuvant to generate an immune response (Shield, et al. (1996) Am.
J. Kidney Dis. 27: 855-864). Rodents with positive immune sera are sacrificed and splenocytes are isolated. Isolated splenocytes are fused to melanomas to produce immortalized cell lines that are then screened for antibody production.
Positive lines are isolated and characterized for antibody production. The direct use of rodent MAbs as human therapeutic agents were confounded by the fact that human anti-rodent antibody (HARA) responses occurred in a significant number of patients treated with the rodent-derived antibody (Khazaeli, et al., (1994) Immunother. 15:42-52). In order to circumvent the problem of HARA, the grafting of the complementarity determining regions (CDRs), which are the critical motifs found within the heavy and light chain variable regions of the immunoglobulin (Ig) subunits making up the antigen binding domain, onto a human antibody backbone found these chimeric molecules are able to retain their binding activity to antigen while lacking the HARA response (Emery and Harris, "Strategies for humanizing antibodies" In:
ANTIBODY
ENGINEERING, C.A.K. Borrebaeck (Ed.) Oxford University Press, NY, 1995. pp.
159-183. A
common problem that exists during the "humanization" of rodent-derived MAbs (referred to hereafter as HAb) is the loss of binding affinity due to conformational changes in the three-dimensional structure of the CDR domain upon grafting onto the human Ig backbone (U. S.
Patent No. 5,530,101 to Queen et al.). To overcome this problem, additional HAb vectors are usually needed to be engineered by inserting or deleting additional amino acid residues within the framework region and/or within the CDR coding region itself in order to recreate high affinity HAbs (U. S. Patent No. 5,530,101 to Queen et al.). This process is a very time consuming procedure that involves the use of expensive computer modeling programs to predict changes that may lead to a high affinity HAb. In some instances the affinity of the HAb is never restored to that of the MAb, rendering them of little therapeutic use.

[0005] Another problem that exists in antibody engineering is the generation of stable, high yielding producer cell lines that is required for manufacturing of the molecule for clinical materials. Several strategies have been adopted in standard practice by those skilled in the art to circumvent this problem. One method is the use of Chinese Hamster Ovary (CHO) cells transfected with exogenous Ig fusion genes containing the grafted human light and heavy chains to produce whole antibodies or single chain antibodies, which are a chimeric molecule containing both light and heavy chains that form an antigen-binding polyp eptide (Reff, M. E.
(1993) Curr. Opin. Biotechnol. 4:573-576). Another method employs the use of human lymphocytes derived from transgenic mice containing a human grafted immune system or transgenic mice containing a human Ig gene repertoire. Yet another method employs the use of monkeys to produce primate MAbs, which have been reported to lack a human anti-monkey response (Neuberger and Gruggermann (1997) Nature 386:25-26). In all cases, the generation of a cell line that is capable of generating sufficient amounts of high affinity antibody poses a major limitation for producing sufficient materials for clinical studies.
Because of these limitations, the utility of other recombinant systems such as plants are currently being explored as systems that will lead to the stable, high-level production of humanized antibodies (Fiedler and Conrad (1995) Bio/Technology 13:1090-1093).

[0006] One strategy to overcome the problem of human reactions against foreign antibodies is to stimulate human immuno globulin-producing cells in vitro. Various attempts to stimulate human antibody production in vitro typically have resulted in low affinity antibodies of the IgM subclass (Zafiropoulos et al. (1997) J. Immunological Methods 200:181-190).

[0007] A method for generating diverse antibody sequences within the variable domain that results in HAbs and MAbs with high binding affinities to antigens would be useful for the creation of more potent therapeutic and diagnostic reagents respectively.
Moreover, the generation of randomly altered nucleotide and polypeptide residues throughout an entire antibody molecule will result in new reagents that are less antigenic and/or have beneficial pharmacokinetic properties. The invention described herein is directed to the use of random genetic mutation throughout an antibody structure in vitro by blocking the endogenous mismatch repair (MMR) activity of a host cell producing immunoglobulins that encode biochemically active antibodies. The invention also relates to methods for repeated in vitro genetic alterations and selection for antibodies with enhanced binding and pharmacokinetic profiles.

[0008] In addition, the ability to develop genetically altered host cells that are capable of secreting increased amounts of antibody also will provide a valuable method for creating cell hosts for product development. The invention described herein is further directed to the creation of genetically altered cell hosts with increased antibody production via the blockade of MNIR. The invention facilitates the generation of high affinity antibodies and the production of cell lines with elevated levels of antibody production derived from hybridoma cells. The invention described herein provides methods for generating antigen-specific monoclonal antibodies (mAbs). Other advantages of the present invention are described in the examples and figures described herein.
SUMMARY OF THE INVENTION

[0009] The invention provides methods for producing hybridoma cells producing high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising:
(a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.

[0010] In some embodiments, the dominant negative allele of a mismatch repair gene comprises a truncation mutation of the PMS2 gene (e.g., a PMS2-134 gene). In some embodiments of the method of the invention, antibodies are screened using an ELISA-based assay or other assays that can measure antibody-antigen binding. In some embodiments, the screening assays screen for hypermutated hybridomas that produce higher affinity antibodies than those produced by the parental hybridomas. In other embodiments, the screening assays screen for hypermutated hybridomas that produce antibodies in higher titers than the parental hybridomas.

[0011] In some embodiments of the method of the invention, the method further comprises inactivation of the dominant negative allele of the mismatch repair gene, thereby stabilizing the genome of said hypermutated hybridoma.

[0012] In some embodiments of the method of the invention, the dominant negative mismatch repair gene is introduced into the hybridoma cell after the fusion of said myeloma with the immunoglobulin-producing cells. In other embodiments, the dominant negative mismatch repair gene is introduced into the myeloma cell prior to the fusion with the immunoglobulin-producing cells. -

[0013] The invention also comprises antibodies produced by the hybridoma cells.

[0014] The invention also comprises methods for producing hybridoma cells that produce high titers of antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor blood cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen of the hypermutated hybridoma cells for antigen-specific antibodies produced in higher titers than that produced by the parental hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by the parental hybridoma cells.

[0015] In some embodiments, the dominant negative allele of a mismatch repair gene comprises a truncation mutation of the PMS2 gene (e.g., a PMS2-134 gene). In some embodiments of the method of the invention, antibodies are screened using an ELISA-based assay. In some embodiments, the screening assays screen for hypermutated hybridomas that produce higher affinity antibodies than those produced by the parental hybridomas. In other embodiments, the screening assays screen for hypermutated hybridomas that produce antibodies in higher titers than the parental hybridomas.

[0016] In some embodiments of the method of the invention, the method further comprising inactivation of the dominant negative allele of the mismatch repair gene, thereby stabilizing the genome of said hypermutated hybridoma.

[0017] In some embodiments of the method of the invention, the dominant negative mismatch repair gene is introduced into the hybridoma cell after the fusion of said myeloma with the immunoglobulin-producing cells. In other embodiments, the dominant negative mismatch repair gene is introduced into the myeloma cell prior to the fusion with the immunoglobulin-producing cells.

[0018] In some embodiments of the method of the invention, the dominant negative allele of the mismatch repair gene is subsequently inactivated in order to restabilize the genome of the cell.

[0019] The dominant negative allele of the mismatch repair gene may be introduced into the myeloma cell prior to fusion with the immunoglobulin producing cells. Thus, the resulting hybridoma cells express the same dominant negative allele of the mismatch repair gene as the myeloma cells. Alternatively, the dominant negative allele of the mismatch repair gene may be introduced into the hybridoma cells.

[0020] The invention also comprises antibodies produced by the hybridoma cells.

[0021] The invention further provides recombinant myeloma cells comprising a polynucleotide sequence encoding a dominant negative mismatch repair protein.
The dominant negative mismatch repair protein may be a dominant negative form of, for example, a PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, or MSH1, and PMSR proteins encoded by homologs of the PMSR genes as described in Nicolaides et al.
(1995) Genomics 30:195-206 and Horii et al. (1994) Biochem. Biophys. Res.
Commun.
204:1257-1264. In some embodiments, the recombinant myeloma cell expresses a polynucleotide encoding a dominant negative allele of a PMS2 gene (e.g., a truncation mutation of the PMS2 gene, such as the PMS2-134 gene).

[0022] In some embodiments, the recombinant myeloma cell is a human cell. In other embodiments, the recombinant myeloma cell does not express immunoglobulin genes and/or Epstein-Barr virus. In other embodiments, the myeloma cells are HAT sensitive.

[0023] The invention also provides a method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; (d) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell, wherein said mammalian expression cell expresses a dominant negative allele of a mismatch repair gene; (e) performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said hybridoma cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0024] In some embodiments, the dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell prior to introduction of the immunoglobulin genes. In other embodiments, the dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell after introduction of said immunoglobulin genes. In other embodiments, the dominant negative allele of a mismatch repair gene is introduced into the mammalian expression cell with the immunoglobulin genes simultaneously.

[0025] The invention also comprises antibodies produced by the mammalian expression cells.

[0026] The invention also provides a method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells, wherein said hybridoma cells express a dominant negative allele of a mismatch repair gene; (c) incubating said parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from said hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
(f) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell;
thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized human immunoglobulin-producing cells.

[0027] In some embodiments, the dominant negative allele of a mismatch repair gene is present in the myeloma cell prior to cell fusion. In other embodiments, the dominant negative allele of the mismatch repair gene is introduced into the hybridoma cell after cell fusion.

[0028] The invention also comprises antibodies produced by the mammalian expression cells.

[0029] The invention also provides a method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from the hybridoma cells to antigen; (d) cloning immunoglobulin genes from the hybridoma into a parental mammalian expression cell, wherein the mammalian expression cell expresses a dominant negative allele of a mismatch repair gene; (e) incubating the parental mammalian expression cell to allow for mutagenesis, thereby forming hypermutated mammalian expression cells; (f) performing a screen of hypermutable mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from the hybridoma cells; and (g) performing a screen of hypermutable mammalian expression cells that secrete higher titers of antibodies than parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0030] In some embodiments, the dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell prior to introduction of the immunoglobulin genes. In other embodiments, the dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell after introduction of said immunoglobulin genes. In other embodiments, the dominant negative allele of a mismatch repair gene is introduced into the mammalian expression cell with the immunoglobulin genes simultaneously.

[0031] The invention also provides antibodies produced by the mammalian expression cells.

[0032] The invention also provides recombinant, hypermutable mammalian expression cells comprising a polynucleotide sequence encoding a dominant negative mismatch repair protein.

[0033] The mismatch repair gene may be a dominant negative mismatch repair gene, including, but not limited to a dominant negative form of PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, or MSHI , and homologs of PMSR genes as described in Nicolaides et al. (1995) Genomics 30:195-206 and Horii et al.
(1994) Biochem.
Biophys. Res. Commun. 204:1257-1264. A non-limiting example includes a dominant negative truncation mutant of PMS2 (e.g., a PMS2-134 gene).

[0034] The invention also provides methods for producing hybridoma cells producing high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) performing a screen for antigen binding for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for the antigen than antibodies produced by said parental hybridoma cells;
thereby producing hybridoma cells producing high-affinity antibodies.

[0035] The invention also comprises antibodies produced by the hybridoma cells.

[0036] The invention also provides methods for producing hybridoma cells that produce high titers of antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) performing a screen of the hypermutated hybridoma cells for antigen-specific antibodies produced in higher titers than that produced by the parental hybridoma cells;
and (e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by said parental hybridoma cells; thereby producing hybridoma cells producing high titers of antibodies.

[0037] In some embodiments of the method of the invention, the hypermutated hybridoma cells also are screened for the production of higher titers of antibodies than that produced by the parental hybridomas. The screening may be using an ELISA-based assay, or any other means to measure antibody-antigen binding.

[0038] The invention also comprises antibodies produced by the hybridoma cells.

[0039] The invention also provides methods for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for antigen binding of antibodies produced from the hybridoma cells; (d) cloning immunoglobulin genes from the hybridoma cells into a mammalian expression cell; (e) incubating the mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair;
(f) performing a screen for mammalian expression cells that secrete antibodies with higher affmity for antigen as compared to antibodies produced from the hybridoma cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0040] In some embodiments of the method of the invention the method may further comprise the removal of the chemical inhibitor from the hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells.

[0041] The invention also comprises antibodies produced by the mammalian expression cells

[0042] The invention also provides methods for producing mammalian expression cells that produce high titers of high affinity antibodies to a selected antigen from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) incubating the hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells; (d) performing a screen for antigen binding for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) cloning immuno globulin genes from the hypermutated hybridoma cells into a mammalian expression cell, thereby forming parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0043] In some embodiments, the parental mammalian expression cell is further incubated in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; and the hypermutated mammalian expression cells are screened for higher production of antibodies than that of the parental mammalian expression cells.

[0044] In some embodiments of the method of the invention the method may further comprise the removal of the chemical inhibitor from the hypermutated hybridoma and/or hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated hybridoma cells and/or hypermutated mammalian expression cells.

[0045] The invention also comprises antibodies produced by the mammalian expression cells.

[0046] The invention also provides a method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; (d) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell; (e) incubating said mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; (f) performing a screen for hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental mammalian expression cells;
and (g) performing a second screen for hypennutated mammalian expression cells that produce higher titers of antibodies than that produced by parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0047] In some embodiments of the method of the invention the method may further comprise the removal of the chemical inhibitor from the hypermutated hybridoma and/or hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated hybridoma cells and/or hypermutated mammalian expression cells.

[0048] The invention also comprises antibodies produced by the mammalian expression cells.

[0049] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair; (b) fusing the immuno globulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.

[0050] The method may further comprise introducing a wild-type gene for mismatch repair into said selected hypermutated hybridoma cell to complement the mismatch repair deficiency, thereby restabilizing the genome of said selected hypermutated hybridoma cell.

[0051] The invention also comprises antibodies produced by the hybridoma cells.

[0052] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immuno globulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.

[0053] The method may further comprise introducing a wild-type gene for mismatch repair into said selected hypermutated hybridoma cell to complement the mismatch repair deficiency, thereby restabilizing the genome of said selected hypermutated hybridoma cell.

[0054] The invention also comprises antibodies produced by the hybridoma cells.

[0055] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells in high titers comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair;
(b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with parental hybridoma cells; (g) selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by the parental hybridoma cells; thereby producing hybridoma cells producing high titers of high-affinity antibodies.

[0056] The method may further comprise introducing a wild-type gene for mismatch repair into said selected hypermutated hybridoma cell to complement the mismatch repair deficiency, thereby restabilizing the genome of said selected hypermutated hybridoma cell.

[0057] The invention also comprises antibodies produced by the hybridoma cells.

[0058] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells in high titers comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with parental hybridoma cells; (g) selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by the parental hybridoma cells;
thereby producing hybridoma cells producing high titers of high-affinity antibodies.

[0059] The method may further comprise introducing a wild-type gene for mismatch repair into said selected hypermutated hybridoma cell to complement the mismatch repair deficiency, thereby restabilizing the genome of said selected hypermutated hybridoma cell.

[0060] The invention also comprises antibodies produced by the hybridoma cells.

[0061] In another embodiment, the invention comprises a method for producing mammalian expression cells that produce high-affinity antibodies in high titers from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair;
(b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) cloning immunoglobulin genes from said hypermutated hybridoma into a mammalian expression cell; thereby producing a mammalian expression cell that produces high titers of high-affinity antibodies in high titer from in vitro immunized immunoglobulin-producing cells.

[0062] In some embodiments, the parental mammalian expression cell is further incubated in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; and the hypermutated mammalian expression cells are screened for higher production of antibodies than that of the parental mammalian expression cells.

[0063] In some embodiments of the method of the invention the method may farther comprise the removal of the chemical inhibitor from the hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells.

[0064] The invention also comprises antibodies produced by the mammalian expression cells.

[0065] In another embodiment, the invention comprises a method for producing mammalian expression cells that produce high-affinity antibodies in high titer from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
and (f) cloning immunoglobulin genes from said hypermutated hybridoma cell into a mammalian expression cell; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

[0066] In some embodiments, the parental mammalian expression cell is further incubated in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; and the hypermutated mammalian expression cells are screened for higher production of antibodies than that of the parental mammalian expression cells.

[0067] In some embodiments of the method of the invention the method may further comprise the removal of the chemical inhibitor from the hypermutated mammalian expression cells, thereby stabilizing the genome of said hypermutated mammalian expression cells.

[0068] The invention also comprises antibodies produced by the hybridoma cells.

[0069] In some embodiments of the methods of the invention, the immunoglobulin-producing cells are mammalian cells, including but not limited to, mouse cells, rat cells, goat cells, cow cells, horse cells, dog cells, cat cells, rabbit cells, bird cells, monkey cells and human cells. In preferred embodiments, the cells are human cells.

[0070] In some embodiments the dominant negative allele of a mismatch repair gene is a dominant negative allele of PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, or MSH1, and homologs of PMSR genes as described in Nicolaides et al.
(1995) Genomics 30:195-206 and Horii et al. (1994) Biochem. Biophys. Res.
Commun.
204:1257-1264. However, the mismatch repair genes are not limit to these examples.

[0071] In some embodiments of the method of the invention, the immunogenic antigen is conjugated to a mitogenic polypeptide comprising at least a portion of a polypeptide including, but not limited to tetanus toxoid, ovalbumin, bovine serum albumen, thyro globulin, diptheria toxoid, BCG, and cholera toxin. In some embodiments, the antigen is generated by denaturing the mature protein.

[0072] In some embodiments of the method of the invention, the antibodies produced have an affinity of at least about 1 x 107 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 108 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 109 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1010 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1011 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1012 M. In other embodiments, the antibodies have an affinity of at least about 1 x 1013 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1014 M-1.

[0073] In some embodiments, the antibodies are produced in a higher titer than the parental cell lines, such as in an amount of at least about 1.5 fold higher than the parental cell line. In other embodiments, the titer is at least about 1.5-3 fold higher than the parental cell line. In other embodiments, the titer is at least about 3-5 fold higher than the parental cell line. In other embodiments, the titer is at least about 5-7 fold higher than the parental cell line. In other embodiments, the titer is at least about 7-9 fold higher than the parental cell line. In other embodiments, the titer is at least about 9-10 fold higher than the parental cell line.

[0074] In some embodiments of the method of the invention, mutation rates are further enhanced by incubating the hybridoma cells with a chemical mutagen, such as, but not limited to N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, procarbazine hydrochloride, chlorambucil, cyclophosphamide, methyl methanesulfonate, ethyl methanesulfonate, diethyl sulfate, acrylamide monomer, triethylene melamin, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, 7,12 dimethylbenz (a) anthracene, ethylene oxide, hexamethylphosphoramide, and bisulfan.

[0075] The chemical inhibitors of mismatch repair used in certain embodiments of the methods of the invention include, but are not limited to, at least one of an anthracene, an ATPase inhibitor, a nuclease inhibitor, an RNA interference molecule, a polymerase inhibitor and an antisense oligonucleotide that specifically hybridizes to a nucleotide encoding a mismatch repair protein. In some embodiments, the chemical inhibitor is an anthracene having the formula:

Rs R10 R4 wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 0-alkyl, S-alkyl, N-alkyl, 0-alkenyl, S-alkenyl, N-alkenyl, 0-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organo silicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said sub stituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups. In certain embodiments, R5 and R6 are hydrogen. In other embodiments, R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. Non-limiting examples of the anthracenes include 1,2-dimethylanthracene, 9,10-dimethylanthracene, 7,8-dimethylanthracene, 9,10-duphenylanthracene, 9,10-dihydroxymethylanthracene, 9-hydroxymethy1-10-methylanthracene, dimethylanthracene-1,2-diol, 9-hydroxymethy1-10-methylanthracene-1,2-diol, 9-hydroxymethy1-10-methylanthracene-3,4-diol, and 9,10-di-m-tolylanthracene.

[0076] The chemical inhibitor may be introduced into the growth medium of the cells. In some embodiments, the chemical inhibitor may be withdrawn from the hypermutated hybridoma cells in order to re-stabilize the genome of the cells.

[0077] The invention also comprises a method for in vitro production of antigen-specific immunoglobulin-producing cells comprising: (a) isolating donor cells from an animal; (b) treating said cells with L-leucyl-L-leucine methy ester hydrobromide; (c) incubating said donor cells with an immunogenic antigen in vitro, at 25-37 C, 5-10% CO2, in medium supplemented with 5-15% serum, and a growth promoting cytokine for 4 days; (d) washing said cells in medium; and (e) culturing said cells in medium supplemented with 5-15% serum an additional 8 days; thereby stimulating the production of antigen-specific immunoglobulin-producing cells.

[0078] In some embodiments, the immunoglobulin-producing cells are human cells.

[0079] In some embodiments of the method of the invention, the immunogenic antigen is conjugated to a mitogenic polypeptide comprising at least a portion of a polypeptide including, but not limited to tetanus toxoid, ovalbumin, bovine serum albumen, thyro globulin, diptheria toxoid, BCG, and cholera toxin. In some embodiments, the antigen is generated by denaturing the mature protein.

[0080] In some embodiments of the method of the invention, the antibodies produced have an affinity of at least about 1 x 107 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 108 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 109M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1010 /\4-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1011 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1012 M. In other embodiments, the antibodies have an affinity of at least about 1 x 1013M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1014M-1.

[0081] In some embodiments, the antibodies are produced in a higher titer than the parental cell lines, such as in an amount of at least about 1.5 fold higher than the parental cell line. In other embodiments, the titer is at least about 1.5-3 fold higher than the parental cell line. In other embodiments, the titer is at least about 3-5 fold higher than the parental cell line. In other embodiments, the titer is at least about 5-7 fold higher than the parental cell line. In other embodiments, the titer is at least about 7-9 fold higher than the parental cell line. In other embodiments, the titer is at least about 9-10 fold higher than the parental cell line.

[0082] In some embodiments of the method of the invention, mutation rates are further enhanced by incubating the hybridoma cells and/or mammalian expression cells with a chemical mutagen, such as, but not limited to N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, procarbazine hydrochloride, chlorambucil, cyclophosphamide, methyl methanesulfonate, ethyl methanesulfonate, diethyl sulfate, acrylamide monomer, triethylene melamin, melphalan, nitrogen mustard, vincristine, dimethylthtrosamine, N-methyl-N'-nitro-nitrosoguanidine, 7,12 dimethylbenz (a) anthracene, ethylene oxide, hexamethylphosphoramide, and bisulfan.

[0083] The mammalian expression cells used in the methods of the invention may include, but are not limited to, Chinese Hamster Ovary, baby hamster kidney cells, human embryonic kidney line 293, normal dog kidney cell lines, normal cat kidney cell lines, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NTH/3T3 cells, LMTK31 cells, mouse sertoli cells, human cervical carcinoma cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.

[0084] These and other embodiments are described more fully in the next section and include certain non-limiting examples.
BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 shows the immune response of PBMCs to antigen stimulation.
PBMCs were cultured in the presence or absence of TT for 4 days then washed with medium and cultured in the presence or absence of TT for an additional eight days. Culture supemates were collected and tested for the presence of antibody reactive to TT. Antibodies bound to TT
pre-coated on the solid phase were detected with HRP-labeled goat anti-human IgG, or HRP-labeled goat anti-human IgM.

[0086] Fig. 2A shows reactivity of donor serum to TT by detection of donor anti-TT IgG.
Fig. 2B shows reactivity of donor serum to TT by detection of donor anti-TT
IgM.

[0087] Figure 3 shows the frequency of the anti-TT response of PBMCs upon in vitro immunization with TT, or with TT in combination with IL-2, or CD4OL.

[0088] Figure 4 shows the intensity of the response of PBMCs upon in vitro immunization with TT, or with TT in combination with IL-2, or CD4OL.

[0089] Figure 5 shows the response of hybridomas expressing anti-TT
antibodies.
10090] Fig. 6A shows the reactivity of unstimulated PBMCs to EGFR. Fig. 6B
shows the reactivity of PBMCs to EGFR after immunization with EGFR-TT. Fig. 6C shows the reactivity of unstimulated PBMCs to EGFR-TT. Fig. 6D shows the reactivity of PBMCs to EGFR-TT after immunization with EGFR-TT.
[0091] Figure 7 shows the response of hybridomas expressing antibodies against human EGFR. Antibodies bound to EGFR or BSA (control) pre-coated on the solid phase were detected with HRP-labeled goat anti-human IgG or HRP-labeled goat anti-human IgM.
[00921 Figure 8 shows the IgG and IgM responses of cells immunized with tumor cells in vitro.
[00931 Figure 9 shows reactivity of clones to GM-CSF, chick ovalbumin (CAB), or keyhole limpet hemocyanin.
[01001 Figure 10 shows inhibitory effect of anti-GM-CSF antibodies on proliferation of TF-1 cells. Shown are the effects of a GM-CSF-specific, blocking antibody; a GM-CSF-specific, non-blocking antibody; and a non-specific antibody.
DETAILED DESCRIPTION OF THE INVENTION
[01011 Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.
Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
[01021 Standard reference works setting forth the general principles of recombinant DNA
technology known to those of skill in the art include, but are not limited to Ausubel et al.
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York (1998);
Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, New York (1989); Kaufman et al., Eds., HANDBOOK
OF
MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, 1RL Press, Oxford (1991).
[01031 The invention provides various embodiments of a method for producing antibody-producing cells and antibodies from in vitro immunized cells with high affinity, and/or increased production. In some embodiments, the cells that produce the antibodies are hybridoma cells, formed by fusing myeloma cells with the lymphoid cells that have been immunized against an antigen in vitro. In other embodiments, the cells that produce the antibodies are mammalian cells that have been transfected with immunoglobulin genes cloned from lymphoid cells that have been immunized against an antigen in vitro. In some embodiments, the method employs both hybridoma cells and mammalian cells. Some basic embodiments of the method of the invention may be described as follows.
[0104] In one embodiment, the invention provides a method for generating hybridoma cells producing high-affinity antibodies from in vitro immunized, immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.
[0105] In another embodiment, the invention provides methods of producing hybridoma cells that produce high liters of antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells express a dominant negative allele of a mismatch repair gene; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
(d) performing a screen of the hypermutated hybridoma cells for antigen-specific antibodies produced in higher titers than that produced by the parental hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by the parental hybridoma cells; thereby producing hybridoma cells that produce high titers of antibodies.
[0106] In another embodiment, the invention provides a method for producing hybridoma cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; (d) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell, wherein said mammalian expression cell expresses a dominant negative allele of a mismatch repair gene;
and (e) performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said hybridoma cells; thereby producing hybridoma cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0107] In another embodiment, the invention provides a method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells are produced by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells, wherein said hybridoma cells express a dominant negative allele of a mismatch repair gene;
(c) incubating said parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from said hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells; and (f) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell; thereby producing high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0108] In yet another embodiment, the invention provides mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells are produced by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from the hybridoma cells to antigen; (d) cloning immunoglobulin genes from the hybridoma into a parental mammalian expression cell, wherein the mammalian expression cell expresses a dominant negative allele of a mismatch repair gene; (e) incubating the parental mammalian expression cell to allow for mutagenesis, thereby forming hypermutated mammalian expression cells; (f) performing a screen of hypermutable mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from the hybridoma cells; and (g) performing a screen of hypermutable mammalian expression cells that secrete higher titers of antibodies than parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0109] In yet another embodiment, the invention provides a method of producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells are produced by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) performing a screen for antigen binding for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for the antigen than antibodies produced by said parental hybridoma cells; thereby producing hybridoma cells that produce high-affinity antibodies.
[0110] In still another embodiment, the invention provides a method of producing hybridoma cells that produce high titers of antibodies from in vitro immunized immunoglobulin-producing cells are produced by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells; (c) incubating the parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells; (d) performing a screen of the hypermutated hybridoma cells for antigen-specific antibodies produced in higher titers than that produced by the parental hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by said parental hybridoma cells; thereby producing hybridoma cells producing high titers of antibodies.
[0111] In another embodiment, the invention provides methods for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immuno globulin-producing cells are produced by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for antigen binding of antibodies produced from the hybridoma cells;
(d) cloning immunoglobulin genes from the hybridoma cells into a mammalian expression cell; (e) incubating the mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair; and (f) performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from the hybridoma cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0112] In yet another embodiment, the invention provides a method for producing mammalian expression cells that produce high affinity antibodies to a selected antigen from in vitro immunized immunoglobulin-producing cells are produced in high titers by: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) incubating the hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells; (d) performing a screen for antigen binding for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for the antigen than antibodies produced by the parental hybridoma cells; and (f) cloning immunoglobulin genes from the hypermutated hybridoma cells into a mammalian expression cell, thereby forming parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0113] In yet another embodiment, the invention also provides methods for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing said immunoglobulin-producing cells with myeloma cells to form hybridoma cells; (c) performing a screen for binding of antibodies produced from said hybridoma cells to antigen; -(d) cloning immunoglobulin genes from said hybridoma into a mammalian expression cell; (e) incubating said mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; (f) performing a screen for hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental mammalian expression cells; and (g) performing a second screen for hypermutated mammalian expression cells that produce higher titers of antibodies that produced by parental mammalian expression cells; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0114] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair; (b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.
[0115] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; and (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; thereby producing hybridoma cells producing high-affinity antibodies.
[0116] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells in high titers comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair;
(b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with parental hybridoma cells; and (g) selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by the parental hybridoma cells; thereby producing hybridoma cells producing high titers of high-affinity antibodies.
[0117] In another embodiment, the invention comprises a method for producing hybridoma cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells in high titers comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; (f) performing a second screen for hypermutated hybridoma cells that produce increased titers of antibodies as compared with parental hybridoma cells; and (g) selecting hypermutated hybridoma cells that produce antibodies in higher titers than produced by the parental hybridoma cells; thereby producing hybridoma cells producing high titers of high-affinity antibodies.
[0118] In another embodiment, the invention comprises a method for producing mammalian expression cells that produce high-affinity antibodies in high titers from in vitro immunized immunoglobulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro, wherein the donor cells are derived from a donor that is naturally deficient in mismatch repair;
(b) fusing the immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells; and (f) cloning immunoglobulin genes from said hypermutated hybridoma into a mammalian expression cell; thereby producing a mammalian expression cell that produce high titers of high-affinity antibodies in high titer from in vitro immunized imm-unoglobulin-producing cells.

[0119] In another embodiment, the invention comprises a method for producing mammalian expression cells that produce high-affinity antibodies in high titer from in vitro immunized immuno globulin-producing cells comprising: (a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro; (b) fusing the immunoglobulin-producing cells with myeloma cells, wherein the myeloma cells are naturally deficient in mismatch repair, thereby forming parental hybridoma cells, wherein the hybridoma cells are deficient in mismatch repair; (c) incubating the parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells; (d) performing a screen for binding of antibodies to antigen for antibodies produced from the hypermutated hybridoma cells; (e) selecting hypermutated hybridoma cells that produce antibodies with enhanced affinity for the antigen than antibodies produced by the parental hybridoma cells;
and (f) cloning immuno globulin genes from said hypermutated hybridoma cell into a mammalian expression cell; thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.
[0120] The invention also provides hybridoma cells, expression cells produced by any of the methods of the invention, as well as antibodies produced by any of the hybridoma cells and expression cells of the invention.
[0121] In still another embodiment, antigen-specific immunoglobulin-producing cells are produced by: (a) isolating donor cells from an animal; (b) treating said cells with L-leucyl-L-leucine methy ester hydrobromide; (c) incubating said donor cells with an immunogenic antigen in vitro, at 25-37 C, 5-10% CO2, in medium supplemented with 5-15%
serum, and a growth promoting cytokine for 4 days; (d) washing said cells in medium; and (e) culturing said cells in medium supplemented with 5-15% serum an additional 8 days; thereby stimulating the production of antigen-specific immunoglobulin-producing cells.
[0122] The blood cells used in the methods of the invention may be derived from any animal that produces antibodies. Preferably, the donor cells are derived from mammals, including, but not limited to humans, monkeys, mice, rats, guinea pigs, hamsters, gerbils, birds, rabbits, sheep, goats, pigs, horses, and cows. The source of blood is not necessarily limited, but may be whole blood or fractions containing lymphocytes. The blood may be donor or cord blood, for example. In some embodiments, the blood cells are preferably human donor cells.
[0123] The myeloma cells used to create the hybridoma cells in the method of the invention may be derived from any species known to have suitable myeloma cells. For example, but not by way of limitation, the myeloma cells may be conveniently derived from humans or mice.
Suitable examples of myeloma cells include, but are not limited to the HuNS1 myeloma as described in U.S. Patent No. 4,720,459 to Winkelhake, and deposited with the American Type Culture Collection (ATCC) as CRL 8644; GM4672; RPMI 8226; and murine myeloma cell lines (e.g., P3-NS1/1-Ag4-1; P3-x63-Ag8.653; Sp2/0-Ag14; NS/0, NS/1, SP2 and S194).
[0124] The mammalian expression cells suitable for use in certain embodiments of the method of the invention include, but are not limited to Chinese Hamster Ovary cells (CHO cells, Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA, 77: 4216), baby hamster kidney (BBK
cells), human embryonic kidney line 293 (HeLa cells, Graham et al., (1977) J.
Gen Virol., 36:
59), normal dog kidney cell line (e.g., MDCK, ATCC CCL 34), normal cat kidney cell line (CRFK cells), monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587), COS (e.g., COS-7) cells, and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246), fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, mouse NTH/3T3 cells, LMTK31 cells, mouse sertoli cells (TM4, Mather, (1980) Biol. Reprod., 23:243-251);
human cervical carcinoma cells (HELA, ATCC CCL 2); buffalo rat liver cells (BRL 3A, ATCC CRL
1442);
human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
mouse mammary tumor cells (MMT 060562, ATCC CCL51), TN cells (Mather et al. (1982) Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and the human hepatoma line (Hep G2).
[0125] As an alternative to mammalian expression cells, other non-mammalian cells may be used to express the cloned immtmoglobulin genes. Such non-mammalian cells include, but are not limited to insect cells (e.g., Spodoptera frugzperda cells and the like).
Vectors and non-mammalian host cells are well known in the art and are continually being optimized and developed. Any host cell system capable of expressing antibodies may be used in the methods of the invention.
[0126] As used herein, "dominant negative allele of a mismatch repair gene"
refers to an allele of a mismatch repair gene that, when expressed, exerts a dominant phenotype in the cell or organism that leads to an inhibition of the mismatch repair system, even in the presence of a wild-type allele. Cells expressing a dominant negative allele of a mismatch repair gene are hypermutable and accumulate mutations at a higher rate than wild-type cells.
Examples of nucleic acid sequences encoding mismatch repair proteins useful in the method of the invention include, but are not limited to the following: PMS1 (SEQ ID NO:1);
PMS2 (SEQ ID
NO:3); PMS2-134 (SEQ ID NO:5); PMSR2 (SEQ ID NO:7); PMSR3 (SEQ ID NO:9); MLH1 (SEQ ID NO:11); MLH3 (SEQ ID NO:13); MSH2 (SEQ ID NO:15); MSH3 (SEQ ID NO:17);

MSH4 (SEQ ID NO:19); MSH5 (SEQ ID NO:21); MSH6 (SEQ ID NO:23); PMSR6 (SEQ ID
NO:25); PMSL9 (SEQ ID NO:27); yeast MLH1 (SEQ ID NO:29); mouse PMS2 (SEQ ID

NO:31); mouse PMS2-134 (SEQ ID NO:33); Arabidopsis thaliana PMS2 (SEQ ID
NO:35); A.
thaliana PMS2-134 (SEQ ID NO:37) A. thaliana PMS1 (SEQ ID NO:39); A. thaliana (SEQ ID NO:41) A. thaliana MSH2 (SEQ ID NO:43); A. thaliana MSH3 (SEQ ID
NO:45); A.
thaliana MSH6-1 (SEQ ID NO:47); and Olyza satvia MLH1 (SEQ ID NO:49). The corresponding amino acid sequences for the listed nucleic acid sequences are:
PMS1 (SEQ ID
NO:2); PMS2 (SEQ ID NO:4); PMS2-134 (SEQ ID NO:6); PMSR2 (SEQ ID NO:8); PMSR3 (SEQ ID NO:10); MLH1 (SEQ ID NO:12); MLH3 (SEQ ID NO:14); MSH2 (SEQ JD
NO:16); MSH3 (SEQ ID NO:18); MSH4 (SEQ ID NO:20); MSH5 (SEQ ID NO:22); MSH6 (SEQ ID NO:24); PMSR6 (SEQ ID NO:26); PMSL9 (SEQ ID NO:28); yeast MLH1 (SEQ ID

NO:30); mouse PMS2 (SEQ ID NO:32); mouse PMS2-134 (SEQ ID NO:34); Arabidopsis thaliana PMS2 (SEQ ID NO:36); A. thaliana PMS2-134 (SEQ ID NO:38); A. thaliana (SEQ ID NO:40); A. thaliana MSH7 (SEQ ID NO:42) A. thaliana MSH2 (SEQ ID
NO:44); A.
thaliana MSH3 (SEQ ID NO:46); A. thaliana MSH6-1 (SEQ ID NO:48); and Ofyza satvia MLH1 (SEQ ID NO:50).
[0127] As used herein, "high titer" refers to an titer of at least about 1.5 fold higher than the parental cell line. In some embodiments, the titer is at least about 1.5-3 fold higher, 3-5 fold higher, 5-7 fold higher, 7-9 fold higher, or 9-10 fold higher than the parental cell line.
[0128] As used herein, "high affinity" refers to a high antibody binding affinity, that may be calculated according to standard methods by the formula Ka = 8/3 (It-TO where "It" is the total molar concentration of inhibitor uptake at 50% tracer and "Tt" is the total molar concentration of tracer. See Muller (1980) J. Immunol. Meth. 34:345-352. , Binding affinity may also be calculated using the formula BIT = 11*NAb*W108 [(V¨Vai)K+Q.W] (See Antoni and Mariani (1985) J Immunol. Meth. 83:61-68). As used herein, "high affinity" is at least about 1 x 107 M-1. In some embodiments, the antibodies have an affinity of at least about 1 x 108 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 109 M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 101 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1011 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1012M-1. In other embodiments, the antibodies have an affinity of at least about 1 x 1013 M-1.
In other embodiments, the antibodies have an affinity of at least about 1 x 1014 M.
[0129] As used herein, "antigen-specific" refers to an interaction between the CDR regions of the immunoglobulin molecule with an epitope of the antigen wherein the CDR
regions of the immunoglobulin molecule binds to the epitope.

[0130] As used herein, "cured" refers to a state of the cells wherein the dominant negative mismatch repair gene has been eliminated from the cell or wherein the expression of the dominant negative allele has been turned off, leading to a stabilized genome, producing stable biological products such as immunoglobulins.
[0131] In some embodiments of the methods of the invention, mismatch repair is inhibited by introducing a dominant negative allele of a mismatch repair gene into a cell.
[0132] In other embodiments of the methods of the invention, mismatch repair is inhibited by exposing cells that express an antibody to a compound that inhibits mismatch repair. In some embodiments, the compound is an ATPase inhibitor. Suitable ATPase inhibitors include, but not limited to ATP analogs that are capable of blocking the ATPase activity necessary for mismatch repair in the cell. Examples of ATP analogs that may be used in the methods of the invention, include, but are not limited to non-hydrolyzable forms of ATP, such as AMP-PNP
and ATPyS, which block mismatch repair activity (Galio et al. (1999) Nucl.
Acids Res.
27:2325-2331; Allen et al. (1997) EMBO J. 16:4467-4476; Bjornson et al. (2000) Biochem.
39:3176-3183). Other suitable ATPase inhibitors may be identified using mismatch repair reporter cells that may be screened with candidate ATPase inhibitors to identify those compounds which effectively block ATPase activity in the cells.
[0133] In other embodiments of the methods of the invention, mismatch repair is inhibited by exposing cells that express an antibody to a nuclease inhibitor. The nuclease inhibitors are capable of blocking exonuclease activity in the mismatch repair biochemical pathway.
Mismatch repair reporter cells may be screened with candidate nuclease inhibitors to identify compounds that effectively block the exonuclease activity of the mismatch repair system.
Suitable nuclease inhibitors which may be used in the methods of the invention include, but are not limited to analogs of N-ethylmaleimide, an endonuclease inhibitor (Huang et al. (1995) Arch. Biochem. Biophys. 316:485); heterodimeric adenosine-chain-acridine compounds, exonuclease III inhibitors (Belmont et al. (2000) Bioorg. Med. Chem Lett.
10:293-295); as well as antibiotic compounds such as heliquinomycin, which have helicase inhibitory activity (Chino et al. (1998) J. Antibiot. (Tokyo) 51:480-486). Other suitable nuclease inhibitors may be identified using mismatch repair reporter cells that may be screened with candidate nuclease inhibitors to identify those compounds which effectively block nuclease activity in the cells.
[0134] In other embodiments of the methods of the invention, mismatch repair is inhibited by exposing the cells producing antibodies to DNA polymerase inhibitors. DNA
polymerase inhibitors are capable of blocking the polymerization of DNA which is required for functional mismatch repair. Examples of suitable DNA polymerase inhibitors include, but are not limited to actinomycin D (Martin et al. (1990)1 Immunol. 145:1859); aphidicolin (Kuwakado et al.
(1993) Biochem. Pharmacol. 46:1909); 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU) (Kukhanova et al. (1998) Biochem. Pharmacol. 55:1181-1187); and 2'3'-dideoxyribonucleoside 5'-triphosphates (ddNTPs) (Ono et al. (1984) Biomed.
Pharmacother. 38:382-389). Other suitable DNA polymerase inhibitors may be identified using mismatch repair reporter cells that may be screened with candidate DNA
polymerase inhibitors to identify those compounds which effectively block DNA polymerase activity in the cells.
[0135] In other embodiments of the methods of the invention, mismatch repair is inhibited by exposing the cells producing antibody to an anthracene. As used herein the term "anthracene"
refers to the compound anthracene. However, when referred to in the general sense, such as "anthracenes," "an anthracene" or "the anthracene," such terms denote any compound that contains the fused triphenyl core structure of anthracene, i.e., SOO
regardless of extent of substitution. The anthracene may be substituted or unsubstituted.
[0136] As used herein, "alkyl" refers to a hydrocarbon containing from 1 to about 20 carbon atoms. Alkyl groups may straight, branched, cyclic, or combinations thereof.
Alkyl groups thus include, by way of illustration only, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, and the like.
Also included within the definition of "alkyl" are fused and/or polycyclic aliphatic cyclic ring systems such as, for example, adamantane. As used herein the term "alkenyl" denotes an alkyl group having at least one carbon-carbon double bond. As used herein the term "alkynyl"
denotes an alkyl group having at least one carbon-carbon triple bond.
[0137] In some preferred embodiments, the alkyl, alkenyl, alkynyl, aryl, aryloxy, and heteroaryl substituent groups described above may bear one or more further substituent groups; that is, they may be "substituted". In some preferred embodiments these substituent groups can include halogens (for example fluorine, chlorine, bromine and iodine), CN, NO2, lower alkyl groups, aryl groups, heteroaryl groups, aralkyl groups, aralkyloxy groups, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino groups. In addition, the alkyl and aryl portions of aralkyloxy, arylalkyl, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, and aryloxycarbonyl groups also can bear such substituent groups. Thus, by way of example only, substituted alkyl groups include, for example, alkyl groups fluoro-, chloro-, bromo- and iodoalkyl groups, amino alkyl groups, and hydroxyalkyl groups, such as hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and the like. In some preferred embodiments such hydroxyalkyl groups contain from 1 to about 20 carbons.
[0138] As used herein the term "awl" means a group having 5 to about 20 carbon atoms and which contains at least one aromatic ring, such as phenyl, biphenyl and naphthyl. Preferred aryl groups include unsubstituted or substituted phenyl and naphthyl groups.
The term "aryloxy" denotes an aryl group that is bound through an oxygen atom, for example a phenoxy group.
[0139] In general, the prefix "hetero" denotes the presence of at least one hetero (i.e., non-carbon) atom, which is in some preferred embodiments independently one to three 0, N, S, P, Si or metal atoms. Thus, the term "heteroaryl" denotes an aryl group in which one or more ring carbon atom is replaced by such a hetero atom. Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, and imidazolyl groups.
[0140] The term "aralkyl" (or "arylalkyl") is intended to denote a group having from 6 to 15 carbons, consisting of an alkyl group that bears an aryl group. Examples of aralkyl groups include benzyl, phenethyl, benzhydryl and naphthylmethyl groups.
[0141] The term "alkylaryl" (or "alkaryl") is intended to denote a group having from 6 to 15 carbons, consisting of an aryl group that bears an alkyl group. Examples of aralkyl groups include methylphenyl, ethylphenyl and methylnaphthyl groups.
[0142] The term "arylsulfonyl" denotes an aryl group attached through a sulfonyl group, for example phenylsulfonyl. The term "alkylsulfonyl" denotes an alkyl group attached through a sulfonyl group, for example methylsulfonyl.
[0143] The term "alkoxycarbonyl" denotes a group of formula -C(=0)-0-R where R
is alkyl, alkenyl, or alkynyl, where the alkyl, alkenyl, or alkynyl portions thereof can be optionally substituted as described herein.
[0144] The term "aryloxycarbonyl" denotes a group of formula -C(=0)-0-R where R is aryl, where the aryl portion thereof can be optionally substituted as described herein.
[0145] The terms "arylalkyloxy" or "aralkyloxy" are equivalent, and denote a group of formula -0-12(-Ril, where R./ is R is alkyl, alkenyl, or alkynyl which can be optionally substituted as described herein, and wherein le denotes a aryl or substituted aryl group.
[0146] The terms "alkylaryloxy" or "alkaryloxy" are equivalent, and denote a group of formula -0-R/-R8, where R/ is an aryl or substituted aryl group, and Rfl is alkyl, alkenyl, or alkynyl which can be optionally substituted as described herein.

[0147] As used herein, the term "aldehyde group" denotes a group that bears a moiety of formula -C(=0)-H. The term "ketone" denotes a moiety containing a group of formula -R-C(=0)-12.=, where R and R= are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or alkaryl, each of which may be substituted as described herein.
[0148] As used herein, the term "ester" denotes a moiety having a group of formula -R-C(=0)-0-R= or -R-O-C(=0)-R= where R and R= are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or alkaryl, each of which may be substituted as described herein.
[0149] The term "ether" denotes a moiety having a group of formula -R-O-R= or where R and R= are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or alkaryl, each of which may be substituted as described herein.
[0150] The term "crown ether" has its usual meaning of a cyclic ether containing several oxygen atoms. As used herein the term "organosulfur compound" denotes aliphatic or aromatic sulfur containing compounds, for example thiols and disulfides. The term "organometallic group" denotes an organic molecule containing at least one metal atom.
[0151] The term "organosilicon compound" denotes aliphatic or aromatic silicon containing compounds, for example alkyl and aryl silanes.
[0152] The term "carboxylic acid" denotes a moiety having a carboxyl group, other than an amino acid.
[0153] Suitable anthracenes that may be used in the method of the invention comprise compounds having the formula:
. 8 f9 1 Rs R10 R4 wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 0-alkyl, S-alkyl, N-alkyl, 0-alkenyl, S-alkenyl, N-alkenyl, 0-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organo sulfur compound, an organometallic group, a carboxylic acid, an organo silicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein WO 204)4/046330 PCT/US24103/036702 said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups. In some embodiments, the R5 and R6 are hydrogen. In other embodiments, R1-R10 are independently hydrogen, hydroxyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl. Suitable anthracenes for use in the methods of the invention include, but are not limited to 1,2-dimethylanthracene, 9,10-dimethylanthracene, 7,8-dimethylanthracene, 9,10-duphenylanthracene, 9,10-dihydroxymethylanthracene, 9-hydroxymethy1-10-methylanthracene, dimethylanthracene-1,2-diol, 9-hydroxymethy1-10-methylanthracene-1,2-diol, 9-hydroxymethy1-10-methylanthracene-3,4-diol, and 9,10-di-m-tolylanthracene.
[0154] Other suitable anthracenes may be identified using mismatch repair reporter cells that may be screened with candidate anthracenes to identify those compounds which effectively block mismatch repair activity in the cells. In some embodiments, the chemical inhibitor of mismatch repair is an RNA interference molecule that is homologous to a mismatch repair gene of the invention. The technique for generating sequence-specific RNA
interference molecules is well-known in the art and may be found in, for example, Sharp et al. (2000) Science 287:2431-2433; Marx (2000) Science 288:1370-1372; Grishok et a/.
(2001) Science 287:2494-2497; and Fire et al. (1998) Nature 391:806-811.
[0155] In other embodiments of the method of the invention, mismatch repair is inhibited by exposing the cells producing antibody to "antisense compounds" which specifically hybridize with one or more nucleic acids encoding a mismatch repair gene. As used herein, the terms "target nucleic acid" and "nucleic acid encoding a mismatch repair gene"
encompass DNA
encoding a mismatch repair gene, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an antisense compound with its target nucleic acid interferes with the normal function of the nucleic acid, such as replication and transcription. The functions of RNA
disrupted by antisense compounds include such functions as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, and splicing of the RNA to yield one or more mRNA species. The antisense compound thereby inhibits the expression or function of a mismatch repair gene.

[0156] It is preferred to target specific nucleic acids for antisense inhibition of mismatch repair in order to reversibly disrupt the function of a given mismatch repair gene. "Targeting"
an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process, beginning with the identification of a nucleic acid sequence whose function is to be modulated. As disclosed herein, there are several mismatch repair genes that may be targeted by an antisense strategy. Among the various mismatch repair genes that may be targeted are PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, or MSH1, and homologs of PMSR genes as described in Nicolaides et al. (1995) Genomics 30:195-206 and Horii et al. (1994) Biochem. Biophys. Res. Commun. 204:1257-1264, including DNA or RNA. The next step of targeting involves the determination of a site or sites within this gene for the antisense interaction to occur, such that inhibition of the function of the mismatch repair gene occurs. In one embodiment, an intragenic site is targeted. An "intragenic site" is a region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon." A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine in eukaryotes. It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. hi the context of the invention, "start codon"
and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an naRNA molecule transcribed from a gene encoding a mismatch repair gene, regardless of the sequence(s) of such codons.
[0157] It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
[0158] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage.
The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region.
[0159] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated.
The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA
splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA
or pre-mRNA.
[0160] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
[0161] In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
"Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA
molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Complementarity of the antisense oligonucleotide is preferably 100%, however, degeneracy may be introduced into the oligonucleotide such that the complementarily, in some embodiments, is 80-85%, 85-90%, 90-95% or 95-100%.
[0162] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are herein below identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary comprise the region of PMS2, for example, which inhibits the translation of the C-terminal portion of the PMS2 protein, effectively forming a truncation mutant. The region targeted comprises a portion of the PMS2 gene that encodes the 134 amino acid of PMS2, for example.
[0163] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
[0164] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e., from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. In some embodiments, the oligonucleotides are at least about 15 nucleotides in length and may be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides in length.
[0165] In some embodiments, the antisense oligonucleotides comprise a sequence that is complementary to a portion of the mismatch repair sequence shown in SEQ ID
NO:1; SEQ ID
NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ
ID
NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25;
SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID
NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47;
or SEQ ID NO:49. In certain embodiments, the oligonucleotide is at least 15-50 nucleotides in length with 85-100% complementarity.
[0166] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA
and DNA is a 3' to 5' phosphodiester linkage.

[01671 Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
[01681 Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalk-ylphosphotriesters, methyl and other alk-yl phosphonates including 3'-alkylene phosphonates, 5'-allcylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoallcylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalk-ylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a,3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most intemucleotide linkage, i.e., a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
[01691 Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.
3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925;
5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361;
5,194,599;
5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, 101701 Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain hetero atomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed -N, 0, S and CH2 component parts.
[0171] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506;
5,166,315;
5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289;
5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437;
5,792,608; 5,646,269 and 5,677,439.
[0172] In other preferred oligonucleotide mimetics, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of PNA
compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.
Further teaching of PNA compounds can be found in Nielsen et al., (1991) Science 254:1497-1500.
[0173] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH2--NH--0¨CH2--, --C117--N(CH3)--0--CH2-- [known as a methylene (methylimino) or MMI backbone], --CH2-0--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)---- and --0--N(CH3)--C112 --CH2-- [wherein the native phosphodiester backbone is represented as --0--P-0--CH2--] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No.
5,034,506.
[01741 Modified oligonucleotides may also contain one or more substituted sugar moieties.
Preferred oligonucleotides comprise one of the following at the 2' position:
OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or 0-alkyl-0-alkyl, wherein the alkyl, alkenyl and alk-yny1 may be substituted or unsubstituted CI to Clo alkyl or C2 to C10 alkenyI
and alkynyl. Particularly preferred are 0RCH2)n ObiCH3, 0(CH2)nOCH3, 0(CH2)nN112, WH2)CH3, 0(CH2)nONH2, and 0(CH2)ONRCH2)õCH3)12, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: CI
to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, 0NO2, NO2, N37 NI12, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2' -methoxyethoxy (2'-0--CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-M0E) (Martin et al. (1995) Helv. Chim.
Acta 78:486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also blown as 2'-DMA0E, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylarninoethoxyethyl or 2'-DMAEOE), i.e., 2'-0--CH2 --0--CH2--N(CH2)2, also described in examples hereinbelow.
[0175] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (¨CH2--)n group bridging the 2' oxygen atom and the 3' or 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
101761 Other preferred modifications include 2'-methoxy 2'-aminopropoxy (2'.
OCH2CH2CH2NH2), 2'-ally1 (2'-CH2--CH=CH2), 2'-0-ally1(2'-0--CH2--CH=CH2) and 2'-fluor (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position.
A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;
5,658,873;
5,670,633; 5,792,747; and 5,700,920.

[0177] Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--CC--CH2) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cyto sines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indo1-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in THE
CONCISE
ENCYCLOPEDIA OF POLYMER SCIENCE AND ENGINEERING, Kroschwitz, (Ed.) John Wiley &
Sons, 1990, pages 858-859, those disclosed by Englisch et al. (1991) Angewandte Chemie (International Edition) 30:613, and those disclosed by Sanghvi, ANTISENSE
RESEARCH AND
APPLICATIONS, Crooke and Lebleu (Eds.), CRC Press, 1993, pages 289-302.
Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C. (Sanghvi, ANTISENSE RESEARCH AND APPLICATIONS, Crooke and Lebleu (Eds.), CRC Press, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[0178] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302;
5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
5,502,177;
5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;
5,750,692;
5,830,653; 5,763,588; 6,005,096; and 5,681,941.
[0179] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyarnides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodatnines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA.
Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger etal. (1989) Proc.
Natl. Acad. Sci. USA 86:6553-6556), cholic acid (Manoharan et al. (1994) Bioorg. Med.
Chem. Let. 4:1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan etal. (1992) Ann.
N.Y. Acad. Sci. 660:306-309; Manoharan etal. (1993) Bioorg. Med. Chem. Let.
3:2765-2770), a thiocholesterol (Oberhauser et al. (1992) Nucl. Acids Res. 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al. (1991) EMBO J.
10:1111-1118; Kabanov etal. (1990) FEBS Lett. 259:327-330; Svinarchuk et al. (1993) Biochimie 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. (1995) Tetrahedron Lett. 36:3651-3654; Shea et a/. (1990) Nud Acids Res. 18:3777-3783), a polyatnine or a polyethylene glycol chain (Manoharan et al. (1995) Nucleosides & Nucleotides 14:969-973), or adamantane acetic acid (Manoharan etal. (1995) Tetrahedron Lett. 36:3651-3654), a palmityl moiety (Mishra et al. (1995) Biochim. Biophys. Acta 1264:229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal. (1996) J. Phannacol. Exp. Ther.
277:923-937.
[0180] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;
4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;
5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;
5,608,046;
4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;
4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;
5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
[0181] It is not necessary for all positions in a given compound to be Uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric"
antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
101821 Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.
5,013,830; 5,149,797;
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355;
5,652,356; and 5,700,922.
10183] As used herein "donor cells comprising inununoglobulin-producing cells"
or "donor cells comprising immunoglobulin-producing cells" sometimes referred to simply as "donor cells" or "donor blood cells" refers to cells that are capable of producing antibodies when immunized with an antigenic compound. Examples of sources of such donor cells suitable for use in the invention include, but are not limited to spleen cells, lymph node cells, bone marrow cells, and immortalizing tumor infiltrating lymphocytes.
[0184] As used herein, the term "amino acid" denotes a molecule containing both an amino group and a carboxyl group. In some preferred embodiments, the amino acids are a-, j3-, y- or 8-amino acids, including their stereoisomers and racemates. As used herein the term "L-amino acid" denotes an a-amino acid having the L configuration around the a-carbon, that is, a carboxylic acid of general formula CH(COOH)(NH2)-(side chain), having the L-configuration.
The term "D-amino acid" similarly denotes a carboxylic acid of general formula CH(COOH)(NH2)-(side chain), having the D-configuration around the a-carbon.
Side chains of L-amino acids include naturally occurring and non-naturally occurring moieties. Non-naturally occurring (i.e., unnatural) amino acid side chains are moieties that are used in place of naturally occurring amino acid side chaing in, for example, amino acid analogs. See, for example, Lehninger, BIOCHEMISTRY, Second Edition, Worth Publishers, Inc., 1975, pages 72-77 (incorporated herein by reference). Amino acid substituents may be attached through their carbonyl groups through the oxygen or carbonyl carbon thereof, or through their amino groups, or through functionalities residing on their sidechain portions.
[0185] As used herein "polynucleotide" refers to a nucleic acid molecule and includes genomic DNA cDNA, RNA, mRNA and the like.
[01861 As used herein "inhibitor of mismatch repair" refers to an agent that interferes with at least one function of the mismatch repair system of a cell and thereby renders the cell more susceptible to mutation.
[0187] As used herein "hypemmtable" refers to a state in which a cell in vitro or in vivo is made more susceptible to mutation through a loss or impairment of the mismatch repair system.

[0188] As used herein "agents," "chemicals," and "inhibitors" when used in connection with inhibition of1VIIMR refers to chemicals, oligonucleotides, RNA interference molecules, analogs of natural substrates, and the like that interfere with normal function of MMR.
[0189] As used herein, "about" refers to an amount within a range of +1- 10%
of the cited value.
[0190] As used herein, "mitogenic polypeptide" refers to a polypeptide when in combination with the antigen provides stimulation of appropriate cells to increase the immune response against the subject antigen.
[0191] As used herein, "hybridoma" refers to the result of a cell fusion between an immunoglobulin-producing cell and a transformed cell, such as a myeloma cell.
[0192] As used herein, "IgG subclass" refers to a category of immunoglobulins comprising IgGl, IgG2, IgG2a, IgG2b, IgG3, and IgG4.
[0193] As used herein, "mismatch repair gene" refers to a gene that encodes one of the proteins of the mismatch repair complex. Although not wanting to be bound by any particular theory of mechanism of action, a mismatch repair complex is believed to detect distortions of the DNA helix resulting from non-complementary pairing of nucleotide bases.
The non-complementary base on the newer DNA strand is excised, and the excised base is replaced with the appropriate base which is complementary to the older DNA strand. In this way, cells eliminate many mutations that occur as a result of mistakes in DNA
replication. Dominant negative alleles cause a mismatch repair defective phenotype even in the presence of a wild-type allele in the same cell. A non-limiting example of a dominant negative allele of a mismatch repair gene is the human gene hPMS2-134, which carries a truncation mutation at codon 134. The mutation causes the product of this gene to abnormally terminate at the position of the 134th amino acid, resulting in a shortened polypeptide containing the N-terminal 133 amino acids. Such a mutation causes an increase in the rate of mutations which accumulate in cells after DNA replication. Thus, expression of a dominant negative allele of a mismatch repair gene results in impairment of mismatch repair activity, even in the presence of the wild-type allele.
[0194] As used herein, "HAT-sensitive" refers to a lethal effect on cells when cultured in medium containing hypoxanthine, aminopterin and thymidine.
[0195] As used herein, "EBV-negative" refers to lack of infection of Epstein-Barr virus in a cell as measured by production of EBNA protein, or detection of EBV nucleic acids.
[0196] As used herein, "Ig-negative" refers to lack of production in a cell of any light or heavy chains of immunoglobulins.

[0197] As used herein, "screening" refers to an assay to assess the genotype or phenotype of a cell or cell product including, but not limited to nucleic acid sequence, protein sequence, protein function (e.g., binding, enzymatic activity, blocking activity, cross-blocking activity, neutralization activity, and the like). The assays include ELISA-based assays, Biacore analysis, and the like.
[0198] As used herein, "isolated" refers to a nucleic acid or protein that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the nucleic acid or protein is purified to greater than 95% by weight of protein. In other embodiments, the nucleic acid or protein is purified to greater than 99% by weight of protein. Determination of protein purity may be by any means known in the art such as the Lowry method, by SDS-PAGE under reducing or non-reducing conditions using a stain such as a Coomassie blue or silver stain.
Purification of nucleic acid may be assessed by any known method, including, but not limited to spectroscopy, agarose or polyacrylamide separation with fluorescent or chemical staining such as methylene blue, for example.
[0199] The invention provides an in vitro immunization method for obtaining antigen-specific immunoglobulin producing cells wherein the cells produce immunoglobulins of the IgG
subclass, and cells produced by this method. The in vitro immunization procedure comprises combining donor cells with an immunogenic antigen in culture. In one embodiment, the buffy coat of donor cells is used. The donor may be from any source, including, but not limited to cord blood, venous blood, and the like. The source of the blood cells may be from any animal producing immune cells, particularly mammals. Non-limiting examples of blood cell sources include, mice, rats, humans, monkeys, dogs, cats, horses, pigs, sheep, goats, rabbits, birds, cows, guinea pigs and fish. The blood or buffy coat may be further enriched for lymphocytes by any known method, such as, but not limited to differential centrifugation, filtration, and the like.
[0200] Donor cells such as peripheral blood mononuclear cells (PBMC) may be incubated in L-leucyl-L-lysine methyl ester hydrobromide (LLOMe). While not wishing to be bound by any particular theory of operation, LLOme is believed to lysosomotropic and specifically kills cytotoxic cells in the PBMC pool such as NK cells, cytotoxic T cells, and CD8+
suppressor T
cells, while not having an effect on B cells, T helper cells accessory cells and fibroblasts (Borrebaeck (1988) Immunol. Today 9(11):355-359). Generally, the PBMCs may be incubated with LLOMe for a period of 1-30 minutes. In some embodiments, the incubation is performed for 10-20 minutes. In other embodiments, the incubation is performed for 15 minutes. The LLOMe is generally a component of culture medium, such as, for example, RPMI 1640, and is provided in a concentration of about 0.10 to 1mM. In some embodiments, LLOMe is provided in an amount of about 0.10 to 0.50 mM. In other embodiments, LLOMe is provided in an amount of about 0.25 mM.
[0201] The antigen may be any antigen provided that it is immunogenic. Whole proteins or peptides may be used. In addition, one may use, for example, membrane preparations (including those from tumors), lymphoma cells, whole cells, single cells, homogenized cells, pathogens, inclusion bodies, cell lysates, protein preparations, and minced tissue (including tumor tissue). Whole proteins may be in native or denatured conformation.
Peptides may be conjugated to carrier molecules to provide immunogenicity. While not wishing to be bound by any particular theory of operation, carrier molecules may provide additional T cell epitopes which may be useful in stimulating a more robust in vitro antibody response.
Examples of carriers that are suitable for use in the method of the invention include tetanus toxoid, diptheria toxin, thyroglobulin, cholera toxin, BCG, bovine serum albumen (BSA), ovalbumin (OVA), and the like. These carriers are referred to herein as "mitogenic polypeptides."
[0202] Antigens may be conjugated to mitogenic polypeptides in any way known in the art.
For example, fusion proteins may be generated by expressing a polypeptide in a recombinant expression system comprising the polynucleotide encoding at least a portion of the antigen joined in-frame to a polynucleotide encoding at least a portion of the mitogenic polypeptide.
The fusion protein may have the mitogenic polypeptide joined at either the amino- or carboxy terminus of the antigen. In some embodiments, more that one antigen may be expressed as a fusion protein in combination with a mitogenic polypeptide. In other embodiments, more than one mitogenic polypeptide may be expressed as a fusion protein with the antigen or antigens.
In other embodiments, more than one mitigenic polypeptide and more than one antigen may be expressed together as a single fusion protein.
[0203] In an alternative embodiment, at least a portion of the mitogenic polypeptide is conjugated to at least a portion of the antigen using chemical cross-linkers.
Examples of chemical cross-linkers include, but are not limited to gluteraldehyde, formaldehyde, 1,1-bis (diazoacety1)-2-phenylethane, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinirnidyl esters such as 3,3'-dithiobis (succinimidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane).
Derivatizing agents such as methyl-3-[(p-azido-phenyl)dithio] propioimidate yield photoactivatable intermediates which are capable of forming cross-links in the presence of light. Alternatively, for example, a lysine residue in the mitogenic polypeptide or antigen may be coupled to a C-terminal or other cysteine residue in the antigen or mitogenic polypeptide, respectively, by treatment with N-y-maleimidobutyryloxy-succinimide (Kitagawa and Aikawa (1976) J. Biochem. 79, 233-236). Alternatively, a lysine residue in the mitogenic polypeptide or antigen may be conjugated to a glutamic or aspartic acid residue in the antigen or mitogenic polypeptide, respectively, using isobutylchloroformate (Thorell and De Larson (1978) RADIOIMMUNOASSAY AND RELATED TECHNIQUES: METHODOLOGY AND CLINICAL
APPLICATIONS, p. 288). Other coupling reactions and reagents have been described in the literature.
[0204] The conditions for the in vitro immunization procedure comprise incubating the cells at about 25-37 C, (preferably 37 C) supplied with about 5-10% CO2. In some embodiments, the incubation is performed with between about 6-9% CO2. In other embodiments the incubation is performed in about 8% CO2. The cell density is between about 2.5 to 5 x 106 cells/m1 in culture medium. In some embodiments, the culture medium is supplemented with about 2-20% FBS. In other embodiments, the culture medium is supplemented with about 5-15%
FBS. In other embodiments, the culture medium is supplemented with about 7-12%
FBS. In other embodiments, the culture medium is supplemented with about 10% FBS.
[0205] The in vitro stimulation culture medium is supplemented with cytokines to stimulate the cells and increase the immune response. In general IL-2 is supplied in the culture medium.
However, other cytokines and additives may also be included to increase the immune response. Such cytokines and factors may include, for example, 11-4 and anti-antibodies.
[0206] The fusion of myeloma cells with the immunoglobulin-producing cells may be by any method known in the art for the creation of hybridoma cells. These methods include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;
and U.S. Patent No. 4,376,110) (see also, Brown et al. (1981) J. Immunol.
127:539-546;
Brown et al. (1980) J. Biol. Chem. 255 (10:4980-4983; Yeh et al. (1976) Proc.
Natl. Acad.
Sci. USA 76:2927-2931; and Yeh et al. (1982) Int. J. Cancer 29:269-275), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.
Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo.

[0207] The technology for producing monoclonal antibody hybridomas is well-known to those of skill in the art and is described, for example in Kenneth, in MONOCLONAL
ANTIBODIES: A
NEW DIMENSION IN BIOLOGICAL ANALYSES, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner (1981) Yale J. Biol. Med., 54:387-402; Ga.lfre et al. (1977) Nature 266:55052;
and Gefter et al. (1977) Somatic Cell Genet. 3:231-236). However, many variations of such methods are possible and would be appreciated by one of skill in the art.
Thus, the techniques for generation of hybridomas is not limited to the disclosures of these references.
[0208] Any myeloma cell may be used in the method of the invention.
Preferably, the myeloma cells are human cells, but the invention is not limited thereto or thereby. In some embodiments, the cells are sensitive to medium containing hypoxanthine, aminopterin, an thymidine (HAT medium). In some embodiments, the myeloma cells do not express immunoglobulin genes. In some embodiments the myeloma cells are negative for Epstein-Barr virus (EBV) infection. In preferred embodiments, the myeloma cells are HAT-sensitive, EBV negative and Ig expression negative. Any suitable myeloma may be used. An example of such a myeloma is that described in U.S. Patent No. 4,720,459 to Winkelhake, and deposited with the American Type Culture Collection (ATCC) as CRL 8644. Murine hybridomas may be generated using mouse myeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/0-Ag14 myeloma lines). These murine myeloma lines are available from the ATCC.
[0209] In some embodiments of the method of the invention, the hybridoma cells and/or mammalian expression cells may be rendered hypermutable by the introduction of a dominant negative allele of a mismatch repair gene. The dominant negative allele of the mismatch repair gene may be introduced into the hybridoma cell (i.e., after the fusion of immtmoglobulin-producing cells with the myeloma cells) or may be introduced into the myeloma cell prior to the fusions. The invention, therefore, also provides hypermutable myeloma cells for use in the generation of hybridoma cells. The dominant negative allele may also be introduced into the mammalian expression cells.
[0210] The dominant negative allele of the mismatch repair gene is in the form of a polynucleotide which may be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide. The polynucleotide can be cloned into an expression vector containing a constitutively active promoter segment (such as, but not limited to, CMV, SV40, EF-1 Dor LTR sequences) or to inducible promoter sequences such as those from tetracycline, or ecdysone/glucocorticoid inducible vectors, where the expression of the dominant negative mismatch repair gene can be regulated. The polynucleotide can be introduced into the cell by transfection.
[0211] Transfection is any process whereby a polynucleotide is introduced into a cell. The process of transfection can be carried out in vitro, e.g., using a suspension of one or more isolated cells in culture. The cell can be any immortalized cell used for creating hybridomas for the production of monoclonal antibodies, or the cell may be the hybridoma itself. The hybridomas may be heterohybridoma cells (e.g. human-mouse cell fusions) or homohybridoma cells (e.g., human-human hybridoma cells and mouse-mouse hybridoma cells).
[0212] In general, transfection will be carried out using a suspension of cells, or a single cell, but other methods can also be applied as long as a sufficient fraction of the treated cells or tissue incorporates the polynucleotide so as to allow transfected cells to be grown and utilized.
The protein product of the polynucleotide may be transiently or stably expressed in the cell.
Techniques for transfection are well known. Available techniques for introducing polynucleotides include but are not limited to electroporation, -transduction, cell fusion, the use of calcium chloride, and packaging of the polynucleotide together with lipid for fusion with the cells of interest. Once a cell has been transfected with the mismatch repair gene, the cell can be grown and reproduced in culture. If the transfection is stable, such that the gene is expressed at a consistent level for many cell generations, then a cell line results.
[0213] The dominant negative allele of the mismatch repair gene may be derived from any known mismatch repair gene including, but not limited to PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1 , GTBP, MSH3, MSH2, MLH3, or MSH1, and homologs of PMSR genes as described in Nicolaides et al. (1995) Genomics 30:195-206 and Horii et al.
(1994) Biochem.
Biophys. Res. Commun. 204:1257-1264 and the like. "Dominant negative alleles"
as used herein, refers to the ability of the allele to confer a hypermutable status to the cell expressing the allele. Any allele which produces such effect can be used in this invention. The dominant negative alleles of a mismatch repair gene can be obtained from the cells of humans, animals, yeast, bacteria, or other organisms. Dominant negative alleles of mismatch repair genes that are suitable for use in the invention have certain functional characteristics associated with structural features. A non-limiting example of a dominant negative mismatch repair gene is the PMS2 truncation mutant, PMS2-134. This gene contains a mutation which truncates the PMS2 protein after amino acid 133. The lack of the C-terminus in the PMS2 protein is believed to interfere with the binding of PMS2 with Screening cells for defective mismatch repair activity can identify such alleles. Cells from animals or humans with cancer can be screened for defective mismatch repair. Cells from colon cancer patients may be particularly useful. Genomic DNA, cDNA, or mRNA from any cell encoding a mismatch repair protein can be analyzed for variations from the wild type sequence. Dominant negative alleles of a mismatch repair gene can also be created artificially, for example, by producing variants of the hPMS2-134 allele or other mismatch repair genes. Various techniques of site-directed mutagenesis can be used. The suitability of such alleles, whether natural or artificial, for use in generating hypermutable cells or animals can be evaluated by testing the mismatch repair activity caused by the allele in the presence of one or more wild-type alleles, to determine if it is a dominant negative allele.
[0214] Dominant negative alleles of such genes, when introduced into cells or transgenic animals, increase the rate of spontaneous mutations by reducing the effectiveness of DNA
repair and thereby render the cells or animals hypermutable. This means that the spontaneous mutation rate of such cells or animals is elevated compared to cells or animals without such alleles. The degree of elevation of the spontaneous mutation rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell or animal. The hypermutable hybridoma cells will accumulate new mutations in gene(s) to produce new output traits within the hybridoma. The hybridoma cells can be screened for desired characteristics and cell lines bearing these characteristics may be expanded.
Furthermore, the hybridoma cells may be "cured" of the mismatch repair defect by eliminating the dominant negative mismatch repair gene in the cell or by turning of its expression, leading to stable biological products consisting of altered genes, RNAs, or polypeptides.
[0215] The dominant negative alleles of the mismatch repair gene may be introduced as part of a vector. The polynucleotide encoding the dominant negative mismatch repair protein allele may be operably linked to a promoter that functions in the cell to drive expression of the dominant negative allele of the mismatch repair gene. Other elements of the vector may include an origin of replication, one or more selectable markers, such as a drug resistance gene that allows the cells to grow in the presence of a growth inhibitory compound.
[0216] In embodiments of the invention that utilize myeloma cells or donor immunoglobulin-producing cells that are naturally deficient in mismatch repair, the invention may further comprise the step of restoring genetic stability of the hybridoma by introducing a wild-type mismatch repair gene into the cell to complement the deficiency and restore genetic stability.
[0217] Another aspect of the invention is the use of cells lacking MMR (either due to defects in endogenous mismatch repair genes, or due to the introduction of a dominant negative MMR
gene) and chemical mutagens to cause an enhanced rate of mutations in a host's genome. The lack of MMR activity has been known to make cells more resistant to the toxic effects of DNA
damaging agents. This invention comprises making proficient MMR cells mismatch repair defective via the expression of a dominant negative MMR gene allele and then enhancing the genomic hypermutability with the use of a DNA mutagen. Chemical mutagens are classifiable by chemical properties, e.g., alkylating agents, cross-linking agents, etc.
The following chemical mutagens are useful, as are others not listed here, according to the invention and may be used to further enhance the rate of mutation in any of the embodiments of the method of the invention: N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), procarbazine hydrochloride, chlorambucil, cyclophosphamide, methyl methanesulfonate (1\4MS), ethyl methanesulfonate (EMS), diethyl sulfate, acrylamide monomer, triethylene melamin (TEM), melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine (MNNG), 7,12 dimethylbenz (a) anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan. In a preferred aspect of the invention, a mutagenesis technique is employed that confers a mutation rate in the range of 1 mutation out of every 100 genes; 1 mutation per 1,000 genes. The use of such combination (MMR deficiency and chemical mutagens will allow for the generation of a wide array of genome alterations (such as but not limited to expansions or deletions of DNA segments within the context of a gene's coding region, a gene's intronic regions, or 5'or 3' proximal and/or distal regions, point mutations, altered repetitive sequences) that are preferentially induced by each particular agent.
[0218] Mutations can be detected by analyzing for alterations in the genotype of the cells or animals, for example by examining the sequence of genomic DNA, cDNA, messenger RNA, or amino acids associated with the gene of interest. Mutations can also be detected by screening the phenotype of the gene. An altered phenotype can be detected by identifying alterations in electrophoretic mobility, spectroscopic properties, or other physical or structural characteristics of a protein encoded by a mutant gene. One can also screen for altered function of the protein in situ, in isolated form, or in model systems. One can screen for alteration of any property of the cell or animal associated with the function of the gene of interest, such as but not limited to measuring protein secretion, chemical-resistance, pathogen resistance, etc.
[0219] In some embodiments of the method of the invention, inducible vectors that control the expression of a dominant negative and normally functioning MMR gene are used.
This strategy restores DNA stability once a host cell or organism exhibiting a new output trait, altered gene, RNA or polypeptide has been generated via trait selection with or without the combination of chemical mutagens to establish a genetically stable version of this cell or organism. In the case of MMR defective cells as a result of ectopically expressing a dominant negative MMR gene allele, the MMR activity is decreased or completely eliminated by removing the inducer molecule from the cell culture or organism's environment.
In addition, the expression of a dominant negative MMR gene can be suppressed by knocking out the MMR gene allele using methods that are standard to those skilled in the art of DNA knockout technology in germ or somatic cells (Waldman etal. (1995) Cancer Res. 55:5187-5190).
[0220] The chiral position of the side chains of the anthracenes is not particularly limited and may be any chiral position and any chiral analog. The anthracenes may also comprise a stereoisomeric form of the anthracenes and include any isomeric analog.
[0221] Examples of hosts are but not limited to cells or whole organisms from human, primate, mammal, rodent, plant, fish, reptiles, amphibians, insects, fungi, yeast or microbes of prokaryotic origin.
102221 A more detailed disclosure of particular embodiments of the invention follows in the specific examples, however, the invention is not limited thereto or thereby.
EXAMPLES
Example I: Generation of Hybridomas Secreting Human Monoclonal Antibodies to Tetanus Toxin (TT) A. Generation and Assaying of TT-specific B lymphocytes 10223] Isolation of lymphocytes from Donor. Lymphocytes were isolated from whole blood by centrifugation through Ficoll-Paque according to the manufacturer's instructions. Isolated lymphocytes were incubated with 0.25mM Leu-Leu methyl ester hydrobromide (LLOMe) prepared in RPM' 1640 medium containing 2% fetal bovine serum (FBS) for 15 minutes at room temperature. The cells were then washed three times with culture medium.
102241 In vitro stimulation of isolated lymphocytes. The cells were incubated at 37 C in a incubator, supplied with 8% CO2, at a density between 2.5 to 5 x 106 cells/ml in culture medium supplemented with 10% FBS and TT and IL-2 at various concentrations.
After four days of culture, the cells were washed four times with medium and the culture was continued for additional eight days.
102251 Measurement of the B cell response. Lymphocyte culture supernatants were collected on day 12 of the culture and tested in an ELISA for the presence of anti-TT
antibodies. Briefly, TT or BSA at 0.5 g/ml in 0.05 M carbonate-bicarbonate buffer was immobilized onto an EIA plate. After blocking with 1% bovine serum albumin (BSA) in PBS
containing 0.05% Tween 20, the supernatant was added to the wells. Antibodies bound to TT
*Trade-mark =

were detected with peroxidase-labeled goat anti-human IgG or anti-human IgM.
TMB was used for color development. The plate was read using a Microplate reader with a 450 nm filter. A supernatant sample that had antibody bound to TT, but not to BSA, and in which the signal was two times the assay background was considered positive. The positive cells were pooled, and used for hybridoma production (Fig. 1).
[0226) Notably, peripheral blood mononuclear cells (PBMCs) from some donors contain a fraction of B cells that secret TT-specific antibodies in culture. This is due to the fact that about 90% of the population in the United States has been vaccinated against TT. Such sera also has a titer of higher than 1000 (Fig. 2). However, the percentage of positive events is greatly increased when PBMCs are immunized in vitro with TT (Fig. 3). The intensity of the PBMC response is also enhanced with the stimulation of TT alone or in combination with IL-2 or CD4OL (Fig. 4). Similar effects were observed with other antigens (data not shown).
[0227] Generation of hybridomas secreting human antibodies. To prepare activated lymphocytes, cells were pooled and cultured in T flasks at 0.5 ¨ 1 x 106 cells/ml in culture medium supplemented with 10% FBS one day prior to the fusion. To prepare the fusion partner, mouse myeloma NSO cells were transfected with human PMS2-134 expression vector as described in Nicolaides et al. (1998) Mot Cell. Biol. 18(3):1635-1641. The cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine (Complete Medium) and the culture was kept in log phase.
102281 Next, lymphocytes were harvested and counted. An equal number of myeloma cells was harvested. Both types of cells were combined and washed three times with RPM! 1640 medium. Polyethylene glycol (PEG) was added dropwise to the loosened cell pellet, and the PEG was subsequently diluted out slowly with 25 ml of RPM! medium in a course of 2.5 minutes. After diluting out the PEG, fused cells were suspended in Complete Medium supplemented with HAT and 20% FBS, and seeded onto 96-well plates.
[0229] Screening and characterization of antigen-specific hybridoma clones.
When the hybridoma cells grew to semi-confluence, supernatants were collected and subjected to an EL1SA for antigen-specific reactivity. As an example, hybridomas derived from TT-immunized lymphocytes were tested. Briefly, TT or BSA at 0.5 ugiml in 0.05 M
carbonate-bicarbonate buffer was immobilized onto the ETA plate. After blocking with 1%
bovine serum albumen in PBS containing 0.05% Tween 20, the cell culture supemate was added to the wells. Antibodies bound to TT were detected with peroxidase-labeled goat anti-human IgG or anti-human IgM. TM33 was used for color development. The plate was read in the Microplatek reader with a 450 nm filter. A cell clone that showed reactivity to IT but not to BSA was * Trade-mark considered positive (Fig. 5). Positive clones were expanded and subcloned by limiting dilution to generate monoclonal cells.
Example 2: Generation of hybridomas secreting human monoclonal antibodies to epidermal growth factor receptor (EGFR) (self antigen) A. Generation of EGFR-specific B lymphocytes 102301 Preparation of Antigen. Human epidermal growth factor receptor (EGFR), purified from A431 cells, was purchased from Sigma. Previous studies found that immune responses to this antigen were very weak, most likely due to tolerance. In order to enhance immunization, we conjugated the EGFR to tetanus toxin C (EGFR-TT) and the conjugate was used as immunogen for in vitro immunization in order to overcome any immunotolerance.
[0231] Preparation of EGFR-TT conjugate. 100 ug of purified EGFR was reconstituted in 100 ul of sterile MilliQ-grade water. 1 mg of purified, lyophilized recombinant tetanus toxin C fragment (TT-C) was dissolved in sterile MilliQ-grade water to yield a 2 mg/ml TT-C
solution. Crosslinking was performed in 50 mM sodium carbonate buffer pH 9.0 at equimolar ratios of EGFR to TT-C, using glutaraldehyde at a final concentration of 0.5%
for 3 hours at room temperature, followed by 4 C overnight. Glutaraldehyde was quenched by addition of a fresh 100 mg/ml solution of sodium borohydride in 50 mM sodium carbonate pH
9.0, under open atmosphere for 1 hour at 4 C. Crosslinked products were dialyzed against Ca2+-, Mg2+-.
free phosphate-buffered saline overnight at 4 C, using 3.5K MWCO Slide-A-Lyzer cassettes.
The reaction was monitored by Western blotting, using commercial anti-EGFR
(mAb-15) and anti-TT-C (Roche) monoclonal antibodies. By this method, greater than 70% of the components are crosslinked, and appear as immunoreactive species of greater MW
than the starting material (data not shown).
102321 In vitro stimulation of peripheral blood mononuclear cells (PBMC).
LLOMe-pretreated PBMC were incubated at a density of 3 x 106 cells/ml in culture medium supplemented with 10% FBS and a stimuli mixture. The stimuli mixture was composed of EGFR-TT at a concentration of 50 ng/ml with or without recombinant human IL-2 at 20111, mouse anti-human CD40 antibody as CD4OL at 0.5 ug/ml (used to enhance IgG
class switching). After four days of culture, the cells were re-fed with complete medium, in the absence of added stimulus, every three or four days. Culture supernatants were collected on days 12-18 and tested for EGFR-specific antibodies.
[0233] Detection of EGFR-specific antibody response. The PBMC response to the stimulation was examined in a EGFR-specific ELISA. Briefly, EGFR, TT, or BSA
at 0.5 ug/ml in 0.05 M carbonate-bicarbonate buffer, pH 9.6, was immobilized onto EIA
plates.
*Trade-mark After blocking the plates with 5% non-fat dry milk in PBS containing 0.05%
Tween 20, the supernatant was added to the wells. Antibodies from the supernatant bound to immobilized antigens were detected with peroxidase-labeled goat anti-human IgG+IgM (H+L).
TMB
substrate kit was used for color development. The plates were read in a Microplate reader with a 450 rim filter. A supernatant sample containing antibody that bound to EGFR, but not to TT and BSA, was considered positive. A robust response was observed in cultures immunized to the EGFR-TT as compared to controls. While anti-EGFR responses were observed in PBMCs for a small fraction of donors, the percentage of positive clones was greatly increased when PBMCs were immunized in vitro with EGFR complexed with TT (Fig.
6). Positive cells were pooled and used for hybridoma production.
[0234] Generation of Hybridomas. To prepare activated lymphocytes, cells were pooled and cultured in T flasks at 0.5 ¨ 1 x 106 cells/ml in culture medium supplemented with 10% FBS
one day prior to the fusion. To prepare the fusion partner, mouse myeloma NSO
cells were transfected with human PMS2-134 expression vector as described in Nicolaides et al. (1998) Mol. Cell. Biol. 18(3):1635-1641. The cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine (Complete Medium) and the culture was kept in log phase.
[0235] Screening and characterization of antigen-specific hybridoma clones.
When the hybridoma cells grew to semi-confluence, supernatants were collected and subjected to an ELISA for antigen-specific reactivity. As an example, hybridomas derived from EGFR-immunized lymphocytes were tested. Briefly, EGFR, TT TNFR1 or BSA at 0.5 ug/ml in 0.05 M carbonate-bicarbonate buffer was immobilized onto the EIA plate. After blocking with 1%
bovine serum albumen in PBS containing 0.05% Tween 20, the cell culture supernate was added to the wells. Antibodies bound were detected with peroxidase-labeled goat anti-human IgG or anti-human IgM. TMB was used for color development. Normal human IgG
(nhIgG) and IgM (nhIgM) were used as controls. The plate was read in the Microplate reader with a 450 rim filter. A cell clone that showed reactivity to EGFR, but not to BSA
was considered positive (Fig. 7).
Example 3 A. Isolation of PBMC from whole blood [0236] Approximately 200m1 of whole blood mixed with 200m1PBS4- was centrifuged through Ficoll-Paque at 2000rpm for 30min. Serum was aspirated, the interface layer containing lymphocytes was collected and diluted 1:3 with PBS' - and centrifuged at 2000 rpm for 10min. The supernatant fluid was aspirated and the pellet was resuspended in 10 ml PBS'. The cell suspension was split into two 50 ml conical tubes and PBS' -was added to each tube to adjust the volume to 35 ml each. The tubes were centrifuged at 800 rpm for 7 minutes to remove the platelets. After aspirating the supernatant fluid, the pellet was resuspended in 10 ml ACK Lysing Buffer and incubated for 5 minutes at room temperature.
Following lysis, 35 ml PBS' - was added to the tubes and the tubes were centrifuged at 1000 rpm for 7 minutes. The cells were then washed with 45 ml RPMI medium.
B. Preparation of Dendritic Cells [0237] Cells were centrifuged at 1000 rpm for 7 minutes and resuspended at 1 x 108 cells per 40 ml cRPMI for a density of 2.5 x 106cells/ml. The cells were incubated at 37 C/8% CO2 for 2 hours. Non-adherent cells were removed for further treatment (see Step C), and the adherent cells were carefully rinsed twice with PBS'. Adherent cells were cultured in cRPMI
supplemented with 400 IU/ml IL-4 and 50 ng/m1 GM-CSF.
C. LLOMe treatment and cryopreservation of non-adherent culture [0238] The non-adherent cell culture was centrifuged at 1000 rpm for 7 minutes. The supernatant fluid was aspirated and the pellet was resuspended in 10 ml RPMI
supplemented with 2% FBS and freshly thawed 85 jug/m1LLOMe. The cells were incubated for 15 minutes at room temperature. The cells were washed twice with cRPMI and resuspended in 45 ml cRPMI. The cells were transferred to an upright T25 flask at a density of 5 x 106 cells/ml in cRPMI supplemented with 2 jig/m1 PHA and incubated at 37 C/8% CO2 for 24 hours. The non-adherent cells were harvested, centrifuged at 1000 rpm for 7 minutes, and the cell pellet was resuspended in 5 ml cold cRPMI/5%DMSO. The tubes containing the cells were wrapped in paper towels and stored at -80 C until needed.
D. Tumor Immunization [0239] On day 6 of the procedure for isolation of dendritic cells, tumor cells were thawed in 2.5 ml pre-warmed medium at 37 C. The flask of dendritic cells was rinsed twice with 10 ml PBS. The dendritic cells were incubated with gentle rocking in 5 ml Cell Dissociation Buffer (Invitrogen Cat. No. 13151-014), and the solution was collected (scraping the remaining cells from the flask. The flask was rinsed with 10 ml cRPMI and the medium was collected. The cells were centrifuged at 1000 rpm for 7 minutes and the pellet was resuspended at 4 x 106 cells/ml cRPMI. Cells were distributed in a culture plate at a density of 1 x 106 cells/well. A tumor sample was chopped into fine pieces of approximately 1-3 mm3.

An aliquot of the tumor suspension was transferred to all but 1 well, titrating the amount of tumor per well. An aliquot of 0.25 ml cRPMI was added to the control well. The total volume in the wells was 0.5m1/well. The dendritic cells and tumor cells were co-cultured at 37 C/8%
CO2 for 24 hours.
E. Co-culture of PBMC with DC
[0240] Frozen PBMC were thawed by adding 40 ml cRPMI/30 IU/ml IL-2/600 IU/ml IL-4/0.75 iu.g/m1CD-40L pre-warmed to 50 C to the frozen cells. When thawed, the cells were incubated for 1-2 hours at 37 C. The cells were centrifuged at 1000 rpm for 7 minutes and the pellet was resuspended in 5 ml of a 2X cocktail of cRPMI/ 60 IU/ml IL-2/1200 IU/ml IL-4/1.5 g/m1 CD-40L. The cell suspension was divided among wells in a tissue culture plate at 0.5 ml/well of suspension and diluted with 0.5 ml medium for a final concentration of 30 IU/ml IL-2, 600 IU/ml IL-4, and 0.75 g/m1 CD-40L. Cells were fed with cRPMI
supplemented with 20 IU/ml IL-2, 400 RI/m1 IL-4, 100 IU/ml IL-10, and 0.5 g/m1 CD-40L.
F. Fusion [0241] Tumor-immunized PBMCs were then fused with A6 myeloma cells to generate hybridomas. Briefly, lymphocytes were harvested from 75% tumor and 100% tumor wells, rinsed with 1 ml RPMI, transfer to conical tubes, and the volume was adjusted to 5 ml with cRPMI. The cells were centrifuged through Ficoll-Paque, and the supernatant fluid was aspirated. Interfaces containing cells from all tubes were combined and the cells were rinsed with cRPMI. The cells were then resuspended in 7.5 ml cRPMI. Viable cells were assessed by trypan blue exclusion. A6 cell viability was also assessed by trypan blue exclusion. A6 cells and tumor-immunized lymphocytes were centrifuged separately at 1200 rpm for 10minutes. The supernatant fluids were aspirated and the cells were washed with 10m1 DPBS
4-Aube. Each cell line was washed three times with 2 ml cold Mannitol Fusion Medium (AVM) (0.3M Marmitol, 0.18mM MgCl2, 0.18mM CaCl2, 1mM Hepes) and the cells were , combined and resuspended in MFM at a density of 3 x 106 A6 cells and 3 x 106 PBMCs in 200 1 for a total of 6 x 106 cells in 200 1. BTX 450 microslides were sterilized with 65 L 100%
EWE and pre-wetted with 6411 MFM. A 400 aliquot of cell suspension was distributed evenly onto a BTX 450-1 microslide. To fuse the cells, the ECM 2001 conditions were set as follows: alignment conditions, 20V for 30 seconds; pulse conditions, 150V for 30 p. seconds (1X); compression conditions, 20V for 9 seconds. After fusion, the cells were transferred to one well of a 24 well plate containing 1 ml phenol red-free cRPMI. The fusion steps were repeated for the remaining cell suspensions, rinsing slide between fusions with 65 j.tL MFM.
The culture plate containing fused cell cultures was incubated overnight at 37 C/8%CO2. The fused cells were cloned and assessed by ELISA for IgG and IgM production. The results are shown in Fig. 8.
Example 4: In vitro immunization Purified GM-CSF from a commercial source is administered in vitro to peripheral blood mononuclear cells (PBMC).
A. Generation and Assaying of GM-CSF-specific B lymphocytes Isolation of lymphocytes from Peripheral Blood.
[0242] Lymphocytes are isolated from whole blood by centrifugation through Ficoll-Paque according to the manufacturer's instructions. Isolated lymphocytes are incubated with 0.25 mM Leu-Leu methyl ester hydrobromide (LLOMe) prepared in RPMI 1640 medium containing 2% fetal bovine serum (FBS) for 15 minutes at room temperature. The cells are then washed three times with culture medium.
In vitro stimulation of isolated lymphocytes.
[0243] The cells are incubated at 37 C in an incubator, supplied with 8% CO2, at a density between 2.5 to 5 x 106 cells/ml in culture medium supplemented with 10% FBS
and GM-CSF
and IL-2 at various concentrations. After four days of culture, the cells are washed four times with medium and the culture was continued for additional eight days.
Measurement of the B cell response.
[0244] Lymphocyte culture supernatants are collected on day 12 of the culture and tested in an ELISA for the presence of anti-GM-CSF antibodies. Briefly, GM-CSF or BSA at 0.5 i.tg/m1 in 0.05 M carbonate-bicarbonate buffer is immobilized onto an ETA plate. After blocking with 1% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20, the supernatant is added to the wells. Antibodies bound to GM-CSF are detected with peroxidase-labeled goat anti-human IgG or anti-human IgM. TMB is used for color development. The plate is read using a Microplate reader with a 450 nm filter. A supernatant sample that had antibody bound to GM-CSF, but not to BSA, and in which the signal was two times the assay background is considered positive. The positive cells are pooled, and used for hybridoma production.
Generation of hybridomas secreting human antibodies.
[0245] To prepare activated lymphocytes, cells are pooled and cultured in T
flasks at 0.5 ¨ 1 x 106 cells/ml in culture medium supplemented with 10% FBS one day prior to the fusion. To prepare the fusion partner, mouse myeloma NSO cells are transfected with human expression vector as described in Nicolaides et aL (1998) MoL Cell. Biol.
18(3):1635-1641.
The cells are cultured in RPMI 1640 supplemented with 10% FBS and 2 mM
glutamine (Complete Medium) and the culture is kept in log phase.
[0246] Next, lymphocytes are harvested and counted. An equal number of myeloma cells is harvested. Both types of cells are combined and washed three times with RPMI

medium. Polyethylene glycol (PEG) is added dropwise to the loosened cell pellet, and the PEG is subsequently diluted out slowly with 25 ml of RPMI medium in a course of 2.5 minutes. After diluting out the PEG, fused cells are suspended in Complete Medium supplemented with HAT and 20% FBS, and seeded onto 96-well plates.
Screening and characterization of antigen-specific hybridoma clones.
[0247] When the hybridoma cells grew to semi-confluence, supernatants are collected and subjected to an ELISA for antigen-specific reactivity. As an example, hybridomas derived from TT-immunized lymphocytes are tested. Briefly, TT or BSA at 0.5 ug/ml in 0.05 M
carbonate-bicarbonate buffer is immobilized onto the EIA plate. After blocking with 1%
bovine serum albumin in PBS containing 0.05% Tween 20, the cell culture supernate is added to the wells. Antibodies bound to GM-CSF are detected with peroxidase-labeled goat anti-human IgG or anti-human IgM. TMB is used for color development. The plate is read in the Microplate reader with a 450 nm filter. A cell clone that showed reactivity to GM-CSF but not to BSA is considered positive. Positive clones are expanded and subcloned by limiting dilution to generate monoclonal cells.
Example 5: Generation of hybridomas secreting human monoclonal antibodies to GM-CSF-KLH
A. Generation of GM-CSF-specific B lymphocytes Preparation of Antigen.
[0248] Human GM-CSF was purchased from a vendor. In order to enhance immunization, GM-CSF was conjugated to keyhole limpet hemocyanin (KLH) (GM-CSF-KLH) and the conjugate was used as immunogen for in vitro immunization in order to overcome any immunotolerance.
Preparation of GM-CSF-KLH conjugate.
[0249] Purified GM-CSF was reconstituted in sterile MilliQ-grade water to yield a lmg/m1 solution. Purified, lyophilized recombinant KLH was dissolved in sterile MilliQ-grade water to yield a 1 mg/ml KLH solution. A 0.2% solution of glutaraldehyde in PBS was prepared.
Crosslinking was performed by combining 25 ul of 1 mg/ml KLH, 25 ul of 1 mg/ml GM-CSF, and 50 ul 0.2% glutaraldehyde in a microcentrifuge tube wrapped in aluminum foil at room temperature, with shaking for 1 hour. Following cross-linking, 25 ul of 1 M
glycine was added to the tube and the solution was incubated an additional 1 hour at room temperature with shaking. Crosslinked products were dialyzed against three changes of 300 ml PBS. The reaction was monitored by Western blotting, using a commercial anti-GM-CSF and anti-KLH
monoclonal antibodies. By this method, greater than 80% of the components are crosslinked, and appeared as immunoreactive species of greater MW than the starting material (data not shown).
In vitro stimulation of peripheral blood mononuclear cells (PBMC).
[0250] LLOMe-pretreated PBMC were incubated at a density of 3 x 106 cells/ml in culture medium supplemented with 10% FBS and a stimuli mixture. The stimuli mixture was composed of GM-CSF-KLH at a concentration of 50 ng/ml with or without recombinant human IL-2 at 20 IU, mouse anti-human CD40 antibody as CD4OL at 0.5 ug/ml (used to enhance IgG class switching). After four days of culture, the cells were re-fed with complete medium, in the absence of added stimulus, every three or four days. Culture supernatants were collected on days 12-18 and tested for GM-CSF-specific antibodies.
Detection of GM-CSF-specific antibody response.

[0251] The PBMC response to the stimulation was examined in a GM-CSF-specific ELISA.
Briefly, GM-CSF, KLH, or chick ovalbumin (CAB) at 0.5 ug/ml in 0.05 M
carbonate-bicarbonate buffer, pH 9.6, was immobilized onto ETA plates. After blocking the plates with 1% BSA containing 0.05% Tween 20, the supernatant were added to the wells.
Antibodies from the supernatant bound to immobilized antigens were detected with peroxidase-labeled goat anti-human IgG+IgM (H+L). TMB substrate kit was used for color development. The plates were read in a Microplate reader with a 450 nm filter. A supernatant sample containing antibody that bound to GM-CSF, but not to KLH and CAB, was considered positive. There was a robust response observed in cultures immunized to the GM-CSF-KLH as compared to controls. While anti-GM-CSF responses were observed in PBMCs for a small fraction of donors, the percentage of positive clones was greatly increased when PBMC were immunized in vitro with GM-CSF complexed with KLH. Positive cells were pooled and used for hybridoma production.
Generation of Hybridomas.
[0252] To prepare activated lymphocytes, cells were pooled and cultured in T
flasks at 0.5 ¨ 1 x 106 cells/ml in culture medium supplemented with 10% FBS one day prior to the fusion. To prepare the fusion partner, mouse myeloma NSO cells were transfected with human PMS2-134 expression vector as described in Nicolaides et al. (1998) Mol. Cell. Biol.
18(3):1635-1641.
The cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM
glutamine (Complete Medium) and the culture was kept in log phase.
Screening and characterization of antigen-specific hybridoma clones.
[0253] When the hybridoma cells grew to semi-confluence, supernatants were collected and subjected to an ELISA for antigen-specific reactivity. As an example, hybridomas derived from GM-CSF-immunized lymphocytes were tested. Briefly, GM-CSF, KLH, or CAB at 0.5 ug/ml in 0.05 M carbonate-bicarbonate buffer was immobilized onto the EIA
plate. After blocking with 1% bovine serum albumin in PBS containing 0.05% Tween 20, the cell culture supernate was added to the wells. Antibodies bound were detected with peroxidase-labeled goat anti-human IgG or anti-human IgM. TMB was used for color development.
Normal human IgG (nhIgG) and IgM (nhIgM) were used as controls. The plate is read in the Microplate reader with a 450 nm filter. A cell clone that showed reactivity to GM-CSF, but not to CAB was considered positive. The results are shown in Fig. 9.
Example 6 Inhibition of Proliferation Assays [0254] TF-1 cells were seeded at 0.2 x 106/m1 in RPMI supplemented with 10%
PBS and 0.5 ng/ml recombinant human GM-CSF. TF-I cells were serum starved for 24 hours in medium containing 0.5% BSA, without rhGM-CSF. Cells were then cultured in the presence of 0.275ng/m1 of GM-CSF for 3 days, with or without 4 ug/ml of various antibodies. Cell proliferation was measured using the ATPLiteassay (Perkin Elmer). In this assay, ATP was released by lysis of viable cells and utilized by the enzyme luciferase to convert luciferin into oxyluciferin. Light was emitted (luminescence) as a result of the reaction, and the intensity of the emission was ultimately proportional to the ATP content and thus to the cell number.
Counts per second (CPS) were obtained by reading the reactions with a huninometer and the percentage of inhibition was calculated according to the formula: 100 - (CPS
no Ab : CPS
with Ab) x 100%. The results are shown in Fig. 10.
[0255] The foregoing examples are merely illustrative of the invention and are not to be construed to limit the scope of the invention in any way. The scope of the invention is defined by the appended claims.
*Trade-mark MOR-0444.ST25.txt SEQUENCE LISTING
<110> Morphotek, Inc.
Grasso, Luigi Liang,Shaohohg Nicolaides, Nicholas C.
Sass, Philip M.
<120> METHODS OF GENERATING HIGH-PRODUCTION OF ANTIBODIES FROM
HYBRIDOMAS CREATED BY IN VITRO IMMUNIZATION
<130> MOR-0444 <150> PCT/US2003/036702 <151> 2003-11-14 <150> US 60/501,650 <151> 2003-09-10 <150> US 60/427,165 <151> 2002-11-15 <160> 50 <170> PatentIn version 3.3 <210> 1 <211> 3063 <212> DNA
<213> Homo sapiens <400> 1 ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60 ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420 ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac 480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780 tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa 840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 Z aftd ZEPIOPPE1P PP16DPEPE6 P1,611.P1.6DP 331.16PPPPP P6a1P1P144 1.1.614Dp613 000E rpalp66e6a ppp111141e 11661Eoppl 65 2e el111661D1 6p61.pp6ppe peule)161a 116614eee6 zleppez1.6e 1.2e6Eveouel 1.262papppl.

leOzepulpe ep6EDDalpz eappPellle DIxD3.11.111 eDDD6D1661 ED1.161616E

6,epplapep6 lEPe6613.16 PDDPAPPE01. PP6PDPI.D1E 11P3P6PPD7 1.E.DE66p6PE
09LZ pEplulaDel 64pD3Duzlp PDP6PDD4P4 3163616RA PP6P666P6P alapal6ppl.
OOLZ p61.6perD6D aDDR62161p p61e1116ee 66e6.pe eftoeeplle lelpoluell D11EPE6PPP ElllE6PD6P 160,61P301 I.PDOD131.61. ZPP10661.PP 666P1PPP
OSSZ
66110E14PP PPE010P11-PP D13.16e66pD DEI.E611eur P1P6PED111. 6666PD
OZSZ P1431.63133 1E6131.610D PlaDEED1P6 616PDE1P6P PPD3P6aP6P APDP61.PPP

Pluzu1.13.46 De6e1.1.1p11. pD1D1E661e p1.113.1.D1.6e 6e6eDeull6 lellupp36e oot?zpep6613e3D 6e6e361Dal 1DEPEleD1P P6E6110143 P6EPPE11.1P 1051DDAPP
OtEZ
6ep6ul.6e6v leleDDlpup 1.4.21.161m. 56P6PDPEPP DDZEDP61PP 1D661ED6lp 64301.1466P DI.D1PPDPDD 1P61.1361-13 DEu61p66rp 666plap P6116PPepp ozzz PPPDPPP6PP 11.14PETZPP PPP41DPPET p61e1D111.1 DDDD1P6PDP 1661pppeal 091Z papeueD16e 66PPEoPPPPP P611PPPD3D 167eD1.1DDI.D ee61e6113e eeeppeeple OOTZ Elplezleal ppeepen16 PEDEAPP6P 333661.11PP 651-P3636PD DPDDDPEPPI.
OVOZ eftruepeft D661e6ppep appDa6apPp Depl6p66ED pe611eDD6e 661pue 0861 Da6e1PeDel p6Dee661.1.D P6PPPZDP1D 66666 1P1PPP610P PPPP66P6PP
0Z61 616p6alpou 6ep661.61DE rftp611pep DP2DPDPPD6 1P66P6Pala 6P1DP6PP1D
098T DaPPPPEIP4P ploala6pD1 D316pleplp 6peD11.6111 14D13616ee D6eD161eDD
0081 Deuefteplu El6p6D1peD 6elapplalp 61e1.1D6EDE 116Eupp661. Dlrepluele OVLT 6papel6leu PDaPPPEPPD PP161PD14P 661PP11D 1PP61PPPDP P61)331PPD
0891 Daellveleu lrulftelft uP1.61EDDel 116PPePPE6 1)D6a6P113. lpPET61.61D
OZ9T DPP641P1PP 66 66.6 Dalepeppla Delpleve66 66pD6p661.6 r61e6p361D
09ST 111PET66D1 lolDePep61 1D166e36see 6eu66e6zev ue666616e6 e61v6elEae 00ST ppefteeleu 3661EPPEET 316P3D3PDP APP146331 lEIPP4P141.1 66 ot'ttPleaeu66ou 6upapapee6 p6661pD1E1 61.ppl6e6lp upallrDp66 pollaPp6ap P6PP1DEDPP PPEZP611P) PP1D11.4PPP 616e16E1.61 leD1661pla 6611.1Dp616 OZET 614P16epap 6PDDPD1PPP 111614E61P 61DP3PPPET P661.31PP61 e6leuluD61 09Z1 P1P61PPPP3 Da4P3311P3 16PD413EZE 63.161PPE31 1P11PP6PEP 66101PDZPP
00ZI 6616Eueleu 1.1.141o6161 e6uDvpv6RD 6PDPEPP16P aloal6Dapp p61D6pD6DD
OVIT
11161.P6PDP PPP1PP1PPP P61.P1.1711P PE3P16PaDD Pl1PDDP661 p1.16113e6D
lxvszIS'VVVO-liow =
moR-0444.ST25.txt <210> 2 <211> 932 <212> PRT
<213> HOW sapiens <400> 2 Met Lys Gin Leu Pro Ala Ala Thr Val Arg Leu Leu ser Ser Ser Gin Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr Ala Ala Asp Asn Phe Ser Thr Gin Tyr Val Leu Asp Gly Ser Gly His Ile Leu Ser Gin Lys Pro Ser His Leu Gly Gin Gly Thr Thr Val Thr Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gin Phe Tyr Ser Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gin Asp Leu Leu Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His Asn Lys Ala Val Ile Trp Gin Lys Ser Arg Val Ser Asp His Lys Met Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser Phe Gin Tyr His Ser Glu Glu Ser Gin Ile Tyr Leu Ser Gly Phe Leu , .
MOR-0444.sT25.txt Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro Glu Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Val His Gin Lys Asp Ile Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser Thr Arg Leu Tyr Pro val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gin Val Leu Leu Gin Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp Thr Ser Val Ile Pro Phe Gin Asn Asp Met His Asn Asp Glu Ser Gly Lys Asn Thr Asp Asp Cys Leu Asn His Gin Ile Ser Ile Gly Asp Phe Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr Lys Asn Ala Phe Gin Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn Ser Gin Thr Glu Tyr Ser Lys Thr cys Phe Ile Ser Ser Val Lys His Thr Gin Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu ser Gly Glu Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser Ala Asp Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly Glu Asn Ile , .
MOR-0444.ST25.txt Glu Pro val Lys Ile Leu val Pro Glu Lys Ser Leu Pro Cys Lys val Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gin Met Asn Leu Asn Glu Asp Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro met Ser Ala Ser Ala Leu Phe val Gin Asp His Arg Pro Gln Phe Leu Ile Glu Asn Pro Lys Thr Ser Leu Glu AS Ala Thr Leu Gin Ile Glu Glu Leu Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gin Met Lys Arg Ala Ile Glu Gin Glu Ser Gin Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro Thr Ser Ala Trp Asn Leu Ala Gin Lys His Lys Leu Lys Thr Ser Leu Ser Asn Gin Pro Lys Leu Asp Glu Leu Leu Gin Ser Gin Ile Glu Lys Arg Arg Ser Gin Asn Ile Lys Met Val Gin Ile Pro Phe Ser Met Lys Asn Leu Lys Ile Asn Phe Lys Lys Gin Asn Lys Val Asp Leu Glu Glu Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp Ala Trp Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro Tyr Arg val Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn MOR-0444.ST25.txt Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gin Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu Ala Val Arg Leu Ser Arg Gin Leu Pro Met Tyr Leu Ser Lys Glu Asp Ile Gin Asp Ile Ile Tyr Arg Met Lys His Gin Phe Gly Asn Glu Ile Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu Pro Glu Thr Thr <210> 3 <211> 2771 <212> DNA
<213> HOMO sapiens <400> 3 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660 L a6ed OOLZ eee11.1.161.1. 1.3.11.DDeelD eD1.1D1.6p6e De6ree61.14 1.61e11411r 6eD6D1x1.11.
Ot9Z 1.661luezer 661.p161DeD 1.6e1.6DDr61. pre6rD1D13. 1PD1616664 DDPPDADIX
08SZ DeDe6p6leD DPPDD6PP6 61.PDDDD461. DPE6613DDD eDDe661e6e 666661rDeD
OZSZ DDeD1r61.DE ue6ee64e6e 6D6PPDPDPP laDaDEaDE6 6611X61P61. 66D16re66D
09vz D61DD6e6rD DaDD614161 p6eD6eeD1.6 e6DDDI.I.DD6 6DD61.61.eD1 66661.DDD6e oovz De6D6e6aD6 1PD11D1P64 DPP6aP6D16 DP66ED3DDE 66)1.1.09E66 loreevezele OE? apreDD61.1D D3.1.1e61.Der rI.D666ver6 zDeD46eDDI. D6leeer6le 6Dze1.16111 08ZZ ze61.1.1.D661 PP6PPP6P14 ZPI.PEE661D TePPEETZP6 1D1161D6PP 61m161D6 OZZZ aDrealDeeD 4D4DP6PD1D DED6PZED13 66e6eD6666 EDDI.D616DD PDED6PDETD
09TZ 61.D61e6e6D laDeezel6e e6e6De66De DD6aeD6eDD u661.6eleD3. zDzezr66e6 OOTZ 1T861DPRED DEEZPUZEla 1P66610DEP 1416PD1661 1PDIXPP661. PPP6PD611.1 OtOZ 64e6Deeeel 6el.e6e6ep Ee6eelDee6 le6e6DD6U APPDIXEEE 6E661DDI.61 0861 1au6erD66 6666 roulaer6eD ev6666ee61. ETPPD6UDPD 6P6lPD1PD
0Z61 ela6eD6eue zee6DueuzD 6e1.1.1.D146e 61-P1-D111.1D P66aDDDD61.
613.6eeefte 0981 leelleere6 461.D6e1.6ze 61.166eD3.33. DD6eD161eD e66eD1Dele ee1.6e11.6re 008T eeD163.14eD e61.D3.3.6eDD 11.1011.PBP6 PEEIEPEPPEZ 1416D6eee D
PDPEPDDDDE
OtLT eD6D1Dzeez DUPDAPDZD D611146e6D zalere161.e 66DDeze6Ee 66eDDereD1 0891 zeDD61Dr66 161x6eDlla 11.D1De6De6 I.Deee61DD6 D6rer6r66e DaD1De661.6 OZ9T leDee66eD6 D1D6666eDe 6666EDDDDa D6eDD66D61. el.6e6D6rD6 eD61DeD1.6e 09ST D666DeDe6r DDDleD6eD2 1.6666e6aDq. le661.6DDal DPDETD66DP D6666Da3P6 00ST 6ee6e661.66 r66D6e6eDe 66DelDDDe6 16eDDDe66D eDa6eDD11.6 e61.6eD66e6 Ott' PPP6PD1DDP 6e61.DDz6D6 6eueDe61.D1. DzeDD6166r DzI.DeD6r1D alD161.D6le 08E13.6666eeee6 EDUE06EZDZD DD6P66PP6P PDDETETDD1 DP6PETDDD6 PDP31DDETP
OZET DPP6P6PDPP DeDPD1633.4 31-331.1.1DD6 6E6eE061.DP 6PDDIal.PDD
1.6a6De6EPP
09ZT PEPPEETP6P MaDEMPP; 1.VD3.1.DDDDI. PED1P66VD6 eeve6e1.661 PDDD6UPPP6 00Z1 61.11.e66D6e D6leD6leve reareazDee 3.66er61161. e661.D61DeD
D6rD6eDlEse OtTT D16apeeaD6 PEOPPD161X 646e4e611.1 666e6 111.DloDe6E vellaz6eD6 0801 5115141306 veee66e6ee Del.D611.1.1e eeD660everz e6eDDI.Dela 6zeeDzele6 OZOT 1.16D61.ve6e Dlar61.161.D lazeDeell.D 146146141.e DDIx16eDae De6Dleezel.
096 61eDeDDelD 3.66e6zee61. 6D4De6eD61 D1.66eeeD6e DDDe6161.1.D D66D66DDee 006 Daellaplal 11.6eDe6eDe 6rDeeD1.1.6e 66 666 66 6D6 leeDepazze Ot8 D1.11.66eDI.D
ozeezeD61.D 1.361.r66D11. 61D6r63.1.3.6 6.665 08L 46463.6DDlo E61.6e1DDDD 061.36PD146 11.11.DDZ1PD ZDO6EUeD61. 1.6PD6e5ED
OZL 66611.1616a DI.D66DIXIX PPP66UPEZP D6UODDAPP 663.66eDeD6 le1661.61.DD
1.xl..szIS'ttt0-110w MOR-0444.ST25.txt atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 <210> 4 <211> 862 <212> PRT
<213> Homo sapiens <400> 4 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser val His Gin Ile Cys Ser Gly Gln Val Val Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gin Glu Phe Ala Asp Leu Thr Gin Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu ser Asp Val Thr Ile Ser Thr Cys His Ala Ser Ala Lys Val Gly Thr Arg Leu Met Phe AS His Asn Gly Lys Ile Ile Gin Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gin Gin Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe Gin Arg Asn Ile Lys Lys Glu Tyr Ala Lys Met Val Gin Val Leu His Ala Tyr Cys Ile Ile Ser Ala Gly Ile Arg val Ser Cys Thr Asn Gin Leu Gly Gin Gly Lys Arg Gin Pro val val Cys Thr Gly Gly Ser Pro Ser Ile Lys . .
MoR-0444.ST25.txt Glu Asn Ile Gly Ser val Phe Gly Gin Lys Gin Leu Gin Ser Leu Ile Pro Phe Val Gin Leu Pro Pro Ser Asp Ser val Cys Glu Glu Tyr Gly Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe Ile Ser Gin Cys Thr His Gly val Gly Arg Ser Ser Thr Asp Arg Gin Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg Leu val Asn Glu val Tyr His met Tyr Asn Arg His Gin Tyr Pro Phe val Val Leu Asn Ile Ser val Asp Ser Glu Cys Val Asp Ile Asn val Thr Pro Asp Lys Arg Gin Ile Leu Leu Gin Glu Glu Lys Leu Leu Leu Ala val Leu Lys Thr Ser Leu Ile Gly met Phe Asp Ser Asp val Asn Lys Leu Asn val Ser Gin Gin Pro Leu Leu Asp Val Glu Gly Asn Leu Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gin Asp Gin Ser Pro Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp val Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly Gin Lys Arg Gly met Leu Ser Ser Ser Thr Ser Gly Ala Ile Ser Asp Lys Gly val Leu Arg Pro Gin Lys Glu Ala Val Ser Ser Ser His Gly Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His . .
MOR-0444.ST25.txt Gly Ser Thr Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly Ser Gin Glu His Val Asp Ser Gin Glu Lys Ala Pro Glu Thr Asp Asp Ser Phe Ser Asp Val Asp Cys His Ser Asn Gin Glu Asp Thr Gly Cys Lys Phe Arg Val Leu Pro Gin Pro Thr Asn Leu Ala Thr Pro Asn Thr Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gin Lys Leu Val Asn Thr Gin Asp Met Ser Ala Ser Gin Val Asp Val Ala Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser Leu Ala Lys Arg Ile Lys Gin Leu His His Glu Ala Gin Gin Ser Glu Gly Glu Gin Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gin Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr met Phe Ala Glu Met Glu Ile Ile Gly Gin Phe Asn Leu Gly Phe Ile Ile Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp Gin His Ala Thr Asp Glu Lys Tyr Asn Phe Glu met Leu Gin Gin His Thr Val Leu Gin Gly Gin Arg Leu Ile Ala Pro Gin Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile MOR-0444.ST25.txt Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gin Asp Val Asp Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser Arg Val Lys Gin Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val Met Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gin Asn <210> 5 <211> 426 <212> DNA
<213> Homo sapiens <400> 5 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 <210> 6 <211> 133 <212> PRT
<213> Homo sapiens <400> 6 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gin Ile Cys Ser Gly Gin Val Val Leu Ser Leu Ser Thr Ala val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly val Asp MOR-0444.ST25.txt Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gin Glu Phe Ala Asp Leu Thr Gin Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser Ala Lys Val Gly Thr <210> 7 <211> 1408 <212> DNA
<213> Homo sapiens <400> 7 ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcct gccgtgcatg 60 ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagac ccggtgacag 120 ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggc gcgtgccaga 180 caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagac accaacgctc 240 ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcgg aatccacgcc 300 aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgtt cggaaaaaac 360 gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcg acctttgaca 420 gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgccc ggaaagggtg 480 gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctg tgggagccgt 540 ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttgga gcgagctgag 600 aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtc agtccatcag 660 atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaagga gttagtagaa 720 aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggacta tggagtggat 780 ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga aggctttact 840 ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggt ggaaactttt 900 ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcac catttctacc 960 tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgg gaaaatcatc 1020 cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagca gttattttct 1080 acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacg tgcctgcttc 1140 cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccat gcttcctgta 1200 MOR-0444.ST25.txt cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctcctt ggaggacaac 1260 gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatg atctgcacaa 1320 atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagt tcctgctgaa 1380 aatccaaaaa aaaaaaaaaa aaaaaaaa 1408 <210> 8 <211> 389 <212> PRT
<213> HOMO sapiens <400> 8 Met Ala Gin Pro Lys Gin Glu Arg Val Ala Arg Ala Arg His Gin Arg Ser Glu Thr Ala Arg His Gin Arg Ser Glu Thr Ala Lys Thr Pro Thr Leu Gly Asn Arg Gin Thr Pro Thr Leu Gly Asn Arg Gin Thr Pro Arg Leu Gly Ile His Ala Arg Pro Arg Arg Arg Ala Thr Thr Ser Leu Leu Thr Leu Leu Leu Ala Phe Gly Lys Asn Ala Val Arg Cys Ala Leu Ile Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pro Leu Thr Glu Pro Leu Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pro Pro Arg Pro Glu Arg val Glu His Asn Val Glu Ser Ser Arg Trp Glu Pro Arg Arg Arg Gly Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro Ser Pro Arg Gly Gly Ser Gly val Ala Ser Leu Glu Arg Ala Glu Asn Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gin Ile Cys Ser Gly Pro Val Val Pro Ser Leu Arg Pro Asn Ala Val Lys Glu Leu val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Val Asp Leu Lys Leu Lys MOR-0444.ST25.txt Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Gly Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Phe Thr Leu Lys His His Thr Cys Lys Ile Gln Glu Phe Ala Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys Arg Val Ser Ala Lys Val Gly Thr Arg Leu Val Phe Asp His Tyr Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Met Thr Val Ser Val Lys Gln Leu Phe Ser Thr Leu Pro Val His His Lys Glu Phe Gln Arg Asn Ile Lys Lys Lys Arg Ala Cys Phe Pro Phe Ala Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Ser Pro Ala met Leu Pro Val Gln Pro Val Glu Leu Thr Pro Arg Ser Thr Pro Pro His Pro Cys Ser Leu Glu Asp Asn Val Ile Thr val Phe Ser Ser val Lys Asn Gly Pro Gly Ser Ser Arg <210> 9 <211> 1785 <212> DNA
<213> Homo sapiens <400> 9 tttttagaaa ctgatgttta ttttccatca accatttttc catgctgctt aagagaatat 60 gcaagaacag cttaagacca gtcagtggtt gctcctaccc attcagtggc ctgagcagtg 120 gggagctgca gaccagtctt ccgtggcagg ctgagcgctc cagtcttcag tagggaattg 180 ctgaataggc acagagggca cctgtacacc ttcagaccag tctgcaacct caggctgagt 240 agcagtgaac tcaggagcgg gagcagtcca ttcaccctga aattcctcct tggtcactgc 300 cttctcagca gcagcctgct cttctttttc aatctcttca ggatctctgt agaagtacag 360 atcaggcatg acctcccatg ggtgttcacg ggaaatggtg ccacgcatgc gcagaacttc 420 MOR-0444.ST25.txt ccgagccagc atccaccaca ttaaacccac tgagtgagct cccttgttgt tgcatgggat 480 ggcaatgtcc acatagcgca gaggagaatc tgtgttacac agcgcaatgg taggtaggtt 540 aacataagat gcctccgtga gaggcgaagg ggcggcggga cccgggcctg gcccgtatgt 600 gtccttggcg gcctagacta ggccgtcgct gtatggtgag ccccagggag gcggatctgg 660 gcccccagaa ggacacccgc ctggatttgc cccgtagccc ggcccgggcc cctcgggagc 720 agaacagcct tggtgaggtg gacaggaggg gacctcgcga gcagacgcgc gcgccagcga 780 cagcagcccc gccccggcct ctcgggagcc ggggggcaga ggctgcggag ccccaggagg 840 gtctatcagc cacagtctct gcatgtttcc aagagcaaca ggaaatgaac acattgcagg 900 ggccagtgtc attcaaagat gtggctgtgg atttcaccca ggaggagtgg cggcaactgg 960 accctgatga gaagatagca tacggggatg tgatgttgga gaactacagc catctagttt 1020 ctgtggggta tgattatcac caagccaaac atcatcatgg agtggaggtg aaggaagtgg 1080 agcagggaga ggagccgtgg ataatggaag gtgaatttcc atgtcaacat agtccagaac 1140 ctgctaaggc catcaaacct attgatcgga agtcagtcca tcagatttgc tctgggccag 1200 tggtactgag tctaagcact gcagtgaagg agttagtaga aaacagtctg gatgctggtg 1260 ccactaatat tgatctaaag cttaaggact atggagtgga tctcattgaa gtttcagaca 1320 atggatgtgg ggtagaagaa gaaaactttg aaggcttaat ctctttcagc tctgaaacat 1380 cacacatgta agattcaaga gtttgccgac ctaactgaag ttgaaacttt cggttttcag 1440 ggggaagctc tgagctcact gtgtgcactg agcgatgtca ccatttctac ctgccacgcg 1500 ttggtgaagg ttgggactcg actggtgttt gatcacgatg ggaaaatcat ccaggaaacc 1560 ccctaccccc accccagagg gaccacagtc agcgtgaagc agttattttc tacgctacct 1620 gtgcgccata aggaatttca aaggaatatt aagaagacgt gcctgcttcc ccttcgcctt 1680 ctgccgtgat tgtcagtttc ctgaggcctc cccagccatg cttcctgtac agcctgcaga 1740 actgtgagtc aattaaacct cttttcttca taaattaaaa aaaaa 1785 <210> 10 <211> 264 <212> PRT
<213> Homo sapiens <400> 10 Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Arg CyS met val Ser Pro Arg Glu Ala Asp Leu Gly Pro Gin Lys Asp Thr Arg Leu Asp Leu Pro Arg Ser Pro Ala Arg Ala Pro Arg Glu Gin Asn Ser Leu Gly Glu Val Asp Arg Arg Gly Pro Arg Glu Gin Thr Arg Ala Pro Ala Thr Ala Ala MOR-0444.ST25.txt Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu Met Asn Thr Leu Gln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pro AS Glu Lys Ile Ala Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly Tyr Asp Tyr His Gln Ala Lys His His His Gly Val Glu Val Lys Glu Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ile Ser Phe Ser Ser Glu Thr Ser His Met <210> 11 <211> 2271 <212> DNA
<213> Homo sapiens <400> 11 atgtcgttcg tggcaggggt tattcggcgg ctggacgaga cagtggtgaa ccgcatcgcg 60 gcgggggaag ttatccagcg gccagctaat gctatcaaag agatgattga gaactgttta 120 gatgcaaaat ccacaagtat tcaagtgatt gttaaagagg gaggcctgaa gttgattcag 180 atccaagaca atggcaccgg gatcaggaaa gaagatctgg atattgtatg tgaaaggttc 240 LT a6Ed TLZZ P
Pla6166PET 6111016PUP peluz)1E61 DD6aDDPPaD 611)6E)61) OZZZ )1ElEEp661 Efte6E)Epa laPDPPPaDD aDD6aDalED PDPD1D6D61 1.))6pEE1E1 )1611E)EDE E661.61)E66 16PPE6aDDa DPETDDlaPD DaD661DD61 6EE616E6E) OOTZ
6E))66E)1) 1)))E6)16E 66)E DE16E)6EE6 6))1E))11E 1)1161E1)6 OVOZ
)61.EEETEE1 6PDaDD6PPP 6aall6aPP6 6EEEE6EE6) E66611EE61 66E61)e))6 El)E6)11.)1 le)11)1E1) )61)E666E6 6411)))))6 1.6aulDEEDE 611e61.)41) )))EllE661. 1E61))EE66 6EE66E61.E6 lapEE66111 )1)111E1)E bE)61.1)61E

6E61)66EE6 EE6Eu61.)14 16E614614E pEzEr61)61 1.)E66EE6Eu E)))1661E6 0081 EE66E6E)E6 61)661.6E6E 6E0)16E1E6 Ela))64106 ae))611))E 6111)1)6)) Ota E)6))666 )1E1.166E)1. )11.61.6611a apv))611.1.1 E61P111E)1 DE1E6E))E1 D11.61.)EE6e E61.6E11)6E PDDPDDPDPP DaDaaDDPaP 116PPDDPPP DaPD6PDPD6 611))6,66z6 p)10)1EE61 6161)6E616 DaaDD1DPDD EulE)61161 E6E666))) 09ST 1166E61E)E 666E)6E61E EllEEEETE6 6=1)16E6 ala1616PaD PDaDDPPlaP

DaP66PPOP6 EIDDDDDDP16 11)6E)61)E 61EuE66Eru 6)))11E6zE 6EE66166zE
OVVT Eu66161E61 )1.1e6EE666 )1E)E6E6EE PETDDDDEPD 6PDDlaDP1D DP66P6P6PP

6aPU P6PDalDP66 66PPPDPPDP 1E66666E66 11.)6E6E)1E EEEE))61)6 OZET
616PP61061 DDDD6PDDD1 DPP6alD6aP 6P66P6aP6P PD6PD66PaD 666E)6616E

1)111E1E6e DE6EE1E66E 6PDPD1611P DDHPDDDDE, PDa6PDDa61 DDDDPPPDEIP

61.)1))6E)6 1)111E)61E 611)6EE6E) EE666)))11 E6E)E16)11 661E6E))E) OTT
))61.E1.)166 EEI.E616E16 EE661)1.1ou 1)14)z6)1.) DE64)16EE) PPDPDDaPPP

11661E6E66 661)z))))6 61)611)E66 E)yel.)611.1 DPETDDDPDa 1)E161E66E
OZOT
DDaDDlaPPD DaD6661DDa D6PPD6P6P6 )1.EDE)6E)6 E)61666)6E 661))4E)6e 6E66E6)u)6 4))11)E)11 ETP6aPDETP PDPDDDDED6 a6aPPlable 6666PD

DDD16PDaPP E6E111.6E)1 ))E161))11 PDDDPDPDPD PUPPPDDDEla laPaDDETD6 lea6a6EDEE E6E1E))6EE EE6E611))1 aDPPDaPPEIP 166131E01P DDPPD1PDal )1)E11.)11) au)616EE6E P616PD1DPa DPPPD6aPPD DaPaPDPlab 64EE6aEEEE
OZL
)11.))6E1)) peueElE66E 6161E6611E Ee6E1E61)E e6E6)16E11 6.)6E66 1.1101PDDaD EalaUlPPDP 66a6DDPPDa DDE0aPPDDDP aDPDP66Pla 61E61)6E16 E)6E666E EDPPPPPPla 6E)1)1116E 11E)66E)61 PPDPDP1.6PD laPlE6PD66 OtS
116alETP66 allaPPET66 6aPaPPEIPP6 a6PPDDaPPP PPPalaDEPP PP6P66P6DP

DD6PaVDPPD E3.41141))E 66E66166)E )1E6E)))E6 66EE)leE)6 61)6161=
0i7PPPaDDIODD D6PPP61DUP PP661P6PDa DPaa6PPD6P 6J D6.6 1D6PDPEPP6 DPPDP1aPaD E1161e)1)6 6161.E))6up zE)6E))661 11)66E6166 E6)111)661 EloyE10111 El6E))6E11 4E66E6141) )16E)6z)Ee Ez6EapelyE
lx1.*szIS'1717170-ww MOR-0444.ST25.txt <210> 12 <211> 756 <212> PRT
<213> HOMO sapiens <400> 12 Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr Val Val Asn Arg Ile Ala Ala Gly Glu Val Ile Gin Arg Pro Ala Asn Ala Ile Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr Ser Ile Gin val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gin Ile Gin Asp Asn Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg Phe Thr Thr Ser Lys Leu Gin Ser Phe Glu Asp Leu Ala Ser Ile Ser Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ala ser Ile Ser His val Ala His Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala Gly Asn Gin Gly Thr Gin Ile Thr Val Glu Asp Leu Phe Tyr Asn Ile Ala Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile Leu Glu val val Gly Arg Tyr Ser val His Asn Ala Gly Ile Ser Phe Ser val Lys Lys Gin Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro Asn Ala Ser Thr val Asp Asn he Arg Ser Ile Phe Gly Asn Ala val Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe MOR-0444.ST25.txt Lys met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys Asn Thr His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln Asn Val Asp val Asn Val His Pro Thr Lys His Glu Val His Phe Leu His Glu Glu Ser Ile Leu Glu Arg Val Gln Gln His Ile Glu Ser Lys Leu Leu Gly Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Thr Leu Leu Pro Gly Leu Ala Gly Pro Ser Gly Glu Met Val Lys Ser Thr Thr Ser Leu Thr Ser Ser Ser Thr Ser Gly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val Arg Thr Asp Ser Arg Glu Gln Lys Leu Asp Ala Phe Leu Gln Pro Leu Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Ile Val Thr Glu Asp Lys Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu Glu Leu Pro Ala Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro Thr Ser Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu Met Val Glu Asp AS Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu moR-0444.5125.txt Ile Asn Glu Gin Gly His Glu Val Leu Arg Glu Met Leu His Asn His Ser Phe Val Gly Cys Val Asn Pro Gin Trp Ala Leu Ala Gin His Gin Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe Tyr Gin Ile Leu Ile Tyr Asp Phe Ala Asn Phe Gly Val Leu Arg Leu Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Met Leu Ala Leu Asp Ser Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp Tyr Phe Ser Leu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe Ile Leu Arg Leu Ala Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys Gin Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gly Gin Gin Ser Glu Val Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu Asp Gly Asn Ile Leu Gin Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg Cys <210> 13 <211> 4895 <212> DNA
<213> Homo sapiens TZ a6Pd 0861 ee6aDreDae PE1DPPPETT 61PPPP3P16 B461PD1DeV 1P111D661D eDD116aela.
0Z61 ereal6e6D6 661e116eal 1116appy6e etee661.61.1 eeD6eaDlee OPEPEETEPE
098T 6e6ree6e6e Dr6eD1D616 eleDua6e66 66aelleDee DeeD6a111D D6aDaDD6eD
008T D6;e66616e e6aDvaD6ap 6epeaale6e 6PPEODPPD1 PPETD41PP6 eaueleeeue OVLI 611D1eDeeD D61D66aeD6 6leela6aDe aDea6eeeete ee661eleee 6e1Dae66eD
0891 66616e6e66 e611aDeD16 1PDDPDEETD 1111=6er 411116leee 66eDDe OZ9T e661616DDa reee6ealaD aD6e16Due6 61DDaaane PEPETIMPP u6e6leeee6 09S1 1661D6ee6P DIXD6P1.611. PPDP6U6UPD eu6e11.61e6 EPPD1PP6PD 1D611D1D6r 00ST DE6PPRDEPP PDP141D1ED D6P6PDE62P PUPD6P1PDD 66rDD16616 6eDlee6lea OVVI 1zeDea.6111. laRD61P61P VPDP1PEPER PEIPDDE1DET e6eDale666 eaD116e6up 08E1 PDEDPRP16D UPPP6ED610 PlDPVPEP6V PUP616106P eueDa6eD61 alee11161e OZEI 6e61e1DD14 u6e11.11.ele eapea6aeD6 re66eDD111 eeD6e66e6r 61e6DDlape 09ZT 6a616D6ee6 eplaDaDeaD 61e61.11eal lbeala1661 euae6eu6a6 ealare66ee 00Z1 laele66e61 66eDaulaxe 661611auaa PPPPE6PED6 UPP1141.161 eveu616e66 OKI ee66pDaleD 61111.614D1 DaDeDe666a Dep6eDalle E611E61010 PPPPDD6PDD
0801 6e661eD616 161e6leaft 63.61DaapED D616eD60161. eualuel6ae leauD66ael OZOT D1DUP6PDDD DeaD166DDe D66D1aD16e eDlaue6lee eD66ea6eDD eDDD1661ev 096 6UPPDDETUD 61eleale16 eee6eue66e elle111.1aD e6aluDaDee eleDeaD6ue 006 eDeMeella 16e1Deftee eDee61611a 61.11116eD6 aelee6eeDe rDelleDeD6 OV8 ee6aDaD6eD arleaD661.6 ealD6e6111 6e6eraele eellalbeel eee6e6peaD
08L 56 eee66611e6 6aelaleeeD 161111e6DD D1161e16De 6UPEDDPEPP
OZL aDDD1D6rD1 1D1166aeDD 1166aDa116 ar6aeee6e6 alaDaD111D alleDDlaDD
099 DeD61eDaDu DaDaDaD6er 6elee6e6eD 66666 e61.1.16e661. De6eaDDDe6 009 61eD6leve6 6e66ee161D D14DETDDPI. 141E1DDRP4 ea6a6eDeel 61DeaDe666 OVS aD6D6eeD6u 6eaDe6161e 6aD6re6161. aD6ree6aDD D6 66.6 r6eD1116aD
08V eee6161141 DPPEP61PPD PHEDUPETP 6eupDa6D11 leue661.61D 616eDD661e OZV De6aD61.1e1 eeeD6611DD 66e6e66e6D Dala661eaa 1.1.66eeDDle e6e6611De6 09E 6eDe166DaD eDD6aree16 eneDlalea 16Daeue666 a6Eee6e6ea 61e61e616e 00E 66661e6661 ale66aueDe 6ele6a6peD al6eeDD11D Deue66auae e61666eD16 OtZ 1D661616av eueD6eu6aD 61B6a1El6 DPEDZDDD63. apuu66e6a1 6666 081 611DD1D6ee aeDD661.116 6aDal6D611. PPEDAPEDP 16ee6116eD 1614)61E4Eu OZT ple61eDDel DDP101-41D1 EDOED4)1.4P 3661.D3616P DDEUE66EP6 e6eD16eDD1 09 alulaueD66 eDDaDe666D 6ee1161ale u6e66D16a6 6a166D66e6 DD16D66Da6 ET <00t>
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= , ZZ a6rd OZOV 16r6reppe6 161.Delp1r6 er66r6r66D 11Drr6zerD D6er6r6rrr 661.6111161 096E elorDDelLe ree6661614 DD1661D1D1 ZE63.6P1.DPD P6EDD111-E1 611.1.EP6210 006E D6661ple6 r661Dareer epeope1161 661e11D1Dr 6r66reeDer 66e6rDe61.6 0.178 rorrle6r6r 1D6DDaDD11 erl.D1DraD4 1D161Drlle ereep66D16 61D1D66erD
08LE rD66eDerp6 ev6e6pelDD lar6ED66r6 D66 66 e6lpeftelD eD6e6lebal OZLE 161DD6lara 116reperze 6E16Eup6rD DI.D1166ED6 1PPD1ZED11. r6lre.66rre 099E DorD3.116D1 elpDpze161 ZDIXDPRDED 1.1.PUPP346E D5P1.3000P5 61D66rDD66 009E 16rDftel6a r61161D614 66r6eDD1r1 16Dpo6alle 16eDDlerpr 6661.Er6rDI.
OVSE D1.161.14D1.6 epl1D6Dlee 6)6e16e116 aDele6ar66 461Deze6e6 eprear66rD
ogvE p6r61r61.61 Dr66E6r6rD 6e6D1D6r6e 6D DiaDD111 DD 11D1461.4 Dpe6D6eu6e OZtE 1111-DDRED1 5166EDE16E D1D1.6661PP 6P634DP3-61 163.E66161.D 66161DETDP
ogEE 61DDr6ruel Dr1.611D6lp 55 6P61DPPDDD D61.1PD11PD PD6PDZDE66 00EE 1De6leureD erD464eall 661re6e66 61DDD6r3.61 E665 D6ED6611E6 otzE ED1161.1616 DEDPPPD16B 666 1D11614D6p 6erD6reulD ler66r616e 081E 6eD4111611 zur661ruur DDr61.66rDr DD61e6re6r 466DDlrr61 rr1161re66 OM 16eDDD1D1D e611D16ur6 6e1P6PDPPD PP6PD1DDET 6rDlealDaz 61.r6rDzlr6 090E ree166DDel 16reelD1.ze elrululDDD 614r166141 DDI.DETP6PD zrzr6161.61 000E DPPETEIPDPE DPaPPD6PDP PlDaleDeDP erelbelrer EneEllaDO llerl.r66rD
0176Z PDPDA1Dal PETDIXOPPD DDIXD16166 lre6r6rDep rere6arD6r rpreDerlal 088Z r13.61e4614 1.11r6reDeD er116161.6r D63.611.6reD 6rDr6ree66 rz6eppale6 OM ED11EPROD1 zDurelleral 146D1.6r6ly 61e66661er DUPPD1DEPP 6P6UPP6D31 09LZ 1666er61De 6e elp1DD6elp Epleeolplx D16eu6e611. DDE661a4DD
OOLZ EPPPET1PPI. alDaD1D1ra 16r66er64D DDr6aelppl 6ee6e6e63. 11.6e1DD6le OV9Z 6rDruDer6r 11.1611r6rr 63.e66epoDa DlarDD111.6 PEETETD4DP 611DDleDrD
08SZ DETEDETDDE eD16146e16 nr6rDlar6 16r;rD6r66 ZE1DEZDPDa UPPU1611.16 OZSZ le61D1Dee6 e6r11D6DDr remrDr66er 63.D61DlleD r61DDrr611 6erD11D16r OW D1D1PPDDP4 Depl6r661e e6666 eleser6rDez e661D1DDar ver61466re 00.17Z 6661EarED6 6e6rr1116r e6e6r114Da e66e1D6eDe Daa6e63.1D6 PE1D6EUE0EU
OVEZ 16DD16olue DDPPEDD3.31 116611.UP41 PET1P6PDET PP6Erea6P1P 61PPDD1146 08ZZ Depp6rzE16 61DDDDlalD 1DODDDIXDP le6101elle leD611.e6ep PPEDED1PPP
OZZZ eveD6r166e r66rD1111D 61D 113.161e eDulellorr APDPaDDEP 61PP6B6P1D
09TZ 6661.r11.6rr leTer6Der6 eD641erree ePPDPVDDD6 lareD1661D lre6rel6r OOTZ ela4DED6rD D6r111r6er PDI.PDPETDD apeer61.16r D44PD46DeD er6!lere66 OVOZ 3-43.PDPPP6P PDDD61.6DED eDDD16601DD r6r116zell reerrllarD 11PDEPETDE
1.XVSZIS.17.17.170-110W

mOR-0444.ST25.txt attgtggagg aatttatccg agaacaactg gagctactcc agaccaccgg aggcatccaa 4080 gggacattgc cactgactgt ccagaaggtg ttggcatccc aagcctgcca tggggccatt 4140 aagtttaatg atggcctgag cttacaggaa agttgccgcc ttattgaagc tctgtcctca 4200 tgccagctgc cattccagtg tgctcacggg agaccttcta tgctgccgtt agctgacata 4260 gaccacttgg aacaggaaaa acagattaaa cccaacctca ctaaacttcg caaaatggcc 4320 caggcctggc gtctctttgg aaaagcagag tgtgatacaa ggcagagcct gcagcagtcc 4380 atgcctccct gtgagccacc atgagaacag aatcactggt ctaaaaggaa caaagggatg 4440 ttcactgtat gcctctgagc agagagcagc agcagcaggt accagcacgg ccctgactga 4500 atcagcccag tgtccctgag cagcttagac agcagggctc tctgtatcag tctttcttga 4560 gcagatgatt cccctagttg agtagccaga tgaaattcaa gcctaaagac aattcattca 4620 tttgcatcca tgggcacaga aggttgctat atagtatcta ccttttgcta cttatttaat 4680 gataaaattt aatgacagtt taaaaaaaaa aaaaaaaaaa attatttgaa ggggtgggtg 4740 atttttgttt ttgtacagtt ttttttcaag cttcacattt gcgtgtatct aattcagctg 4800 atgctcaagt ccaaggggta gtctgccttc ccaggctgcc cccagggttt ctgcactggt 4860 cccctctttt cccttcagtc ttcttcactt ccctt 4895 <210> 14 <211> 1429 <212> PRT
<213> HOMO sapiens <400> 14 Met Ile Lys Cys Leu Ser Val Glu Val Gin Ala Lys Leu Arg Ser Gly Leu Ala Ile Ser Ser Leu Gly Gin Cys Val Glu Glu Leu Ala Leu Asn Ser Ile Asp Ala Glu Ala Lys Cys Val Ala Val Arg Val Asn Met Glu Thr Phe Gin val Gin val Ile Asp Asn Gly Phe Gly met Gly ser Asp Asp val Glu Lys val Gly Asn Arg Tyr Phe Thr Ser Lys Cys His Ser val Gin Asp Leu Glu Asn Pro Arg Phe Tyr Gly Phe Arg Gly Glu Ala Leu Ala Asn Ile Ala Asp Met Ala Ser Ala Val Glu Ile Ser Ser Lys Lys Asn Arg Thr met Lys Thr Phe Val Lys Leu Phe Gin Ser Gly Lys moR-0444.ST25.txt Ala Leu Lys Ala Cys Glu Ala Asp Val Thr Arg Ala Ser Ala Gly Thr Thr Val Thr Val Tyr Asn Leu Phe Tyr Gin Leu Pro Val Arg Arg Lys Cys Met Asp Pro Arg Leu Glu Phe Glu Lys Val Arg Gin Arg Ile Glu Ala Leu Ser Leu Met HIS Pro Ser Ile Ser Phe Ser Leu Arg Asn Asp Val Ser Gly Ser Met Val Leu Gin Leu Pro Lys Thr Lys Asp Val Cys Ser Arg Phe Cys Gin Ile Tyr Gly Leu Gly Lys Ser Gin Lys Leu Arg Glu Ile Ser Phe Lys Tyr Lys Glu Phe Glu Leu Ser Gly Tyr Ile Ser Ser Glu Ala HiS Tyr Asn Lys Asn Met Gin Phe Leu Phe Val Asn Lys Arg Leu Val Leu Arg Thr Lys Leu His Lys Leu Ile Asp Phe Leu Leu Arg Lys Glu ser Ile Ile Cys Lys Pro Lys Asn Gly Pro Thr Ser Arg Gin Met Asn Ser Ser Leu Arg His Arg Ser Thr Pro Glu Leu Tyr Gly Ile Tyr Val Ile Asn Val Gin Cys Gin Phe Cys Glu Tyr Asp Val Cys met Glu Pro Ala Lys Thr Leu Ile Glu Phe Gin Asn Trp Asp Thr Leu Leu Phe Cys Ile Gin Glu Gly val Lys met Phe Leu Lys Gin Glu Lys Leu Phe Val Glu Leu Ser Gly Glu Asp Ile Lys Glu Phe ser Glu Asp Asn Gly Phe ser Leu Phe Asp Ala Thr Leu Gin Lys Arg Val Thr Ser Asp Glu Arg Ser Asn Phe Gin Glu Ala Cys Asn Asn Ile Leu Asp Ser . .
MOR-0444.ST25.txt Tyr Glu met Phe Asn Leu Gin Ser Lys Ala val Lys Arg Lys Thr Thr Ala Glu Asn Val Asn Thr Gin Ser Ser Arg Asp Ser Glu Ala Thr Arg Lys Asn Thr Asn Asp Ala Phe Leu Tyr Ile Tyr Glu Ser Gly Gly Pro Gly His Ser Lys Met Thr Glu Pro Ser Leu Gin Asn Lys Asp Ser Ser Cys Ser Glu Ser Lys Met Leu Glu Gin Glu Thr Ile Val Ala Ser Glu Ala Gly Glu Asn Glu Lys His Lys Lys Ser Phe Leu Glu Arg Ser Ser Leu Glu Asn Pro Cys Gly Thr Ser Leu Glu Met Phe Leu Ser Pro Phe Gin Thr Pro Cys His Phe Glu Glu Ser Gly Gin Asp Leu Glu Ile Trp Lys Glu Ser Thr Thr Val Asn Gly Met Ala Ala Asn Ile Leu Lys Asn Asn Arg Ile Gin Asn Gin Pro Lys Arg Phe Lys Asp Ala Thr Glu Val Gly Cys Gin Pro Leu Pro Phe Ala Thr Thr Leu Trp Gly Val His Ser Ala Gin Thr Glu Lys Glu Lys Lys Lys Glu Ser Ser Asn Cys Gly Arg Arg Asn val Phe Ser Tyr Gly Arg val Lys Leu Cys Ser Thr Gly Phe Ile Thr His val Val Gin Asn Glu Lys Thr Lys Ser Thr Glu Thr Glu His Ser Phe Lys Asn Tyr Val Arg Pro Gly Pro Thr Arg Ala Gin Glu Thr Phe Gly Asn Arg Thr Arg His Ser val Glu Thr Pro Asp Ile Lys Asp Leu Ala Ser Thr Leu Ser Lys Glu Ser Gly Gin Leu Pro Asn Lys MOR-0444.ST25.txt Lys Asn Cys Arg Thr Asn Ile Ser Tyr Gly Leu Glu Asn Glu Pro Thr Ala Thr Tyr Thr Met Phe Ser Ala Phe Gin Glu Gly Ser Lys Lys Ser Gin Thr Asp Cys Ile Leu Ser Asp Thr Ser Pro Ser Phe Pro Trp Tyr Arg His Val ser Asn Asp Ser Arg Lys Thr Asp Lys Leu Ile Gly Phe Ser Lys Pro Ile Val Arg Lys Lys Leu Ser Leu Ser ser Gin Leu Gly ser Leu Glu Lys Phe Lys Arg Gin Tyr Gly Lys Val Glu Asn Pro Leu Asp Thr Glu Val Glu Glu Ser Asn Gly val Thr Thr Asn Leu Ser Leu Gin Val Glu Pro Asp Ile Leu Leu Lys Asp Lys Asn Arg Leu Glu Asn Ser Asp Val Cys Lys Ile Thr Thr met Glu His Ser Asp Ser Asp Ser Ser Cys Gin Pro Ala Ser His Ile Leu Asp Ser Glu Lys Phe Pro Phe Ser Lys Asp Glu Asp Cys Leu Glu Gin Gin Met Pro Ser Leu Arg Glu Ser Pro Met Thr Leu Lys Glu Leu Ser Leu Phe Asn Arg Lys Pro Leu Asp Leu Glu Lys Ser ser Glu Ser Leu Ala Ser Lys Leu Ser Arg Leu Lys Gly Ser Glu Arg Glu Thr Gin Thr Met Gly Met Met Ser Arg Phe Asn Glu Leu Pro Asn Ser Asp Ser Ser Arg Lys Asp Ser Lys Leu Cys Ser val Leu Thr Gin Asp Phe Cys Met Leu Phe Asn Asn Lys His Glu Lys Thr Glu Asn Gly Val Ile Pro Thr Ser Asp Ser Ala Thr Gin Asp MoR-0444.ST25.txt Asn Ser Phe Asn Lys Asn Ser Lys Thr His Ser Asn Ser Asn Thr Thr Glu Asn Cys val Ile Ser Glu Thr Pro Leu val Leu Pro Tyr Asn Asn ser Lys val Thr Gly Lys Asp Ser Asp Val Leu Ile Arg Ala Ser Glu Gin Gin Ile Gly Ser Leu Asp Ser Pro Ser Gly Met Leu met Asn Pro val Glu Asp Ala Thr Gly Asp Gin Asn Gly Ile Cys Phe Gin Ser Glu Glu Ser Lys Ala Arg Ala Cys Ser.Glu Thr Glu Glu Ser Asn Thr Cys Cys Ser Asp Trp Gin Arg His Phe Asp val Ala Leu Gly Arg met Val Tyr val Asn Lys Met Thr Gly Leu Ser Thr Phe Ile Ala Pro Thr Glu Asp Ile Gin Ala Ala Cys Thr Lys Asp Leu Thr Thr val Ala Val Asp Val Val Leu Glu Asn Gly ser Gin Tyr Arg Cys Gin Pro Phe Arg Ser Asp Leu val Leu Pro Phe Leu Pro Arg Ala Arg Ala Glu Arg Thr Val Met Arg Gin Asp Asn Arg Asp Thr Val Asp Asp Thr val Ser Ser Glu ser Leu Gin Ser Leu Phe ser Glu Trp Asp Asn Pro Val Phe Ala Arg Tyr Pro Glu val Ala val Asp val Ser Ser Gly Gin Ala Glu Ser Leu Ala Val Lys Ile His Asn Ile Leu Tyr Pro Tyr Arg Phe Thr Lys Gly Met Ile His Ser Met Gin val Leu Gin Gin Val Asp Asn Lys Phe Ile Ala Cys Leu MOR-0444.ST25.txt Met Ser Thr Lys Thr Glu Glu Asn Gly Glu Ala Asp Ser Tyr Glu Lys Gin Gin Ala Gin Gly Ser Gly Arg Lys Lys Leu Leu Ser Ser Thr Leu Ile Pro Pro Leu Glu Ile Thr Val Thr Glu Glu Gin Arg Arg Leu Leu Trp Cys Tyr His Lys Asn Leu Glu Asp Leu Gly Leu Glu Phe Val Phe Pro Asp Thr Ser Asp Ser Leu Val Leu Val Gly Lys Val Pro Leu Cys Phe Val Glu Arg Glu Ala Asn Glu Leu Arg Arg Gly Arg Ser Thr val Thr Lys Ser Ile Val Glu Glu Phe Ile Arg Glu Gin Leu Glu LeU Leu Gin Thr Thr Gly Gly Ile Gin Gly Thr Leu Pro Leu Thr Val Gin Lys Val Leu Ala Ser Gin Ala Cys His Gly Ala Ile Lys Phe Asn Asp Gly Leu Ser Leu Gin Glu Ser Cys Arg Leu Ile Glu Ala Leu Ser Ser Cys Gin Leu Pro Phe Gin Cys Ala His Gly Arg Pro Ser Met Leu Pro Leu Ala Asp Ile Asp His Leu Glu Gin Glu Lys Gin Ile Lys Pro Asn Leu Thr Lys Leu Arg Lys met Ala Gin Ala Trp Arg Leu Phe Gly Lys Ala Glu Cys Asp Thr Arg Gin ser Leu Gin Gin Ser met Pro Pro Cys Glu Pro Pro <210> 15 <211> 3145 6Z a6Pd 0Z61 D1161.DDeD6 e661ppeD16 1.6DpDaD6ql. 1.D6pD1.61.16 1D61.p6p1D6 pD1D6p1161 0981 61P64peol.D pDp6pD6lep Dyep6p161p 1D66pD11.D1 11.plupD161 1.PpapPpll 0081 611.pDD61.p6 6PDDAPP6P p61plep6pD peppleppee DDElp16p6p poleppal1D
OLT 11Du611ppe D6POPPODP1 lleer1161.6 64p6pp6pD Dlpzp6p161 Dp1.6p111Dp 0891 PEPPI.PPDPP 16)11DD16P PPPP6PP66P PlEaDDPP16 1.6D141Dp11 ple661.116p 0Z91 DeD616pDD1 1p661Dpvel 1.p6pDpeuD6 61.DDDe6611 D6611D1p6p 6pDp6eD616 09ST UP1PPI4PDE eD16pD61e6 Pr6PreP661 loPbaue6le eleep6p6Pu 1.1eP616PD1 00S1 Dlpploplp6 lalpplaDop epp163.1aDa lpp61pDpup pe66166pD1 p661el.p6p1 Oi'tI11Deppeep6 ulaluup66 eollatuen 1Dlloaloz 16plaDlal 08E1 p6a61.111.1.6 eD6614614P alepappDp Depep66pp6 1PpeepPu66 1D1D66eDel OZET P1161PP1DD P1DPPD1PPP 1P3.666PD1P 1D1DP6DDPI. 1611.P6VPDP llOPPPAPD
09ZT opeDefteED 11.1.6uP6PPD D603.1De6DDp elaDl.p6eDD 011P6016D1 0OZT 6ppDp1.11.De 6pD66p614p p6pD61p6PP 6p161111D6 pP6616P111 pp611p6p6p OTT 66p6p1pp6p opp6pv1p66 1pD1DI.DD6p D6PP11p661 6pDDET1161. 3.Dp6eppDp6 OZOT 611.64D41.66 6pD1.11.1.2DD erl1DDD6P6 PD16eD6pD6 11p1p6611e ppEolplez6p 096 DD6pollop6 13.11.Delop6 lopp61116p De66111.Dpe DD11p61.p6p D1p11D1Dep 006 6E113.146pp Dapp166D61. D161oppl.PD 11.16pD6116 6pDlpp6p66 lppp6eDD61 OV8 1p161D616p lpp61.p6pD6 p6p666eppp pD66ppe611 611.66DDeeD 1DDE66eDle 08L 114PDp6ppp eDuDD11.1aD P61D6PEPPP PE6PPP6PDP D1P61D11pp 6666pep OZL Dllppl.e6pD p6p6loppp6 6661pD6p6 61D61.DP6e6 e66e66DDDe 1111616q.ep 099 66ppeDDp66 11p6pDpapp 1DD1D1D66e 611D1PeDD1 D116eDle61 ep1p61DDD1 009 leP61.6161D p66p1Dvep6 6e6pDpleDD 11.P661.6ael. 6661.16E661 166pDp6p6p DD663.p61.16 PADD161PP PP11616661 611616611P DD11D6PD3.6 1.el.P61peDe 0817 p3.66111D1D 1.1pDp6pe61 116pD1D1D1 DzpeD661.DD lol1D66pel. pleD66111p On, 16613.P61pp 6P66PeDDI.P D66PP1PPP6 666P1PP 6PP1P1116P u6116u6P1P
09E 16eD1.6o116 61D1.1.D1.1.D1. P6PreP161.1 1101.PP6114 1Pal.epppl.
6pz1D61611.
00E 616p6eD61D lpe6peeD6e 66eD66 66 661.eDel.6eP D1e6166666 EDDDPETPD1 OVZ 16166p666D DADD661D6 I.D6D6Dp66p 6D66DPD6D6 6Del.elolaD p6D6666DDe 081 6D111.1DD6D 616PDEDDPD DaDDETau 66DAleD66 6ED111)11D 6D616D1aD6 OZT 60166P6DD6 6D6D6e6e66 146pD61D6D p6p66pp6DD 6pD61.66D66 leDu6D111.6 09 6p66p6q.66p 66E0DPE011 D1111eD6D6 D166661.616 6616P1.1D6p D666066 SI <00V>
sual.des owoH <ETZ>
Nom <ZTZ>
lAvszIS'VVVO-nw mOR-0444.ST25.txt catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 <210> 16 <211> 934 <212> PRT
<213> Homo sapiens <400> 16 Met Ala val Gin Pro Lys Glu Thr Leu Gin Leu Glu Ser Ala Ala Glu Val Gly Phe Val Arg Phe Phe Gin Gly Met Pro Glu Lys Pro Thr Thr Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly val Ile Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gin Ser Val Val Leu . .
MOR-0444.ST25.txt Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp Leu Leu Leu val Arg Gin Tyr Arg Val Glu val Tyr Lys Asn Arg Ala Gly Asn Lys Ala Ser Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys Ala Ser Pro Gly Asn Leu Ser Gin Phe Glu Asp Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser Ile Gly Val Val Gly Val Lys met Ser Ala Val Asp Gly Gin Arg Gin Val Gly Val Gly Tyr val Asp Ser Ile Gin Arg Lys Leu Gly Leu Cys Glu Phe Pro Asp Asn Asp Gin Phe Ser Asn Leu Glu Ala Leu Leu Ile Gin Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly Asp Met Gly Lys Leu Arg Gin Ile Ile Gin Arg Gly Gly Ile Leu Ile Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gin Asp Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gin met Asn Ser Ala Val Leu Pro Glu Met Glu Asn Gin Val Ala Val Ser Ser Leu Ser Ala 260 . 265 270 Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gin Phe Glu Leu Thr Thr Phe Asp Phe Ser Gin Tyr Met Lys Leu Asp Ile Ala Ala Val Arg Ala Leu Asn Leu Phe Gin Gly Ser val Glu Asp Thr Thr Gly Ser Gin Ser Leu Ala Ala Leu Leu Asn Lys Cys Lys Thr Pro Gin Gly Gin Arg Leu Val Asn Gin Trp Ile Lys Gin Pro Leu Met Asp MOR-0444.51-25.txt Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu Val Glu Ala Phe Val Glu Asp Ala Glu Leu Arg Gin Thr Leu Gin Glu Asp Leu Leu Arg Arg Phe Pro Asp Leu Asn Arg Leu Ala Lys Lys Phe Gin Arg Gin Ala Ala Asn Leu Gin Asp Cys Tyr Arg Leu Tyr Gin Gly Ile Asn Gin Leu Pro Asn Val Ile Gin Ala Leu Glu Lys His Glu Gly Lys His Gin Lys Leu Leu Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser Lys Phe Gin Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gin Val Glu Asn His Glu Phe Leu Val Lys Pro Ser Phe AS Pro Asn Leu Ser Glu Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gin Ser Thr Leu Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gin Ile Lys Leu Asp Ser Ser Ala Gin Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile Gin Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr ser Leu Asn Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala Gin Asp Ala Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly Tyr Val Glu Pro met Gin Thr Leu Asn Asp Val Leu Ala Gin Leu Asp Ala Val Val Ser Phe Ala His Val Ser Asn Gly Ala Pro val Pro Tyr Val Arg Pro Ala Ile MOR-0444.ST25.txt Leu Glu Lys Gly Gin Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala Cys Val Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr Phe Glu Lys Asp Lys Gin Met Phe His Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Ile Arg Gin Thr Gly Val Ile Val Leu Met Ala Gin Ile Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gin Leu Lys Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe His Glu Leu Thr Ala Leu Ala Asn Gin Ile Pro Thr Val Asn Asn Leu His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gin val Lys Lys Gly Val Cys Asp Gin Ser Phe Gly Ile His val Ala Glu Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala Lys Gin Lys Ala Leu Glu Leu Glu Glu Phe Gin Tyr Ile Gly Glu Ser Gin Gly Tyr Asp Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg Glu Gin Gly Glu Lys Ile Ile Gin Glu Phe Leu Ser Lys val Lys Gin Met Pro Phe MOR-0444.ST25.txt Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Lys Leu Lys Gin Leu Lys Ala Glu Val Ile Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser Arg Ile Lys Val Thr Thr <210> 17 <211> 4374 <212> DNA
<213> HOMO sapiens <400> 17 gggcacgagc cctgccatgt ctcgccggaa gcctgcgtcg ggcggcctcg ctgcctccag 60 ctcagcccct gcgaggcaag cggttttgag ccgattcttc cagtctacgg gaagcctgaa 120 atccacctcc tcctccacag gtgcagccga ccaggtggac cctggcgctg cagcggccgc 180 agcgccccca gcgcccgcct tcccgcccca gctgccgccg cacgtagcta cagaaattga 240 cagaagaaag aagagaccat tggaaaatga tgggcctgtt aaaaagaaag taaagaaagt 300 ccaacaaaag gaaggaggaa gtgatctggg aatgtctggc aactctgagc caaagaaatg 360 tctgaggacc aggaatgttt caaagtctct ggaaaaattg aaagaattct gctgcgattc 420 tgcccttcct caaagtagag tccagacaga atctctgcag gagagatttg cagttctgcc 480 aaaatgtact gattttgatg atatcagtct tctacacgca aagaatgcag tttcttctga 540 agattcgaaa cgtcaaatta atcaaaagga cacaacactt tttgatctca gtcagtttgg 600 atcatcaaat acaagtcatg aaaatttaca gaaaactgct tccaaatcag ctaacaaacg 660 gtccaaaagc atctatacgc cgctagaatt acaatacata gaaatgaagc agcagcacaa 720 agatgcagtt ttgtgtgtgg aatgtggata taagtataga ttctttgggg aagatgcaga 780 gattgcagcc cgagagctca atatttattg ccatttagat cacaacttta tgacagcaag 840 tatacctact cacagactgt ttgttcatgt acgccgcctg gtggcaaaag gatataaggt 900 gggagttgtg aagcaaactg aaactgcagc attaaaggcc attggagaca acagaagttc 960 actcttttcc cggaaattga ctgcccttta tacaaaatct acacttattg gagaagatgt 1020 gaatccccta atcaagctgg atgatgctgt aaatgttgat gagataatga ctgatacttc 1080 taccagctat cttctgtgca tctctgaaaa taaggaaaat gttagggaca aaaaaaaggg 1140 caacattttt attggcattg tgggagtgca gcctgccaca ggcgaggttg tgtttgatag 1200 tttccaggac tctgcttctc gttcagagct agaaacccgg atgtcaagcc tgcagccagt 1260 agagctgctg cttccttcgg ccttgtccga gcaaacagag gcgctcatcc acagagccac 1320 atctgttagt gtgcaggatg acagaattcg agtcgaaagg atggataaca tttattttga 1380 atacagccat gctttccagg cagttacaga gttttatgca aaagatacag ttgacatcaa 1440 moR-0444.sT25.txt aggttctcaa attatttctg gcattgttaa cttagagaag cctgtgattt gctctttggc 1500 tgccatcata aaatacctca aagaattcaa cttggaaaag atgctctcca aacctgagaa 1560 ttttaaacag ctatcaagta aaatggaatt tatgacaatt aatggaacaa cattaaggaa 1620 tctggaaatc ctacagaatc agactgatat gaaaaccaaa ggaagtttgc tgtgggtttt 1680 agaccacact aaaacttcat ttgggagacg gaagttaaag aagtgggtga cccagccact 1740 ccttaaatta agggaaataa atgcccggct tgatgctgta tcggaagttc tccattcaga 1800 atctagtgtg tttggtcaga tagaaaatca tctacgtaaa ttgcccgaca tagagagggg 1860 actctgtagc atttatcaca aaaaatgttc tacccaagag ttcttcttga ttgtcaaaac 1920 tttatatcac ctaaagtcag aatttcaagc aataatacct gctgttaatt cccacattca 1980 gtcagacttg ctccggaccg ttattttaga aattcctgaa ctcctcagtc cagtggagca 2040 ttacttaaag atactcaatg aacaagctgc caaagttggg gataaaactg aattatttaa 2100 agacctttct gacttccctt taataaaaaa gaggaaggat gaaattcaag gtgttattga 2160 cgagatccga atgcatttgc aagaaatacg aaaaatacta aaaaatcctt ctgcacaata 2220 tgtgacagta tcaggacagg agtttatgat agaaataaag aactctgctg tatcttgtat 2280 accaactgat tgggtaaagg ttggaagcac aaaagctgtg agccgctttc actctccttt 2340 tattgtagaa aattacagac atctgaatca gctccgggag cagctagtcc ttgactgcag 2400 tgctgaatgg cttgattttc tagagaaatt cagtgaacat tatcactcct tgtgtaaagc 2460 agtgcatcac ctagcaactg ttgactgcat tttctccctg gccaaggtcg ctaagcaagg 2520 agattactgc agaccaactg tacaagaaga aagaaaaatt gtaataaaaa atggaaggca 2580 ccctgtgatt gatgtgttgc tgggagaaca ggatcaatat gtcccaaata atacagattt 2640 atcagaggac tcagagagag taatgataat taccggacca aacatgggtg gaaagagctc 2700 ctacataaaa caagttgcat tgattaccat catggctcag attggctcct atgttcctgc 2760 agaagaagcg acaattggga ttgtggatgg cattttcaca aggatgggtg ctgcagacaa 2820 tatatataaa ggacggagta catttatgga agaactgact gacacagcag aaataatcag 2880 aaaagcaaca tcacagtcct tggttatctt ggatgaacta ggaagaggga cgagcactca 2940 tgatggaatt gccattgcct atgctacact tgagtatttc atcagagatg tgaaatcctt 3000 aaccctgttt gtcacccatt atccgccagt ttgtgaacta gaaaaaaatt actcacacca 3060 ggtggggaat taccacatgg gattcttggt cagtgaggat gaaagcaaac tggatccagg 3120 cgcagcagaa caagtccctg attttgtcac cttcctttac caaataacta gaggaattgc 3180 agcaaggagt tatggattaa atgtggctaa actagcagat gttcctggag aaattttgaa 3240 gaaagcagct cacaagtcaa aagagctgga aggattaata aatacgaaaa gaaagagact 3300 caagtatttt gcaaagttat ggacgatgca taatgcacaa gacctgcaga agtggacaga 3360 ggagttcaac atggaagaaa cacagacttc tcttcttcat taaaatgaag actacatttg 3420 tgaacaaaaa atggagaatt aaaaatacca actgtacaaa ataactctcc agtaacagcc 3480 MOR-0444.ST25.txt tatctttgtg tgacatgtga gcataaaatt atgaccatgg tatattccta ttggaaacag 3540 agaggttttt ctgaagacag tctttttcaa gtttctgtct tcctaacttt tctacgtata 3600 aacactcttg aatagacttc cactttgtaa ttagaaaatt ttatggacag taagtccagt 3660 aaagccttaa gtggcagaat ataattccca agcttttgga gggtgatata aaaatttact 3720 tgatattttt atttgtttca gttcagataa ttggcaactg ggtgaatctg gcaggaatct 3780 atccattgaa ctaaaataat tttattatgc aaccagttta tccaccaaga acataagaat 3840 tttttataag tagaaagaat tggccaggca tggtggctca tgcctgtaat cccagcactt 3900 tgggaggcca aggtaggcag atcacctgag gtcaggagtt caagaccagc ctggccaaca 3960 tggcaaaacc ccatctttac taaaaatata aagtacatct ctactaaaaa tacgaaaaaa 4020 ttagctgggc atggtggcgc acacctgtag tcccagctac tccggaggct gaggcaggag 4080 aatctcttga acctgggagg cggaggttgc aatgagccga gatcacgtca ctgcactcca 4140 gcttgggcaa cagagcaaga ctccatctca aaaaagaaaa aagaaaagaa atagaattat 4200 caagctttta aaaactagag cacagaagga ataaggtcat gaaatttaaa aggttaaata 4260 ttgtcatagg attaagcagt ttaaagattg ttggatgaaa ttatttgtca ttcattcaag 4320 taataaatat ttaatgaata cttgctataa aaaaaaaaaa aaaaaaaaaa aaaa 4374 <210> 18 <211> 1128 <212> PRT
<213> Homo sapiens <400> 18 Met Ser Arg Arg Lys Pro Ala Ser Gly Gly Leu Ala Ala Ser Ser Ser Ala Pro Ala Arg Gin Ala val Leu Ser Arg Phe Phe Gin Ser Thr Gly ser Leu Lys Ser Thr Ser Ser Ser Thr Gly Ala Ala Asp Gin Val Asp Pro Gly Ala Ala Ala Ala Ala Ala Pro Pro Ala Pro Ala Phe Pro Pro Gin Leu Pro Pro His val Ala Thr Glu Ile Asp Arg Arg Lys Lys Arg Pro Leu Glu Asn Asp Gly Pro Val Lys Lys Lys Val Lys Lys val Gin Gin Lys Glu Gly Gly Ser Asp Leu Gly Met Ser Gly Asn Ser Glu Pro Lys Lys Cys Leu Arg Thr Arg Asn Val Ser Lys Ser Leu Glu Lys Leu . .
MOR-0444.ST25.txt Lys Glu Phe Cys Cys Asp Ser Ala Leu Pro Gin Ser Arg val Gin Thr Glu Ser Leu Gin Glu Arg Phe Ala Val Leu Pro Lys Cys Thr Asp Phe Asp Asp Ile Ser Leu Leu His Ala Lys Asn Ala Val Ser Ser Glu Asp ser Lys Arg Gin Ile Asn Gin Lys Asp Thr Thr Leu Phe Asp Leu Ser Gin Phe Gly Ser Ser Asn Thr Ser His Glu Asn Leu Gin Lys Thr Ala Ser Lys Ser Ala Asn Lys Arg Ser Lys Ser Ile Tyr Thr Pro Leu Glu Leu Gin Tyr Ile Glu Met Lys Gin Gin His Lys Asp Ala Val Leu Cys Val Glu Cys Gly Tyr Lys Tyr Arg Phe Phe Gly Glu Asp Ala Glu Ile Ala Ala Arg Glu Leu Asn Ile Tyr cys His Leu Asp His Asn Phe Met Thr Ala Ser Ile Pro Thr His Arg Leu Phe Val His Val Arg Arg Leu Val Ala Lys Gly Tyr Lys Val Gly Val Val Lys Gin Thr Glu Thr Ala Ala Leu Lys Ala Ile Gly Asp Asn Arg Ser Ser Leu Phe Ser Arg Lys Leu Thr Ala Leu Tyr Thr Lys Ser Thr Leu Ile Gly Glu Asp Val Asn Pro Leu Ile Lys Leu Asp Asp Ala val Asn val Asp Glu Ile Met Thr Asp Thr Ser Thr Ser Tyr Leu Leu Cys Ile Ser Glu Asn Lys Glu Asn Val Arg Asp Lys Lys Lys Gly Asn Ile Phe Ile Gly Ile Val Gly Val Gin Pro Ala Thr Gly Glu val val Phe Asp Ser Phe Gin Asp Ser Ala moR-0444.sT25.txt Ser Arg Ser Glu Leu Glu Thr Arg Met Ser Ser Leu Gin Pro Val Glu Leu Leu Leu Pro Ser Ala Leu Ser Glu Gin Thr Glu Ala Leu Ile His Arg Ala Thr Ser Val Ser val Gin Asp Asp Arg Ile Arg Val Glu Arg Met Asp Asn Ile Tyr Phe Glu Tyr Ser His Ala Phe Gin Ala val Thr Glu Phe Tyr Ala Lys Asp Thr Val Asp Ile Lys Gly Ser Gin Ile Ile Ser Gly Ile Val Asn Leu Glu Lys Pro Val Ile Cys Ser Leu Ala Ala Ile Ile Lys Tyr Leu Lys Glu Phe Asn Leu Glu Lys Met Leu Ser Lys Pro Glu Asn Phe Lys Gin Leu Ser Ser Lys Met Glu Phe Met Thr Ile Asn Gly Thr Thr Leu Arg Asn Leu Glu Ile Leu Gin Asn Gin Thr Asp Met Lys Thr Lys Gly Ser Leu Leu Trp Val Leu Asp His Thr Lys Thr Ser Phe Gly Arg Arg Lys Leu Lys Lys Trp Val Thr Gin Pro Leu Leu Lys Leu Arg Glu Ile Asn Ala Arg Leu Asp Ala Val Ser Glu Val Leu His Ser Glu Ser Ser val Phe Gly Gin Ile Glu Asn His Leu Arg Lys Leu Pro Asp Ile Glu Arg Gly Leu Cys Ser Ile Tyr His Lys Lys Cys Ser Thr Gin Glu Phe Phe Leu Ile Val Lys Thr Leu Tyr His Leu Lys Ser Glu Phe Gin Ala Ile Ile Pro Ala val Asn Ser His Ile Gin Ser Asp Leu Leu Arg Thr val Ile Leu Glu Ile Pro Glu Leu Leu Ser Pro mOR-0444.ST25.txt Val Glu His Tyr Leu Lys Ile Leu Asn Glu Gin Ala Ala Lys Val Gly Asp Lys Thr Glu Leu Phe Lys Asp Leu Ser Asp Phe Pro Leu Ile Lys Lys Arg Lys Asp Glu Ile Gin Gly Val Ile Asp Glu Ile Arg Met His Leu Gin Glu Ile Arg Lys Ile Leu Lys Asn Pro Ser Ala Gin Tyr val Thr Val Ser Gly Gin Glu Phe Met Ile Glu Ile Lys Asn Ser Ala Val Ser Cys Ile Pro Thr Asp Trp Val Lys Val Gly Ser Thr Lys Ala Val Ser Arg Phe His Ser Pro Phe Ile Val Glu Asn Tyr Arg His Leu Asn Gin Leu Arg Glu Gin Leu Val Leu Asp Cys Ser Ala Glu Trp Leu Asp Phe Leu Glu Lys Phe Ser Glu His Tyr His Ser Leu Cys Lys Ala Val His His Leu Ala Thr Val Asp Cys Ile Phe Ser Leu Ala Lys Val Ala Lys Gin Gly Asp Tyr Cys Arg Pro Thr Val Gin Glu Glu Arg Lys Ile Val Ile Lys Asn Gly Arg His Pro Val Ile Asp Val Leu Leu Gly Glu Gln Asp Gin Tyr val Pro Asn Asn Thr Asp Leu Ser Glu Asp Ser Glu Arg val met Ile Ile Thr Gly Pro Asn met Gly Gly Lys Ser Ser Tyr Ile Lys Gin Val Ala Leu Ile Thr Ile Met Ala Gln Ile Gly Ser Tyr Val Pro Ala Glu Glu Ala Thr Ile Gly Ile Val Asp Gly he Phe Thr Arg met Gly Ala Ala Asp Asn Ile Tyr Lys Gly Arg Ser Thr Phe met MOR-0444.5125.txt Glu Glu Leu Thr Asp Thr Ala Glu Ile Ile Arg Lys Ala Thr Ser Gin Ser Leu Val Ile Leu Asp Glu Leu Gly Arg Gly Thr Ser Thr His Asp Gly Ile Ala Ile Ala Tyr Ala Thr Leu Glu Tyr Phe Ile Arg Asp Val Lys Ser Leu Thr Leu Phe Val Thr His Tyr Pro Pro Val Cys Glu Leu Glu Lys Asn Tyr Ser His Gin Val Gly Asn Tyr His Met Gly Phe Leu Val Ser Glu Asp Glu Ser Lys Leu Asp Pro Gly Ala Ala Glu Gin Val Pro Asp Phe Val Thr Phe Leu Tyr Gin Ile Thr Arg Gly Ile Ala Ala Arg Ser Tyr Gly Leu Asn Val Ala Lys Leu Ala Asp val Pro Gly Glu Ile Leu Lys Lys Ala Ala His Lys Ser Lys Glu Leu Glu Gly Leu Ile Asn Thr Lys Arg Lys Arg Leu Lys Tyr Phe Ala Lys Leu Trp Thr Met His Asn Ala Gin Asp Leu Gin Lys Trp Thr Glu Glu Phe Asn Met OU Glu Thr Gin Thr Ser Leu Leu His <210> 19 <211> 3095 <212> DNA
<213> HOMO sapiens <400> 19 cagaaacctc atacttctcg ggtcagggaa ggtttgggag gatgctgagg cctgagatct 60 catcaacctc gccttctgcc ccggcggttt cccccgtcgt cggagaaacc cgctcacctc 120 agggtccccg ctacaatttc ggactccagg agactccaca gagccgccct tcggtccagg 180 tggtctctgc atccacctgt cctggcacgt caggagctgc gggcgaccgg agcagcagca 240 gcagcagcct tccctgcccc gcgccaaact cccggccagc tcaaggttca tactttggaa 300 acaaaagagc ttatgcagaa aacacagttg catcaaattt tacttttggt gcaagctcat 360 mOR-0444.ST25.txt cttctgcacg agatactaat tatcctcaaa cacttaaaac tccattgtct actggaaatc 420 ctcagagatc aggttataag agctggacac cacaagtggg atattcagct tcatcctcat 480 ctgcgatttc tgcacactcc ccatcagtta ttgtagctgt tgtagaaggg agaggacttg 540 ccagaggtga aataggaatg gcaagtattg atttaaaaaa cccccaaatt atactatccc 600 agtttgcaga caacacaaca tatgcaaagg tgatcactaa acttaaaatt ttatcacctt 660 tggaaataat aatgtcaaat actgcttgtg ctgtggggaa ttccaccaag ttgttcactc 720 tgatcacaga aaatttcaag aatgttaatt tcactactat ccaaaggaaa tacttcaatg 780 aaacaaaagg attagagtac attgaacagt tatgcatagc agaattcagc actgtcctaa 840 tggaggttca gtccaagtat tactgccttg cagctgttgc agctttgtta aaatatgttg 900 aatttattca aaattcagtt tatgcaccaa aatcactgaa gatttgtttc cagggtagtg 960 aacagacagc catgatagat tcatcatcag cccaaaacct tgaattgtta attaataatc 1020 aagactatag gaataatcac actctctttg gtgttctaaa ttatactaag actcctggag 1080 ggagtagacg acttcgttct aatatattag agcctctagt tgatattgaa accattaaca 1140 tgcgcttaga ttgtgttcaa gaactacttc aagatgagga actatttttt ggacttcaat 1200 cagttatatc aagatttctt gatacagagc agcttctttc tgttttagtc caaattccag 1260 agcaagacac ggtcaatgct gctgaatcaa agataacaaa tttaatatac ttaaaacata 1320 ccttggaact tgtggatcct ttaaagattg ctatgaagaa ctgtaacaca cctttattaa 1380 gagcttacta tggttccttg gaagacaaga ggtttggaat catacttgaa aagattaaaa 1440 cagtaattaa tgatgatgca agatacatga aaggatgcct aaacatgagg actcagaagt 1500 gctatgcagt gaggtctaac ataaatgaat ttcttgacat agcaagaaga acatacacag 1560 agattgtaga tgacatagca ggaatgatat cacaacttgg agaaaaatat agtctacctt 1620 taaggacaag tcttagctct gttcgaggat ttttcatcca gatgactaca gattgtatag 1680 ccctacctag tgatcaactt ccttcagaat ttattaagat ttctaaagtg aaaaattctt 1740 acagctttac atcagcagat ttaattaaaa tgaatgaaag atgccaagaa tctttgagag 1800 aaatctatca catgacttat atgatagtgt gcaaactgct tagtgagatt tatgaacata 1860 ttcattgctt atataaacta tctgacactg tgtcaatgct ggatatgcta ctgtcatttg 1920 ctcatgcctg cactctttct gactatgttc gaccagaatt tactgatact ttagcaatca 1980 aacagggatg gcatcctatt cttgaaaaaa tatctgcgga aaaacctatt gccaacaata 2040 cctatgttac agaagggagt aattttttga tcataactgg accaaacatg agtggaaaat 2100 ccacatattt aaaacagatt gctctttgtc agattatggc ccagattgga tcatatgttc 2160 cagcagaata ttcttccttt agaattgcta aacagatttt tacaagaatt agtactgatg 2220 atgatatcga aacaaattca tcaacattta tgaaagaaat gaaagagata gcatatattc 2280 tacataatgc taatgacaaa tcgctcatat taattgatga acttggcaga ggtactaata 2340 cggaagaagg tattggcatt tgttatgctg tttgtgaata tctactgagc ttaaaggcat 2400 MOR-0444.ST25.txt ttacactgtt tgctacacat ttcctggaac tatgccatat tgatgccctg tatcctaatg 2460 tagaaaacat gcattttgaa gttcaacatg taaagaatac ctcaagaaat aaagaagcaa 2520 ttttgtatac ctacaaactt tctaagggac tcacagaaga gaaaaattat ggattaaaag 2580 ctgcagaggt gtcatcactt ccaccatcaa ttgtcttgga tgccaaggaa atcacaactc 2640 aaattacgag acaaattttg caaaaccaaa ggagtacccc tgagatggaa agacagagag 2700 ctgtgtacca tctagccact aggcttgttc aaactgctcg aaactctcaa ttggatccag 2760 acagtttacg aatatattta agtaacctca agaagaagta caaagaagat tttcccagga 2820 ccgaacaagt tccagaaaag actgaagaat aatcacaatt ctaatgtaat aatatatctt 2880 aattcaagga acctagaatt tatttttctc cttagagata aggaaaataa catttgccaa 2940 atttcatatt ttaattgaaa attacattat attaacatca caattgtcat ctatatattc 3000 tatatgaaaa atatttatta taacttaaca aatgagaact acttaaagga atggttttta 3060 tgttaggaga aaatacaata caccacaaaa aaaaa 3095 <210> 20 <211> 936 <212> PRT
<213> Homo sapiens <400> 20 Met Leu Arg Pro Glu Ile Ser Ser Thr Ser Pro Ser Ala Pro Ala Val Ser Pro Val Val Gly Glu Thr Arg Ser Pro Gin Gly Pro Arg Tyr Asn Phe Gly Leu Gin Glu Thr Pro Gin Ser Arg Pro Ser Val Gin Val Val Ser Ala Ser Thr Cys Pro Gly Thr Ser Gly Ala Ala Gly Asp Arg Ser Ser Ser Ser Ser Ser Leu Pro Cys Pro Ala Pro Asn Ser Arg Pro Ala Gin Gly Ser Tyr Phe Gly Asn Lys Arg Ala Tyr Ala Glu Asn Thr Val Ala Ser Asn Phe Thr Phe Gly Ala Ser Ser Ser Ser Ala Arg Asp Thr Asn Tyr Pro Gin Thr Leu Lys Thr Pro Leu Ser Thr Gly Asn Pro Gin Arg Ser Gly Tyr Lys Ser Trp Thr Pro Gin Val Gly Tyr Ser Ala Ser MOR-0444.ST25.txt Ser Ser Ser Ala Ile Ser Ala His Ser Pro Ser Val Ile val Ala val Val Glu Gly Arg Gly Leu Ala Arg Gly Glu Ile Gly met Ala Ser Ile Asp Leu Lys Asn Pro Gin Ile Ile Leu Ser Gln Phe Ala Asp Asn Thr Thr Tyr Ala Lys Val Ile Thr Lys Leu Lys Ile Leu Ser Pro Leu Glu Ile Ile Met Ser Asn Thr Ala Cys Ala Val Gly Asn Ser Thr Lys Leu Phe Thr Leu Ile Thr Glu Asn Phe Lys Asn Val Asn Phe Thr Thr Ile Gin Arg Lys Tyr Phe Asn Glu Thr Lys Gly Leu Glu Tyr Ile Glu Gin Leu Cys Ile Ala Glu Phe Ser Thr Val Leu met Glu val Gin Ser Lys Tyr Tyr Cys Leu Ala Ala Val Ala Ala Leu Leu Lys Tyr Val Glu Phe Ile Gin Asn Ser Val Tyr Ala Pro Lys Ser Leu Lys Ile Cys Phe Gin Gly Ser Glu Gin Thr Ala Met Ile Asp Ser Ser Ser Ala Gin Asn Leu Glu Leu Leu Ile Asn Asn Gin Asp Tyr Arg Asn Asn His Thr Leu Phe Gly Val Leu Asn Tyr Thr Lys Thr Pro Gly Gly Ser Arg Arg Leu Arg Ser Asn Ile Leu Glu Pro Leu Val Asp Ile Glu Thr Ile Asn Met Arg Leu Asp Cys Val Gin Glu Leu Leu Gin Asp Glu Glu Leu Phe Phe Gly Leu Gin Ser Val Ile Ser Arg Phe Leu Asp Thr Glu Gin Leu Leu Ser Val Leu Val Gin Ile Pro Glu Gin Asp Thr Val Asn Ala Ala Glu Ser MOR-0444.ST25.txt Lys Ile Thr Asn Leu Ile Tyr Leu Lys His Thr Leu Glu Leu Val Asp Pro Leu Lys Ile Ala met Lys Asn Cys Asn Thr Pro Leu Leu Arg Ala Tyr Tyr Gly Ser Leu Glu Asp Lys Arg Phe Gly Ile Ile Leu Glu Lys Ile Lys Thr Val Ile Asn Asp Asp Ala Arg Tyr Met Lys Gly Cys Leu Asn Met Arg Thr Gin Lys Cys Tyr Ala Val Arg Ser Asn Ile Asn Glu Phe Leu Asp Ile Ala Arg Arg Thr Tyr Thr Glu Ile Val Asp Asp Ile Ala Gly Met Ile Ser Gin Leu Gly Glu Lys Tyr Ser Leu Pro Leu Arg Thr Ser Leu Ser Ser Val Arg Gly Phe Phe Ile Gin met Thr Thr Asp Cys Ile Ala Leu Pro Ser Asp Gin Leu Pro Ser Glu Phe Ile Lys Ile Ser Lys Val Lys Asn Ser Tyr Ser Phe Thr Ser Ala Asp Leu Ile Lys Met Asn Glu Arg Cys Gin Glu ser Leu Arg Glu Ile Tyr His Met Thr Tyr met Ile Val cys Lys Leu Leu Ser Glu Ile Tyr Glu His Ile His Cys Leu Tyr Lys Leu Ser Asp Thr Val Ser Met Leu Asp Met Leu Leu Ser Phe Ala His Ala Cys Thr Leu Ser Asp Tyr val Arg Pro Glu Phe Thr Asp Thr Leu Ala Ile Lys Gin Gly Trp His Pro Ile Leu Glu Lys Ile Ser Ala Glu Lys Pro Ile Ala Asn Asn Thr Tyr Val Thr Glu Gly Ser Asn Phe Leu Ile Ile Thr Gly Pro Asn Met Ser Gly Lys Ser Thr MOR-0444.ST25.txt Tyr Leu Lys Gin Ile Ala Leu Cys Gin Ile Met Ala Gin Ile Gly Ser Tyr Val Pro Ala Glu Tyr Ser Ser Phe Arg Ile Ala Lys Gin Ile Phe Thr Arg Ile Ser Thr Asp Asp Asp Ile Glu Thr Asn Ser Ser Thr Phe Met Lys Glu Met Lys Glu Ile Ala Tyr Ile Leu His Asn Ala Asn Asp Lys Ser Leu Ile Leu Ile Asp Glu Leu Gly Arg Gly Thr Asn Thr Glu Glu Gly Ile Gly Ile Cys Tyr Ala Val Cys Glu Tyr Leu Leu Ser Leu Lys Ala Phe Thr Leu Phe Ala Thr His Phe Leu Glu Leu Cys His Ile Asp Ala Leu Tyr Pro Asn Val Glu Asn Met His Phe Glu Val Gin His Val Lys Asn Thr Ser Arg Asn Lys Glu Ala Ile Leu Tyr Thr Tyr Lys Leu Ser Lys Gly Leu Thr Glu Glu Lys Asn Tyr Gly Leu Lys Ala Ala Glu Val Ser Ser Leu Pro Pro Ser Ile Val Leu Asp Ala Lys Glu Ile Thr Thr Gin Ile Thr Arg Gin Ile Leu Gin Asn Gin Arg Ser Thr Pro Glu Met Glu Arg Gin Arg Ala Val Tyr His Leu Ala Thr Arg Leu val Gin Thr Ala Arg Asn Ser Gin Leu AS Pro Asp Ser Leu Arg Ile Tyr Leu Ser Asn Leu Lys Lys Lys Tyr Lys Glu Asp Phe Pro Arg Thr Glu Gin Val Pro Glu Lys Thr Glu Glu <210> 21 <211> 2726 <212> DNA
<213> Homo sapiens gv DE.Pd 31.pleD31.ve 636136P3.ED llEPEIDEDP3 laparpo6De 6e16ED6666 1.1EEE6DD66 E66E6E36E3 D64611.1.36e 366u1.661.D3 D663.Epllep Eple611366 e6eD
098T 1DDP1P1ED6 PETP666P31. PD1DEP3DDP 661DEDI.E33. 6ETED4666e 666EEEDE66 0081 66166161.ER 6PDP3D3OPP 33361.61113 DPE6D3D616 1013PPE061E 61D1D31X3E
OVLT
6E3661EE6E pplEE6Dr1.6 66611.DD1.6E PDED3DD1DE 11.636 766U PD4DP1D661 0891 EzDE666DDD 62D616e3D6 lloaD663.D6 1DD3.6DE66z DADDD1DD6 11.DDE661.1E
OZ9T 16P6DDDPP1 aD1601D6ED6 E6DED661.D6 166PDD616P DP1D6PDDP1 61P61361D6 09ST DE6E66E.DDE 666DDle6e6 361DED6aDD p666661.D61. leD61.e66aD EIPMETDDET

6D3D646e3.6 D4E1.DED64D 6Eu6e66e6p DaDI.D11461 Eplapp661D E661EellE6 OVVT
E61.11DE61.6 EDD66e6E1.6 61.EDDlaDD6 app6DDDDI.I. EaD11.1D1.1D D11D6611E6 NET
171DDDIXDP apl.ED1.61.6r 361uplapp1. aul6DoDaDE 6666 666pED
OZET
6oDD61166E 61DEI.I.DD11 1.6EDDD41.De 6663.P61.DP6 ER6D6PEPPE 6E6aP614Ee E6applE6E1 PDRPDDDD1D D1.6PDEDlaD 6DIXPPP61D 61.1DD6PD66 6P6111DE66 16el6ee66 641.PDZDAP 3D6D4P1PDD PDEaDDe61E 61D1D1.16V6 PEDDD614PD
ovtIE666D113.D1 DETDDIXDDI. EreDEIDDEaDD D1D6DD61.DD 63.E666e61D D66613Dp61.

6EDE1.61613 P6PPDBaD1D 11.66PD661D P6D6P01.66P PDDPDEDDD1 6146PE61eD
OZOT
6DEEE61Dal E61.D1DD616 DPEETUDIXD PD1.6661.DDI. DE6DZUDEI1.D 61.E6ED1D66 zEDE661D4E PETDDDDEaD EaD1141145 PD14PDZSDE 661D1.6D1.D1 3.6eDaD6E66 6664DDE6ar D1DP6D016D PDED1.1651.E 1D66POZDEa DETE6E6E66 6616EE1.61D

ED363.E6EDE EDI.DDlee66 1.1.1.DaDD6ED 1D6666u66e E61.3e661.6E DD661.6peep e3.616E31.3D ppeD1D16e6 1.6E6EE1144 le6EDEI.D11. 616EDElape DEETEDZPET
OZL
1.EDEE61661 31E33.3E611 6e6 Ee3.11)6663. DDleD33D16 D6E0163.PEZ

66. DEE6116666 3zEE6EE6EE 633.6663.333. lfte613613 666e66e D6E6D116E3 Ep1.pploD61 DE614.13331 lEzlEDDalp 4DIOD11D1D DlEPPOP61 OVS
DP3D61.3E61 EDADPEIEDD 33.P33.1DDIO P1DPEE661.3 11133136D PPDPPEAPP
08V 4E6E664D16 61441E6646 16PPD36141 1.1B4PD1PPP 613De6 ozv eD6e66EDDD 1DD611D6EE E.6661DallE 6D4DE6aplE e6p6aE66ED EpeDD616E6 461461.D 1.6PDDDDIPP DI.E6E6106 1D1.1.6e6E6E Dp4D11D6EE 31DD6E6E6D

PDDE6EDODD 61X6EDD6IX D1.1.DEDDI.E1 DEDDI.DE61.6 EI.DEI.E61.E1 DEI.DD61.1ED
oz 66613.DelE6 666. 61.D61.61616 1DIXDOIX6E 6D)661.D6E6 6E66E66E66 e6D16ee66e 66e66r6DD6 6E666EDDDD 666DD61.6ED DDD66D3D6E DDDD11.D66D
OZT
DI.D31.D366p 66661DDE6E 6DDE666ED6 DDRDE66P66 PEDDDPU6D6 a6PlaDD1D

D661.ED1D6E EDDI.DD6E6E D1.DE6D6E16 1DDPODDI.D1. 16D6666D6P D166D166)6 TZ <00b>
axl.szis'VVV0-110w MOR-0444.ST25.txt ccttggcctc tccaccttca tgatcgacct caaccagcag gtggcgaaag cagtgaacaa 2040 tgccactgca cagtcgctgg tccttattga tgaatttgga aagggaacca acacggtgga 2100 tgggctcgcg cttctggccg ctgtgctccg acactggctg gcacgtggac ccacatgccc 2160 ccacatcttt gtggccacca actttctgag ccttgttcag ctacaactgc tgccacaagg 2220 gcccctggtg cagtatttga ccatggagac ctgtgaggat ggcaacgatc ttgtcttctt 2280 ctatcaggtt tgcgaaggtg ttgcgaaggc cagccatgcc tcccacacag ctgcccaggc 2340 tgggcttcct gacaagcttg tggctcgtgg caaggaggtc tcagacttga tccgcagtgg 2400 aaaacccatc aagcctgtca aggatttgct aaagaagaac caaatggaaa attgccagac 2460 attagtggat aagtttatga aactggattt ggaagatcct aacctggact tgaacgtttt 2520 catgagccag gaagtgctgc ctgctgccac cagcatcctc tgagagtcct tccagtgtcc 2580 tccccagcct cctgagactc cggtgggctg ccatgccctc tttgtttcct tatctccctc 2640 agacgcagag tttttagttt ctctagaaat tttgtttcat attaggaata aagtttattt 2700 tgaagaaaaa aaaaaaaaaa aaaaaa 2726 <210> 22 <211> 835 <212> PRT
<213> Homo sapiens <400> 22 met Ala Ser Leu Gly Ala Asn Pro Arg Arg Thr Pro Gin Gly Pro Arg Pro Gly Ala Ala Ser Ser Gly Phe Pro Ser Pro Ala Pro Val Pro Gly Pro Arg Glu Ala Glu Glu Glu Glu Val Glu Glu Glu Glu Glu Leu Ala Glu Ile His Leu Cys Val Leu Trp Asn Ser Gly Tyr Leu Gly Ile Ala Tyr Tyr Asp Thr Ser Asp Ser Thr Ile His Phe met Pro Asp Ala Pro Asp His Glu Ser Leu Lys Leu Leu Gin Arg Val Leu Asp Glu Ile Asn Pro Gin Ser val Val Thr Ser Ala Lys Gin Asp Glu Asn met Thr Arg Phe Leu Gly Lys Leu Ala Ser Gin Glu His Arg Glu Pro Lys Arg Pro Glu Ile Ile Phe Leu Pro Ser Val Asp Phe Gly Leu Glu Ile Ser Lys MOR-0444.ST25.txt Gin Arg Leu Leu Ser Gly Asn Tyr Ser Phe Ile Pro Asp Ala Met Thr Ala Thr Glu Lys Ile Leu Phe Leu Ser Ser Ile Ile Pro Phe Asp Cys Leu Leu Thr val Arg Ala Leu Gly Gly Leu Leu Lys Phe Leu Gly Arg Arg Arg Ile Gly val Glu Leu Glu Asp Tyr Asn Val Ser Val Pro Ile Leu Gly Phe Lys Lys Phe Met Leu Thr His Leu Val Asn Ile Asp Gin Asp Thr Tyr Ser Val Leu Gin Ile Phe Lys Ser Glu Ser His Pro Ser Val Tyr Lys val Ala Ser Gly Leu Lys Glu Gly Leu Ser Leu Phe Gly Ile Leu Asn Arg Cys His Cys Lys Trp Gly Glu Lys Leu Leu Arg Leu Trp Phe Thr Arg Pro Thr His Asp Leu Gly Glu Leu Ser Ser Arg Leu Asp val Ile Gin Phe Phe Leu Leu Pro Gin Asn Leu Asp Met Ala Gin Met Leu His Arg Leu Leu Gly His Ile Lys Asn Val Pro Leu Ile Leu Lys Arg Met Lys Leu Ser His Thr Lys Val Ser Asp Trp Gin Val Leu Tyr Lys Thr Val Tyr Ser Ala Leu Gly Leu Arg Asp Ala Cys Arg Ser Leu Pro Gin Ser Ile Gin Leu Phe Arg Asp Ile Ala Gin Glu Phe ser Asp Asp Leu His His Ile Ala Ser Leu Ile Gly Lys Val val Asp Phe Glu Gly Ser Leu Ala Glu Asn Arg Phe Thr val Leu Pro Asn Ile Asp Pro Glu Ile Asp Glu Lys Lys Arg Arg Leu Met Gly Leu Pro Ser Phe . .
MOR-0444.ST25.txt Leu Thr Glu val Ala Arg Lys Glu Leu Glu Asn Leu Asp Ser Arg Ile Pro Ser Cys Ser Val Ile Tyr Ile Pro Leu Ile Gly Phe Leu Leu Ser Ile Pro Arg Leu Pro Ser Met Val Glu Ala Ser Asp Phe Glu Ile Asn Gly Leu Asp Phe Met Phe Leu Ser Glu Glu Lys Leu His Tyr Arg Ser Ala Arg Thr Lys Glu Leu Asp Ala Leu Leu Gly Asp Leu His Cys Glu Ile Arg Asp Gin Glu Thr Leu Leu met Tyr Gin Leu Gin Cys Gin Val Leu Ala Arg Ala Ala Val Leu Thr Arg Val Leu Asp Leu Ala Ser Arg Leu Asp Val Leu Leu Ala Leu Ala Ser Ala Ala Arg Asp Tyr Gly Tyr Ser Arg Pro Arg Tyr Ser Pro Gin Val Leu Gly Val Arg Ile Gin Asn Gly Arg His Pro Leu Met Glu Leu Cys Ala Arg Thr Phe val Pro Asn Ser Thr Glu Cys Gly Gly Asp Lys Gly Arg Val Lys Val Ile Thr Gly Pro Asn Ser Ser Gly Lys Ser Ile Tyr Leu Lys Gin Val Gly Leu Ile Thr Phe Met Ala Leu Val Gly Ser Phe val Pro Ala Glu Glu Ala Glu Ile Gly Ala Val Asp Ala Ile Phe Thr Arg Ile His Ser Cys Glu Ser Ile Ser Leu Gly Leu Ser Thr Phe Met Ile Asp Leu Asn Gin Gin Val Ala Lys Ala val Asn Asn Ala Thr Ala Gin Ser Leu val Leu Ile Asp Glu Phe Gly Lys Gly Thr Asn Thr Val Asp Gly Leu Ala Leu Leu Ala . .
MOR-0444.ST25.txt Ala Val Leu Arg His Trp Leu Ala Arg Gly Pro Thr Cys Pro His Ile Phe Val Ala Thr Asn Phe Leu Ser Leu Val Gin Leu Gin Leu Leu Pro Gin Gly Pro Leu Val Gin Tyr Leu Thr Met Glu Thr Cys Glu Asp Gly Asn Asp Leu Val Phe Phe Tyr Gin Val Cys Glu Gly Val Ala Lys Ala Ser His Ala Ser His Thr Ala Ala Gin Ala Gly Leu Pro Asp Lys Leu Val Ala Arg Gly Lys Glu Val Ser Asp Leu Ile Arg Ser Gly Lys Pro Ile Lys Pro Val Lys Asp Leu Leu Lys Lys Asn Gin Met Glu Asn Cys Gin Thr Leu Val Asp Lys Phe Met Lys Leu Asp Leu Glu Asp Pro Asn Leu Asp Leu Asn Val Phe Met Ser Gin Glu Val Leu Pro Ala Ala Thr Ser Ile Leu <210> 23 <211> 4264 <212> DNA
<213> Homo sapiens <400> 23 atttcccgcc agcaggagcc gcgcggtaga tgcggtgctt ttaggagctc cgtccgacag 60 aacggttggg ccttgccggc tgtcggtatg tcgcgacaga gcaccctgta cagcttcttc 120 cccaagtctc cggcgctgag tgatgccaac aaggcctcgg ccagggcctc acgcgaaggc 180 ggccgtgccg ccgctgcccc cggggcctct ccttccccag gcggggatgc ggcctggagc 240 gaggctgggc ctgggcccag gcccttggcg cgatccgcgt caccgcccaa ggcgaagaac 300 ctcaacggag ggctgcggag atcggtagcg cctgctgccc ccaccagttg tgacttctca 360 ccaggagatt tggtttgggc caagatggag ggttacccct ggtggccttg tctggtttac 420 aaccacccct ttgatggaac attcatccgc gagaaaggga aatcagtccg tgttcatgta 480 cagttttttg atgacagccc aacaaggggc tgggttagca aaaggctttt aaagccatat 540 acaggttcaa aatcaaagga agcccagaag ggaggtcatt tttacagtgc aaagcctgaa 600 TS D6Ed 0179Z APDEIMP1 DPEP6PP61E, 161ErlEI.D6 66ED6EDE6E DDDPDDEE6P D15PETP6aD
085? DDD1D16661. 461.pplED11. PETP16PD3.3 EaD66E6E61 1D1P6PDD11 DETP6ETTaD
11D6P6e461 16eP63D1D1 PPPPDP61DD 61614661PD ZDDPETP6P1 PDD61P6P1D
09VZ 1.6DI.E61EE1 1E1D61ellE DDEE161D1D ED=6461.1. 1D661rED6E EE1DD1D66D
00VZ 6E.E4.661111 DDI.DP1PDA 11DE1E6116 66E6E6E4DE aDDDPPNEE 666 017E? leuppep661 EE61D11114. E6E6614Dee DevelarDE6 1.6ED61.E6E1 D61.661EE6D
08ZZ reDzuaDD6E eeDDED1101. EaD61661D1 E6EEDE1DED 6ED16EDEDE 61.Da1E661.1 OZZZ D301.1Elelp efte64111E P10661EPD1 E411106E66 EDae611E11. DD61PPRPEP
091Z DI.DDE3.311D 1.616116616 6v1D1D61D1 DI.DDD6614.E E616EEEE6E 6E66EDDeDe OOTZ 61166611ED D11E61D16E 6ED11De6lu 166EEE3.4D6 1.66EDDDDE1 161E616666 OVOZ 1.1ED664E61. 6pplApppe 666. EE6EE66E61 30D1D1DPE6 P613.1DeRPP
0861 DD1.ED61p66 614146 PDDD 1D66DODPIX 6.6666 'n1.10101.16 1DD1611ED1 0Z61 46E6EruaD1. areDeurv4D PPPMEPEOZ DZDZPEE66E BUEP64,11E1 14.16EEDE16 098T PDDDDD1p1D PDPD66a6U1 DI.DPEIET141 E6E6D11.63.1 EDD6D1e61E 66ED
0081 I-Mel:EDI-11 laftEE6661 DED11DEI.E6 14.61.41D616 4.61664.E1.ED
61.6D1DeleD
OLT D661014D11 EftE66E6EE Eupftepplp DETlaD41D1 el6Ev16EDE 1Der6E61D1 0891 DDD4E6166E E661D61.616 PDP41DE6ED PDPa666PPD De1.1.ED1E66 u461D1E6E6 OZ9T 66E66E61.66 16E6E1E64E 16rEDD1Elp lEDED661E6 EEE6E161v6 DED66E661.E
ogst 61EvE6EDD1 De6p61DE6E DEE6616E6D ED6E16ErE4 E1D666EE6E D6166aDDDI.
00ST 1E6ED1.1E11 6DD66141ED 61.1.ErE64DD 1.11D661D11. EDDD6661DE
ED66EEE61.v.
Ott Dale1.664D6 6661DEE616 ED46E661.1.E 1.1D1.D61E66 4EDEDDE461 D6E61E1141 08E1 eeE6666166 EEDE4164D4 ED161.1DI.E6 141DEp6ED1 D16Erlav6r D6646616EE
OZET 66E61E6661 DD1DE1611D 41EED1DD11 ae66e61DD6 161E1DaDED ElD1ED61.E6 09ZT 1111E6DDDD ED1E61DD66 E66E66EDED 6e61.p6E6eu 6E6EEEE.66e 66EE11D661 00ZT eu6E111DEE E61ED1E166 111.64DE1DD D6D16E16ED E6 1E61661.6 6E6616E116 OTT DEDDDEMeDD D1PP61D4IX PReDaDDDA aD1D111D6P 6E61.1.1Dele EfteDDPPRE, 0801 EDI.ED144.ED 6E1DEED6EE DPBRDDR3D6 PD1DDADPR E66ee66E1D lo6eeE66e OZOT EvE11D1D1D 661EEE661D E6166lue6e 6eE66D6Eeu 6D1D611.6re ED161DDD6E
096 DEE61.DD66E E616e6E61.6 v1E6666646 E6616ED6ee leee64E616 ED6eE66EE6 006 6P66pplppo v6vDD6EE1.1 1.EE66161E6 1D1D661661 1EDE616e6e 61D11.E6ED1 ovg EaeD1.666ee 6DPBPPEEE1 EPPDDEIDD6E 3.6EE6D66E1 D1E66EEDED P6PE1DDETD
08L Pl6PrE0ETET v616E6E611 vee6zue1e6 EE6Eu616E6 Ev1E6EDvel. 6DP11DPRDE
OZL D66E466E66 3.E6E6re6EE 66E6EE6EDD 6e6ED1.DDD6 E61E616414 BED6611EE6 099 11.D66E6EE4 av6EEDEesee ulEEE44DD6 EE61E6vD61 6DEED61.uvo 6E6E61DEle zxvszi.S-VVVO-bOw m a6Pd ST OT
PLV cmd .JS sAl 0Jd 4d Ld JS JAI nal J41 JaS uLD 6JV JaS laW
VZ <0017>
sual.des ow0H <ETZ>
12Id <m>
09E1 <LIZ>
tZ <OTZ>
t9Zt eble 09Z17 eppp111114 PlalpEpple ezDaP6aP11 PDPPDP6Ppl lppe166166 ppeDDe6101 00Zt 10e6116e61 1106pe6611 epelppEapp 666 ep1.1P61.14D v61D6a4ppp OtTt lepp1.61D6u p61D6zeoez 61DueD166u pe616e1D66 13D6z116pp 666D114pla 080t p6pplopp46 EozepEllebe eft6lalue6 p6eD6ppee 6P1PDP666p Pepollp116 OZOV PP66e6eDDD 1Dleplo611 D66peD6pD6 lepalaD661 plAppppl.) 3161.1D6p66 096E ftellPplau PelP1313D4 16oullelop 6p66PD6PD DpoP6pP646 leu6lpupu6 Pa56leD6ap D663x4pDp6 666 lo61.151euu eplolaplau 6pp6u1.6e11.
Ot8E Pplleppelo Pp1.pPeD411 lel1PDP16D 161epppapz Dp6p6aD61.1 Dpp6ppp146 08LE 116ED6apee D6pleeD66a e6661x6111 uppeD6aDel 66e6ee66e1 leu61r6616 OZLE
laD61663.31 DalepeD6pD epp61pD6ze DI.DeleD6p3 D6loppe61.6 pEllue6116 1111PDPI. 6ppe6466pD 161ppleP6p Dp6pD4DD61. 6641op6ezp el.13.616r6r 1PEolleppDp DED4)66eD6 4616ep6136 1DD346Dell 611.6661x6p DDD661up16 OtSE 106elaulaD
66666 e6ullo6DE aolfteD666 6661ElepED De661Dp116 08tE
14D6161611 plopfteepp6 61epep66pD 6p66p5e66 e61.61066e4 pelolzpDp6 OZVE am.Dpalel 111e61p6e6 61114111Dp 6ep6Dellpp 611DDleDD6 Depap66ePp 1306p6plap zloppppoDo ple6er66aD 61161311pe 16eDDD6316 161eaDD166 le6466666e 6Da6p1plov paD66aDD6a 551, e66 1461.6e) 6566 OtZE P161o61D16 eD664Dp66e epellreepp ap6111Depl elD1161Dp6 )66D6luD61 08TE Dr66ue6lze DzE161E666 v66Duv6pe6 1D6leeelep apluelD661 16pe6eep6 OZTE
11.P4Depepp De661.Dp1P6 Dpep461D66 6pp6pepppa pleue6116P 65e6 090E PDD611.1epp 6D1DPD3PD1 llep6p613D zlppe664D6 epDp11.6DDp p66p1661.1p 000E 6666634elp 1.6e1EDDe66 U16135611P P6P3PRAD6 epeue6p6pa Dpplee661D
0176Z plop6e6EDE p6apepp6r6 urleDp63.36 110106UeDD pftplapolo 1DE63.11366 088Z R6PPeD371. DP11P41DE6 610P6ETP67 aD66PPPV61 PDDE6141DD ETDPaP6663.
onz u6opee63.1e e6e1.54De61 11u6loplla 1.6D166ee61 DDIXPETPPD P6P3610101 Dzepa66eD6 pplappl.ppe plpaftuala 3.661e61o61 1.6euftE661 uplp666ple OOLZ laPepe1616 lpel6pEppl le66pr661D 4D6101.1.1D1 lalP6a1P11 PftebeEfte lxvszIS'tttO-liow MOR-0444.ST25.txt Leu ser Asp Ala Asn Lys Ala Ser Ala Arg Ala ser Arg Glu Gly Gly Arg Ala Ala Ala Ala Pro Gly Ala Ser Pro Ser Pro Gly Gly Asp Ala Ala Trp Ser Glu Ala Gly Pro Gly Pro Arg Pro Leu Ala Arg Ser Ala Ser Pro Pro Lys Ala Lys Asn Leu Asn Gly Gly Leu Arg Arg Ser Val Ala Pro Ala Ala Pro Thr Ser Cys Asp Phe Ser Pro Gly Asp Leu val Trp Ala Lys Met Glu Gly Tyr Pro Trp Trp Pro Cys Leu Val Tyr Asn His Pro Phe Asp Gly Thr Phe Ile Arg Glu Lys Gly Lys Ser Val Arg Val His Val Gln Phe Phe Asp Asp Ser Pro Thr Arg Gly Trp Val Ser Lys Arg Leu Leu Lys Pro Tyr Thr Gly Ser Lys Ser Lys Glu Ala Gln Lys Gly Gly His Phe Tyr Ser Ala Lys Pro Glu Ile Leu Arg Ala Met Gln Arg Ala Asp Glu Ala Leu Asn Lys Asp Lys Ile Lys Arg Leu Glu Leu Ala Val Cys Asp Glu Pro Ser Glu Pro Glu Glu Glu Glu Glu Met Glu val Gly Thr Thr Tyr Val Thr Asp Lys Ser Glu Glu Asp Asn Glu Ile Glu Ser Glu Glu Glu Val Gln Pro Lys Thr Gln Gly Ser Arg Arg Ser Ser Arg Gln Ile Lys Lys Arg Arg Val Ile Ser Asp Ser Glu Ser Asp Ile Gly Gly Ser Asp Val Glu Phe Lys Pro Asp Thr Lys Glu Glu Gly Ser Ser Asp Glu Ile Ser Ser Gly Val Gly Asp Ser Glu Ser Glu MOR-0444.ST25.txt Gly Leu Asn Ser Pro Val Lys Val Ala Arg Lys Arg Lys Arg Met Val Thr Gly Asn Gly Ser Leu Lys Arg Lys Ser Ser Arg Lys Glu Thr Pro Ser Ala Thr Lys Gln Ala Thr Ser Ile Ser Ser Glu Thr Lys Asn Thr Leu Arg Ala Phe Ser Ala Pro Gin Asn Ser Glu Ser Gin Ala His val Ser Gly Gly Gly Asp Asp Ser Ser Arg Pro Thr Val Trp Tyr His Glu Thr Leu Glu Trp Leu Lys Glu Glu Lys Arg Arg Asp Glu His Arg Arg Arg Pro Asp His Pro Asp Phe Asp Ala Ser Thr Leu Tyr Val Pro Glu Asp Phe Leu Asn Ser Cys Thr Pro Gly Met Arg Lys Trp Trp Gin Ile Lys Ser Gin Asn Phe Asp Leu Val Ile Cys Tyr Lys Val Gly Lys Phe Tyr Glu Leu Tyr His met Asp Ala Leu Ile Gly Val Ser Glu Leu Gly Leu Val Phe Met Lys Gly Asn Trp Ala His Ser Gly Phe Pro Glu Ile Ala Phe Gly Arg Tyr Ser Asp Ser Leu Val Gin Lys Gly Tyr Lys Val Ala Arg Val Glu Gln Thr Glu Thr Pro Glu Met Met Glu Ala Arg Cys Arg Lys met Ala His Ile Ser Lys Tyr Asp Arg Val Val Arg Arg Glu Ile Cys Arg Ile Ile Thr Lys Gly Thr Gin Thr Tyr Ser val Leu Glu Gly Asp Pro Ser Glu Asn Tyr Ser Lys Tyr Leu Leu Ser Leu Lys Glu Lys Glu Glu Asp Ser Ser Gly His Thr Arg Ala Tyr Gly Val Cys Phe . .
MOR-0444.ST25.txt val Asp Thr Ser Leu Gly Lys Phe Phe Ile Gly Gin Phe Ser Asp Asp Arg His Cys Ser Arg Phe Arg Thr Leu Val Ala His Tyr Pro Pro Val Gin Val Leu Phe Glu Lys Gly Asn Leu Ser Lys Glu Thr Lys Thr Ile Leu Lys Ser Ser Leu Ser Cys Ser Leu Gin Glu Gly Leu Ile Pro Gly Ser Gin Phe Trp Asp Ala Ser Lys Thr Leu Arg Thr Leu Leu Glu Glu Glu Tyr Phe Arg Glu Lys Leu Ser Asp Gly Ile Gly Val Met Leu Pro Gin Val Leu Lys Gly Met Thr Ser Glu Ser Asp Ser Ile Gly Leu Thr Pro Gly Glu Lys Ser Glu Leu Ala Leu Ser Ala Leu Gly Gly Cys Val Phe Tyr Leu Lys Lys Cys Leu Ile Asp Gin Glu Leu Leu Ser Met Ala Asn Phe Glu Glu Tyr Ile Pro Leu Asp Ser Asp Thr Val Ser Thr Thr Arg Ser Gly Ala Ile Phe Thr Lys Ala Tyr Gin Arg Met Val Leu Asp Ala Val Thr Leu Asn Asn Leu Glu Ile Phe Leu Asn Gly Thr Asn Gly Ser Thr Glu Gly Thr Leu Leu Glu Arg Val Asp Thr Cys His Thr Pro Phe Gly Lys Arg Leu Leu Lys Gin Trp Leu Cys Ala Pro Leu Cys Asn His Tyr Ala Ile Asn Asp Arg Leu Asp Ala Ile Glu Asp Leu Met Val val Pro Asp Lys Ile Ser Glu Val Val Glu Leu Leu Lys Lys Leu Pro Asp Leu Glu Arg Leu Leu Ser Lys Ile His Asn Val Gly Ser Pro Leu . .
mOR-0444.ST25.txt Lys Ser Gin Asn His Pro Asp Ser Arg Ala Ile met Tyr Glu Glu Thr Thr Tyr Ser Lys Lys Lys Ile Ile Asp Phe Leu Ser Ala Leu Glu Gly Phe Lys Val Met Cys Lys Ile Ile Gly Ile Met Glu Glu Val Ala Asp Gly Phe Lys Ser Lys Ile Leu Lys Gin Val Ile Ser Leu Gin Thr Lys Asn Pro Glu Gly Arg Phe Pro Asp Leu Thr Val Glu Leu Asn Arg Trp Asp Thr Ala Phe Asp His Glu Lys Ala Arg Lys Thr Gly Leu Ile Thr Pro Lys Ala Gly Phe Asp Ser Asp Tyr Asp Gin Ala Leu Ala Asp Ile Arg Glu Asn Glu Gin Ser Leu Leu Glu Tyr Leu Glu Lys Gin Arg Asn Arg Ile Gly Cys Arg Thr Ile Val Tyr Trp Gly Ile Gly Arg Asn Arg , Tyr Gin Leu Glu Ile Pro Glu Asn Phe Thr Thr Arg Asn Leu Pro Glu Glu Tyr Glu Leu Lys Ser Thr Lys Lys Gly Cys Lys Arg Tyr Trp Thr Lys Thr Ile Glu Lys Lys Leu Ala Asn Leu Ile Asn Ala Glu Glu Arg Arg Asp Val Ser Leu Lys Asp Cys Met Arg Arg Leu Phe Tyr Asn Phe Asp Lys Asn Tyr Lys Asp Trp Gin Ser Ala Val Glu Cys Ile Ala val Leu Asp Val Leu Leu Cys Leu Ala Asn Tyr Ser Arg Gly Gly Asp Gly Pro Met Cys Arg Pro Val Ile Leu Leu Pro Glu Asp Thr Pro Pro Phe Leu Glu Leu Lys Gly Ser Arg His Pro Cys . .
mOR-0444.ST25.txt Ile Thr Lys Thr Phe Phe Gly Asp Asp Phe Ile Pro Asn Asp Ile Leu Ile Gly Cys Glu Glu Glu Glu Gin Glu Asn Gly Lys Ala Tyr Cys Val Leu val Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Leu Met Arg Gin Ala Gly Leu Leu Ala Val Met Ala Gin Met Gly Cys Tyr Val Pro Ala Glu Val Cys Arg Leu Thr Pro Ile Asp Arg val Phe Thr Arg Leu Gly Ala Ser Asp Arg Ile Met Ser Gly Glu Ser Thr Phe Phe Val Glu Leu Ser Glu Thr Ala Ser Ile Leu Met His Ala Thr Ala His Ser Leu val Leu Val Asp Glu Leu Gly Arg Gly Thr Ala Thr Phe Asp Gly Thr Ala Ile Ala Asn Ala Val Val Lys Glu Leu Ala Glu Thr Ile Lys Cys Arg Thr Leu Phe Ser Thr His Tyr His Ser Leu Val Glu Asp Tyr Ser Gin Asn Val Ala Val Arg Leu Gly His Met Ala Cys met Val Glu Asn Glu Cys Glu Asp Pro Ser Gin Glu Thr Ile Thr Phe Leu Tyr Lys Phe Ile Lys Gly Ala Cys Pro Lys Ser Tyr Gly Phe Asn Ala Ala Arg Leu Ala Asn Leu Pro Glu Glu Val Ile Gin Lys Gly His Arg Lys Ala Arg Glu Phe Glu Lys Met Asn Gin Ser Leu Arg Leu Phe Arg Glu val Cys Leu Ala Ser Glu Arg Ser Thr val Asp Ala Glu Ala Val His Lys Leu MOR-0444.ST25.txt Leu Thr Leu Ile Lys Glu Leu <210> 25 <211> 1445 <212> DNA
<213> Homo sapiens <400> 25 tttttttttt tgatgttctc cagtgcctca gtggcagcag aactggccct gtatcaggcc 60 gctaccgcca ctccatgacc aacctccctg catacccccc cccccagcac ccctcccaca 120 ggaccgcttc tgtgtttggg acccaccagg cctttgcacc atacaacaaa ccctcactct 180 ccggggcccg gtctgcgccc aggctgaaca ccacgaacgc ctgggacgca gctcctcctt 240 ccctggggag ccagcccctc taccgctcca gcctctccca cctgggaccg cagcacctgc 300 ccccaggatc ctccacctcc ggtgcagtca gtgcctccct ccccagcggt ccctcaagca 360 gcccaggcga gcgtccctgc cactgtgccc atgcagatgc caagccagca gagtcagcag 420 gcgctcgctg gagcgacccg aagccagagc agagcagagc aggtcataaa actacacgga 480 agagctgaaa gtgcccccag atgaggactg catcatctgc atggagaagc tgtccgcagc 540 gtctggatac agcgatgtga ctgacagcaa ggcaatgggg cccctggctg tgggctgcct 600 caccaagtgc agccacgcct tccacctgct gtgcctcctg gccatgtact gcaacggcaa 660 taagggccct gagcacccca atcccggaaa gccgttcact gccagagggt ttcccgccag 720 tgctaccttc cagacaacgc cagggccgca agcctccagg ggcttccaga acccggagac 780 actggctgac attccggcct ccccacagct gctgaccgat ggccactaca tgacgctgcc 840 cgtgtctccg gaccagctgc cctgtgacga ccccatggcg ggcagcggag gcgcccccgt 900 gctgcgggtg ggccatgacc acggctgcca ccagcagcca cgtatctgca acgcgcccct 960 ccctggccct ggaccctatc gtacagaacc tgctaaggcc atcaaaccta ttgatcggaa 1020 gtcagtccat cagatttgct ctgggccagt ggtactgagt ctaagcactg cagtgaagga 1080 gttagtagaa aacagtctgg atgctggtgc cactaatatt gatctaaagc ttaaggacta 1140 tggaatggat ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga 1200 aggcttaatg atgtcaccat ttctacctgc cacgtctcgg cgaaggttgg gactcgactg 1260 gtgtttgatc acgatgggaa aatcatccag aagaccccct acccccaccc cagagggacc 1320 acagtcagcg tgaagcagtt attttctacg ctacctgtgc gccataagga atttcaaagg 1380 aatattaaga agaaacatgc tgcttcccct tcgccttctg ccgtgattgt cagttttaac 1440 cggaa 1445 <210> 26 <211> 270 <212> PRT
<213> HOMO sapiens <400> 26 . .
MOR-0444.ST25.txt met Glu Lys Leu Ser Ala Ala Ser Gly Tyr Ser Asp val Thr Asp Ser Lys Ala Met Gly Pro Leu Ala Val Gly Cys Leu Thr Lys Cys Ser His Ala Phe His Leu Leu Cys Leu Leu Ala Met Tyr Cys Asn Gly Asn Lys Gly Pro Glu His Pro Asn Pro Gly Lys Pro Phe Thr Ala Arg Gly Phe Pro Ala Ser Ala Thr Phe Gin Thr Thr Pro Gly Pro Gin Ala Ser Arg Gly Phe Gin Asn Pro Glu Thr Leu Ala Asp Ile Pro Ala Ser Pro Gin Leu Leu Thr Asp Gly His Tyr Met Thr Leu Pro Val Ser Pro Asp Gin Leu Pro Cys Asp Asp Pro Met Ala Gly Ser Gly Gly Ala Pro Val Leu Arg Val Gly His Asp His Gly Cys His Gin Gin Pro Arg Ile Cys Asn Ala Pro Leu Pro Gly Pro Gly Pro Tyr Arg Thr Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gin Ile Cys Ser Gly Pro val Val Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly met Asp Leu Ile Glu Val Ser Gly Asn Gly Cys Gly val Glu Glu Glu Asn Phe Glu Gly Leu Met met Ser Pro Phe Leu Pro Ala Thr Ser Arg Arg Arg Leu Gly Leu Asp Trp Cys Leu Ile Thr Met Gly Lys Ser Ser Arg Arg Pro Pro Thr Pro Thr Pro Glu Gly Pro Gin Ser Ala mOR-0444.sT25.txt <210> 27 <211> 795 <212> DNA
<213> Homo sapiens <400> 27 atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag ggaggcggat 60 ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg ggcccctcgg 120 gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gcgcgcgcca 180 gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc ggagccccag 240 gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat gaacacattg 300 caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gtggcggcaa 360 ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta cagccatcta 420 gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga ggtgaaggaa 480 gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca acatagtcca 540 gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat ttgctctggg 600 ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tctggatgct 660 ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tgaagtttca 720 gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt cagctctgaa 780 acatcacaca tgtaa 795 <210> 28 <211> 260 <212> PRT
<213> Homo sapiens <400> 28 Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Arg Cys Met Val Ser Pro Arg Glu Ala Asp Leu Gly Pro Gin Lys Asp Thr Arg Leu Asp Leu Pro Arg ser Pro Ala Arg Ala Pro Arg Glu Gin Asn Ser Leu Gly Glu val Asp Arg Arg Gly Pro Arg Glu Gin Thr Arg Ala Pro Ala Thr Ala Ala Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gin Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Phe Gin Glu Gin Gin Glu Met Asn Thr Leu Gin Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp MOR-0444.ST25.txt Phe Thr Gin Glu Glu Trp Arg Gin Leu Asp Pro Asp Glu Lys Ile Ala Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly Tyr Asp Tyr His Gin Ala Lys His His His Gly Val Glu Val Lys Glu Val Glu Gin Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys Gin His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gin Ile Cys Ser Gly Pro Val Val Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ile Ser Phe Ser Ser Glu <210> 29 <211> 3218 <212> DNA
<213> Saccharomyces cerevisiae <400> 29 aaataggaat gtgatacctt ctattgcatg caaagatagt gtaggaggcg ctgctattgc 60 caaagacttt tgagaccgct tgctgtttca ttatagttga ggagttctcg aagacgagaa 120 attagcagtt ttcggtgttt agtaatcgcg ctagcatgct aggacaattt aactgcaaaa 180 ttttgatacg atagtgatag taaatggaag gtaaaaataa catagaccta tcaataagca 240 atgtctctca gaataaaagc acttgatgca tcagtggtta acaaaattgc tgcaggtgag 300 atcataatat cccccgtaaa tgctctcaaa gaaatgatgg agaattccat cgatgcgaat 360 gctacaatga ttgatattct agtcaaggaa ggaggaatta aggtacttca aataacagat 420 aacggatctg gaattaataa agcagacctg ccaatcttat gtgagcgatt cacgacgtcc 480 aaattacaaa aattcgaaga tttgagtcag attcaaacgt atggattccg aggagaagct 540 ttagccagta tctcacatgt ggcaagagtc acagtaacga caaaagttaa agaagacaga 600 Z9 abed 0179Z ell-13)341D 3361E1616e ee611zeD11 1.61641e616 q.6e61D3114 P111613111 Beeeppelee 161D661111 6Deeeelllp ee116166e6 e61.14116eP epelelple6 OZSZ
e3D1133PED 36P4EUe661. 6D1.61e66up DzplzeppDp 5lopap661 DD1166PP6D

PUED1E1611 DDD1101.D11 6DEDUeErelD Pl1P71DDIX ZeDPDPP66P PPEIPZEPUlE
OOIZ1-116e3DAE PEEP6DP6PE 6PD153.16D1 PD61E6D1DP DP1P6D16PP u6DD1.1.661e 0.17EZ
1P6appelel UlD1DU11PD 611E6P6P6P Pl411x1661. P6P101.61.6P ETEDEP61P6 6e6661.1e61 aftufteel6 661DADlel PlellIalle Dpellbeepl 661.D1.31eDD
Ozu lauDelD66u UUE3411DPI. DPDDP1D1D1 eueel.D6ve6 1.61D16erez loe63.erle6 elD1661e61. ee616614ue ftelP))41P1 Dez6e6lere I.D61.e1.6eD6 e61rDe6661 OOTZ P1PPPP16P1 1PP1PUPEPP 6EPPDD1.106 1E6DPEaPET 1.46P6DP61.1 4PPETD1P1D
OVOZ DIOZEP4P1.6 11416elele 61x6rD161.6 leeppelbe6 PDPaDDETP1 efte466111 0861 Derep6plap e6eDe64116 611e6eplea Dllel.D6e61 r4D616461D le66Delle6 PluP1.1.1111 1D6UPE14DP 61.EDEreDllP 1D6DD6r1.1e 6eu6ee66e 61e6e46116 6661.16)P3.1 eP6111.PPPD 63.1414PDP6 PDPP1DPP6P 6D1PDP1P6D 42P61e6P16 008I Peuee616D6 llEPPETEDa Pl6P6DP11D 1PPal6leel 1.6e6D6e66e elppel6e6e lzel.e1666D P6PPPE011a P6DDDI.DPD6 TrE6pepe6D e61e6DD611 ep6eD3116 0891 perzell.r66 666146reee DPEPErePETP 4DDETD1P66 ee63.13e6ze E6leeDle 0Z91 PpraerDD61 e61.6D6Dpee pee6DEleer6 leeelpepe6 1D6eeee6e3 66e6uepppl 61Deepe636 ueepelD16) 3.e66reb3.14 00ST DPED116PDP PD16PPDaDD
6Delle1eel Doeepeplap 61e6elee6e 01717T Da6elpeeel ee6e6eepe6 eeee636eee 66eezpeepa. 6ezuepp6up e6Delelepl 08E1 lee6e6e166 a6eDDD6ee De6D31D16e 6e66e6 6eze616eee 66 OZET
luealleppe aP61.1P316P 6e3D6eeDER PDEEDIalEP Dl1011DEIET ED11.1DP16D
09ZT Pplapele61. leDD6131e1 lee6D363ED 611EeDlEED AD1PPPEET 566 00ZT 2e6eepp6e6 13D1.116D11 66e6e6e6ue eDU6DDDPD1 16DPB1161P 64161D66D6 OTT
6pple6elel 161lee6661 13.e11.1e111 1DDESEDZIO 666ueepp61 Dlelleeppl 0801 De4116D6e) ee611136e6 PU6EE10113 le61.6ZEDP6 16P1DP6PlE eleelleD11 OZOT
eeDllzeD31.6e PPEEDDlEle olalee61.14 ee161.61.66e ue661e61.1.6 aftee661D3 emalefte 656e DIXZEDalal DV11PP111P P6D110661.6 1DTERP1PPD 1161.613P66 PllP66Ple6 55BDEZ PlEDZIODPP P11.6PD1P14 lollelleel ploe6P66D1 afoereue163. 1D1114D661 lepe66veDD 11PD1lVD36 Dule6D666D 161161e6ea zezereplol. Delee61e61 euzuppp3.66 r61.1.3D666e OZL
Pale6eloll DDlleleepa 1.11.1110DP6 ee61.16e1DD le6peppel.6 6De6Peer66 1061.161Dye pEoppApeu 661161P6EP 1.66PP5P361 ele31.11.6e6 e661eD6161 lAVSZIS'117170-110W

. .
MOR-0444.ST25.txt acgattcatc cgcgagattt caaaggatat gaaatatggt tgcagttagg aaagtatgtc 2700 agaaatgtat attcggattg aaactcttct aatagttctg aagtcacttg gttccgtatt 2760 gttttcgtcc tcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gtaccaaaga 2820 cccgagtcct tttatgagag aaaacatttc atcatttttc aactcaatta tcttaatatc 2880 attttgtagt attttgaaaa caggatggta aaacgaatca cctgaatcta gaagctgtac 2940 cttgtcccat aaaagtttta atttactgag cctttcggtc aagtaaacta gtttatctag ttttgaaccg aatattgtgg gcagatttgc agtaagttca gttagatcta ctaaaagttg 3060 tttgacagca gccgattcca caaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt 3120 aatggtttgc atcaccatcg gatgtctgtt gaaaaactca ctttttgcat ggaagttatt 3180 aacaataaga ctaatgatta ccttagaata atgtataa <210> 30 <211> 769 <212> PRT
<213> Saccharomyces cerevisiae <400> 30 Met Ser Leu Arg Ile Lys Ala Leu Asp Ala Ser Val Val Asn Lys Ile Ala Ala Gly Glu Ile Ile Ile Ser Pro Val Asn Ala Leu Lys Glu met Met Glu Asn Ser Ile Asp Ala Asn Ala Thr Met Ile Asp Ile Leu Val Lys Glu Gly Gly Ile Lys Val Leu Gin Ile Thr Asp Asn Gly Ser Gly Ile Asn Lys Ala Asp Leu Pro Ile Leu Cys Glu Arg Phe Thr Thr Ser Lys Leu Gin Lys Phe Glu Asp Leu Ser Gin Ile Gin Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala Arg val Thr Val Thr Thr Lys Val Lys Glu Asp Arg Cys Ala Trp Arg Val Ser Tyr Ala Glu Gly Lys Met Leu Glu Ser Pro Lys Pro Val Ala Gly Lys Asp Gly Thr Thr Ile Leu Val Glu Asp Leu Phe Phe Asn Ile Pro Ser Arg Leu . .
MoR-0444.ST25.txt Arg Ala Leu Arg Ser His Asn Asp Glu Tyr Ser Lys Ile Leu Asp Val val Gly Arg Tyr Ala Ile His Ser Lys Asp Ile Gly Phe Ser Cys Lys Lys Phe Gly Asp Ser Asn Tyr Ser Leu Ser Val Lys Pro Ser Tyr Thr val Gin Asp Arg Ile Arg Thr val Phe Asn Lys Ser Val Ala Ser Asn Leu Ile Thr Phe His Ile Ser Lys Val Glu Asp Leu Asn Leu Glu ser Val Asp Gly Lys Val Cys Asn Leu Asn Phe Ile Ser Lys Lys Ser Ile Ser Leu Ile Phe Phe Ile Asn Asn Arg Leu Val Thr Cys Asp Leu Leu Arg Arg Ala Leu Asn Ser Val Tyr Ser Asn Tyr Leu Pro Lys Gly Phe Arg Pro Phe Ile Tyr Leu Gly Ile val Ile Asp Pro Ala Ala val Asp Val Asn Val His Pro Thr Lys Arg Glu Val Arg Phe Leu Ser Gin Asp Glu Ile Ile Glu Lys Ile Ala Asn Gin Leu His Ala Glu Leu Ser Ala Ile Asp Thr Ser Arg Thr Phe Lys Ala Ser Ser Ile Ser Thr Asn Lys Pro Glu Ser Leu Ile Pro Phe Asn Asp Thr Ile Glu Ser Asp Arg Asn Arg Lys Ser Leu Arg Gin Ala Gin Val val Glu Asn Ser Tyr Thr Thr Ala Asn Ser Gin Leu Arg Lys Ala Lys Arg Gin Glu Asn Lys Leu Val Arg Ile Asp Ala Ser Gin Ala Lys Ile Thr Ser Phe Leu Ser Ser Ser Gin Gin Phe Asn Phe Glu Gly Ser Ser Thr Lys Arg Gin Leu Ser Glu MOR-0444.ST25.txt Pro Lys Val Thr Asn Val Ser His Ser Gin Glu Ala Glu Lys Leu Thr Leu Asn Glu Ser Glu Gin Pro Arg Asp Ala Asn Thr Ile Asn Asp Asn Asp Leu Lys Asp Gin Pro Lys Lys Lys Gin Lys Leu Gly Asp Tyr Lys Val Pro Ser Ile Ala Asp Asp Glu Lys Asn Ala Leu Pro Ile Ser Lys Asp Gly Tyr Ile Arg Val Pro Lys Glu Arg Val Asn Val Asn Leu Thr Ser Ile Lys Lys Leu Arg Glu Lys Val Asp Asp Ser Ile His Arg Glu Leu Thr Asp Ile Phe Ala Asn Leu Asn Tyr Val Gly Val Val Asp Glu Glu Arg Arg Leu Ala Ala Ile Gin His Asp Leu Lys Leu Phe Leu Ile Asp Tyr Gly Ser Val Cys Tyr Glu Leu Phe Tyr Gin Ile Gly Leu Thr Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gin Ser Thr Asn Val Ser Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Glu Phe Asp Glu Leu Asn Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Lys Ile Trp Asp Met Ser Ser met Leu Asn Glu Tyr Tyr Ser Ile Glu Leu Val Asn Asp Gly Leu Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Ser Leu Pro Leu Leu Leu Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pro Phe Phe Ile Tyr Arg Leu Gly Lys Glu val Asp Trp Glu Asp Glu Gin Glu Cys Leu Asp Gly Ile Leu Arg Glu Ile Ala Leu Leu Tyr Ile Pro Asp Met Val Pro Lys . .
MOR-0444.ST25.txt val Asp Thr Leu Asp Ala Ser Leu Ser Glu Asp Glu Lys Ala Gin Phe Ile Asn Arg Lys Glu His Ile Ser Ser Leu Leu Glu His Val Leu Phe Pro Cys Ile Lys Arg Arg Phe Leu Ala Pro Arg His Ile Leu Lys Asp Val Val Glu Ile Ala Asn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg Cys <210> 31 <211> 3056 <212> DNA
<213> Mus musculus <400> 31 gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120 gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg 180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta 360 aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480 cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg 540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600 ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg 660 tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa 720 aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct 1260 19 a6Pd ZE <00t>
sninDsnw snw <ETZ>
JId <ZTZ>
658 <TTZ>
ZE <OTZ>

PEIPPETTETP PPPDPHIPPD laePplpeft PD1.1.E.Dep APEIDDI.DUD DPET61.11.DP 66E.DDDET61. 61pDaD6p66 6166ppl6l Ot6Z
6661.1DEI.D6 pp1D6r1D1D D13.616p661 P64pD66eD6 6m.1646eD fteuellleD
088Z D2P6610P16 1UP1.11D61D 61PD6P11ET PETDP46446 D1Pa4P61D1 eulfte1111 66pp6p6ppr D611.1.66Daz 611e6pDpal Pall6r6rar D6e1.611DDD DPDPDEtaDP
09LZ P6pDapapap D161p661D1. peDD6116Dp D666 DD poDo6eo66) EDDDD3610E
OOLZ e66 DD
DP66aP6P64 6664PDEDD3 PDZED16PP 6ee61e6e6D 6e6D6zeepa 0179Z D6D66Dpe66 au6ap646e pl6ep66D3.6 1DD6e6eppl 136111.61e6 epe6eD16e6 08SZ DED4DDDE6D DE01.61.PD466 66apppempu 616eull6le llaple61De e6eee OZSZ 6PPDDDDP66 laaDoe661.D PPPpeaftap enDellDDI. lluolleeel D666yee61D
09tZ
pplEmppap6 le66p61.E.61. app1.6111De 613.1D661.ey 6pey6pplle zuuE661Dle 00tZ PPPET4u64D e6e6. eeD3.61o61D vel.lpee61D 1De6PDDDDE DEDZED1D56 OtEZ e6PD6D6ETD 31D61.66DeD ep6eD6eD61 Alube61.11 DeeDE16eu6 r61y66361.D
08ZZ 61p6pppe6 6a66app1lp loDe66e6ee e6e 5e 6664pDpela OZZZ
1.6PD16661a Dle6e664p6 e6eD611161 e6D1veel.6e 11.e6e6EEEP 6eplopp61.P

6PP6PAPD6 PP3DPPPP6P 661DDD61.1.1 efteDD666p 11.1reue6ep palfteolpee OOTZ 61PDPPEOPP 6pp6D66eep 1DDPARDP1 1.6eD6eu6le u6D6eelp6e ap1011.6e61 OVOZ D4D1D146p6 DaDD1D616D leuftbeezu eoleppeelv DAeq.61e6D 1.66r61D6ED
0861 6PDaDDPAP 6eD1DD1661 Dp63.1p6pee plollappee D161EePplo Dpeftue66u Ettopftepal D6a6eepp61 PPRDeDDDED 3.61)16D1.06 ElDIODP101. AppepEoler 098T plpluftlapa pe6ee6eDa6 pe61.1ppl6e De66eou166 upplppl6DD 61DE661Dou 616646appe eleapepe66 uplollope6 epefte6elp Dp4D6E61.6D pElelpy63.6 OLT Pa6plalp6e 1.6eaD663.6e aleD3DDED6 eD11.6e6eu6 5e6 6b.
DETD1DoeD6 0891 yo6pD43666 eplaameep aseleeeeft 6e6eDe661p .5 e6.66. DD6PDP6E0 OZ91 eppol6e63.6 611eeepe66 eD6D1D6626 DDI.DD66v6E Dellplpzup 16Dp6pollo Dauzlozpal Pla616D666 fteee6e616 evpoll.116u Ei6pepe6q.De E61.D61DE6E
00ST 6pDp16666E 1D3.6eeDze6 U6EUE1DE17 D1P014D1D1 1410D66PET 6E61.366rDD
Ott lplupDlepel el66fteue6 u6DE6ED6ep eD6p6ue61D eD1.1DD1DaD velE6eeD6E

ee66eD3616 appempeeft I.DEE6ep6lo el.pD61Delep el6plappul. 6bpp611.61e OZET 6e1D61peDD 6eD6uppueD
ppeEeD61e6 16eDv61.1.16 lpe66pau61 lxvsZis*tttO-NOW

mOR-0444.ST25.txt Met Glu Gin Thr Glu Gly Val Ser Thr Glu Cys Ala Lys Ala Ile Lys Pro Ile Asp Gly Lys Ser Val His Gin Ile Cys Ser Gly Gin Val Ile Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn ser Val Asp Ala Gly Ala Thr Thr Ile AS Leu Arg Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gin Glu Phe Ala Asp Leu Thr Gin Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp val Thr Ile Ser Thr Cys His Gly Ser Ala Ser Val Gly Thr Arg Leu Val Phe Asp His Asn Gly Lys Ile Thr Gin Lys Thr Pro Tyr Pro Arg Pro Lys Gly Thr Thr val Ser val Gin His Leu Phe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gin Arg Asn Ile Lys Lys Glu Tyr Ser Lys met Val Gin Val Leu Gin Ala Tyr Cys Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gin Leu Gly Gin Gly Lys Arg His Ala val Val Cys Thr Ser Gly Thr Ser Gly Met Lys Glu Asn Ile Gly Ser Val Phe Gly Gin Lys Gin Leu Gin Ser Leu Ile Pro Phe val Gin Leu Pro Pro Ser Asp Ala Val Cys Glu Glu Tyr Gly Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr Phe Arg Ala Ser MOR-0444.ST25.txt Phe His Ser Ala Arg Thr Ala Pro Gly Gly val Gin Gin Thr Gly Ser Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gin Gin Arg Ser Leu Ser Leu Ser Met Arg Phe Tyr His met Tyr Asn Arg His Gin Tyr Pro Phe Val val Leu Asn Val Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val Thr Pro Asp Lys Arg Gin Ile Leu Leu Gin Glu Glu Lys Leu Leu Leu Ala val Leu Lys Thr Ser Leu Ile Gly met Phe Asp Ser Asp Ala Asn Lys Leu Asn val Asn Gin Gin Pro Leu Leu Asp Val Glu Gly Asn Leu Val Lys Leu His Thr Ala Glu Leu Glu Lys Pro Val Pro Gly Lys Gin Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val Ala Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro Ser Glu Lys Arg Gly val Leu Ser Ser Tyr Pro Ser Asp val Ile Ser Tyr Arg Gly Leu Arg Gly Ser Gin Asp Lys Leu Val Ser Pro Thr Asp Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu he Ser Thr Pro Glu Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu Asp Arg Pro Ser Gin Glu Thr Ile Asn Cys Gly Asp Leu Asp Cys Arg Pro MOR-0444.ST25.txt Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp HiS Gly Tyr Gln Cys Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg Phe Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro Gly Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys His Glu Leu Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu met Glu Ile Leu Gly Gln Phe Asn Leu Gly Phe Ile val Thr Lys Leu Lys Glu Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr Asn Phe Glu met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val Ile Asp Glu Asp Ala Pro val Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Ile Asp Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser Arg Val Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser val Met Ile Gly moR-0444.ST25.txt Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu Ile Thr His met Gly Glu met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr met Arg His Val Ala Asn Leu Asp Val Ile Ser Gin Asn <210> 33 <211> 399 <212> DNA
<213> Mus musculus <400> 33 atggagcaaa ccgaaggcgt gagtacagaa tgtgctaagg ccatcaagcc tattgatggg 60 aagtcagtcc atcaaatttg ttctgggcag gtgatactca gtttaagcac cgctgtgaag 120 gagttgatag aaaatagtgt agatgctggt gctactacta ttgatctaag gcttaaagac 180 tatggggtgg acctcattga agtttcagac aatggatgtg gggtagaaga agaaaacttt 240 gaaggtctag ctctgaaaca tcacacatct aagattcaag agtttgccga cctcacgcag 300 gttgaaactt tcggctttcg gggggaagct ctgagctctc tgtgtgcact aagtgatgtc 360 actatatcta cctgccacgg gtctgcaagc gttgggact 399 <210> 34 <211> 133 <212> PRT
<213> Mus musculus <400> 34 Met Glu Gin Thr Glu Gly val Ser Thr Glu Cys Ala Lys Ala Ile Lys Pro Ile Asp Gly Lys Ser val His Gin Ile Cys Ser Gly Gin Val Ile Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn Ser Val Asp Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly val Asp Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gin Glu Phe Ala Asp Leu Thr Gin Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser MOR-0444.ST25.txt Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Gly Ser Ala Ser Val Gly Thr <210> 35 <211> 3099 <212> DNA
<213> Arabidopsis thaliana <400> 35 gtcttcttct tcatccttgt ctcaccttcg attttggcgg caaaacataa accctaaggg 60 ttttctcact ctctctctct cttctcacac acacagtccc agagtacggt ggtgttgatt 120 cgattgagga gattcatctg tttatagggt ttagcaaatg caaggagatt cttctccgtc 180 tccgacgact actagctctc ctttgataag acctataaac agaaacgtaa ttcacagaat 240 ctgttccggt caagtcatct tagacctctc ttcggccgtc aaggagcttg tcgagaatag 300 tctcgacgcc ggcgccacca gtatagagat taacctccga gactacggcg aagactattt 360 tcaggtcatt gacaatggtt gtggcatttc cccaaccaat ttcaaggttc ttgcacttaa 420 gcatcatact tctaaattag aggatttcac agatcttttg aatttgacta cttatggttt 480 tagaggagaa gccttgagct ctctctgtgc attgggaaat ctcactgtgg aaacaagaac 540 aaagaatgag ccagttgcta cgctcttgac gtttgatcat tctggtttgc ttactgctga 600 aaagaagact gctcgccaaa ttggtaccac tgtcactgtt aggaagttgt tctctaattt 660 acctgtacga agcaaagagt ttaagcggaa tatacgcaaa gaatatggga agcttgtatc 720 tttattgaac gcatatgcgc ttattgcgaa aggagtgcgg tttgtctgct ctaacacgac 780 tgggaaaaac ccaaagtctg ttgtgctgaa cacacaaggg aggggttcac ttaaagataa 840 tatcataaca gttttcggca ttagtacctt tacaagtcta cagcctgtaa gtatatgtgt 900 atcagaagat tgtagagttg aagggtttct ttccaagcct ggacagggta ctggacgcaa 960 tttagcagat cgacagtatt tctttataaa tggtcggcct gtagatatgc caaaagtcag 1020 caagttggtg aatgagttat ataaagatac aagttctcgg aaatatccag ttaccattct 1080 ggattttatt gtgcctggtg gagcatgtga tttgaatgtc acgcccgata aaagaaaggt 1140 gttcttttct gacgagactt ctgttatcgg ttctttgagg gaaggtctga acgagatata 1200 ttcctccagt aatgcgtctt atattgttaa taggttcgag gagaattcgg agcaaccaga 1260 taaggctgga gtttcgtcgt ttcagaagaa atcaaatctt ttgtcagaag ggatagttct 1320 ggatgtcagt tctaaaacaa gactagggga agctattgag aaagaaaatc catccttaag 1380 ggaggttgaa attgataata gttcgccaat ggagaagttt aagtttgaga tcaaggcatg 1440 tgggacgaag aaaggggaag gttctttatc agtccatgat gtaactcacc ttgacaagac 1500 acctagcaaa ggtttgcctc agttaaatgt gactgagaaa gttactgatg caagtaaaga 1560 cttgagcagc cgctctagct ttgcccagtc aactttgaat acttttgtta ccatgggaaa 1620 moR-0444.ST25.txt aagaaaacat gaaaacataa gcaccatcct ctctgaaaca cctgtcctca gaaaccaaac 1680 ttctagttat cgtgtggaga aaagcaaatt tgaagttcgt gccttagctt caaggtgtct 1740 cgtggaaggc gatcaacttg atgatatggt catctcaaag gaagatatga caccaagcga 1800 aagagattct gaactaggca atcggatttc tcctggaaca caagctgata atgttgaaag 1860 acatgagaga gaacatgaaa agcctataag gtttgaagaa ccaacatcag ataacacact 1920 caccaagggg gatgtggaaa gggtttcaga ggacaatcca cggtgcagtc agccactgcg 1980 atctgtggcc acagtgctgg attccccagc tcagtcaacc ggtcctaaaa tgttttccac 2040 attagaattt agtttccaaa acctcaggac aaggaggtta gagaggctgt cgagattgca 2100 gtccacaggt tatgtatcta aatgtatgaa tacgccacag cctaaaaagt gctttgccgc 2160 tgcaacatta gagttatctc aaccggatga tgaagagcga aaagcaaggg ctttagctgc 2220 agctacttct gagctggaaa ggctttttcg aaaagaggat ttcaggagaa tgcaggtact 2280 cgggcaattc aatcttgggt tcatcattgc aaaattggag cgagatctgt tcattgtgga 2340 tcagcatgca gctgatgaga aattcaactt cgaacattta gcaaggtcaa ctgtcctgaa 2400 ccagcaaccc ttactccagc ctttgaactt ggaactctct ccagaagaag aagtaactgt 2460 gttaatgcac atggatatta tcagggaaaa tggctttctt ctagaggaga atccaagtgc 2520 tcctcccgga aaacacttta gactacgagc cattccttat agcaagaata tcacctttgg 2580 agtcgaagat cttaaagacc tgatctcaac tctaggagat aaccatgggg aatgttcggt 2640 tgctagtagc tacaaaacca gcaaaacaga ttcgatttgt ccatcacgag tccgtgcaat 2700 gctagcatcc cgagcatgca gatcatctgt gatgatcgga gatccactca gaaaaaacga 2760 aatgcagaag atagtagaac acttggcaga tctcgaatct ccttggaatt gcccacacgg 2820 acgaccaaca atgcgtcatc ttgtggactt gacaacttta ctcacattac ctgatgacga 2880 caatgtcaat gatgatgatg atgatgatgc aaccatctca ttggcatgaa cactcaaaag 2940 tcttaacgta tttagatgtg agaatcctta agattaacat tgaggaacac tcggttataa 3000 ctacaatcgt aaatgtaaat tgtcttagtc tatatgatct ttttggtcac aacaggtaat 3060 ttcattttcc tttgattact tctcgtgaaa aaacaaatt 3099 <210> 36 <211> 923 <212> PRT
<213> Arabidopsis thaliana <400> 36 Met Gin Gly Asp Ser Ser Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu Ile Arg Pro Ile Asn Arg Asn val Ile His Arg Ile Cys Ser Gly Gin val Ile Leu Asp Leu Ser Ser Ala val Lys Glu Leu val Glu Asn Ser MOR-0444.ST25.txt Leu Asp Ala Gly Ala Thr ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly Glu Asp Tyr Phe Gin Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr Asn Phe Lys Val Leu Ala Leu Lys His His Thr Ser Lys Leu Glu Asp Phe Thr Asp Leu Leu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val Ala Thr Leu Leu Thr Phe Asp His Ser Gly Leu Leu Thr Ala Glu Lys Lys Thr Ala Arg Gin Ile Gly Thr Thr Val Thr Val Arg Lys Leu Phe Ser Asn Leu Pro Val Arg Ser Lys Glu Phe Lys Arg Asn Ile Arg Lys Glu Tyr Gly Lys Leu Val Ser Leu Leu Asn Ala Tyr Ala Leu Ile Ala Lys Gly Val Arg Phe val Cys Ser Asn Thr Thr Gly Lys Asn Pro Lys Ser Val Val Leu Asn Thr Gin Gly Arg Gly Ser Leu Lys Asp Asn Ile Ile Thr Val Phe Gly Ile Ser Thr Phe Thr Ser Leu Gln Pro Val Ser Ile Cys Val Ser Glu Asp Cys Arg val Glu Gly Phe Leu Ser Lys Pro Gly Gin Gly Thr Gly Arg Asn Leu Ala Asp Arg Gin Tyr Phe Phe Ile Asn Gly Arg Pro Val Asp Met Pro Lys val Ser Lys Leu Val Asn Glu Leu Tyr Lys Asp Thr Ser Ser Arg Lys Tyr Pro Val Thr Ile Leu Asp Phe Ile Val Pro Gly Gly Ala Cys Asp Leu Asn . .
mOR-0444.ST25.txt Val Thr Pro Asp Lys Arg Lys val Phe Phe Ser Asp Glu Thr Ser val Ile Gly Ser Leu Arg Glu Gly Leu Asn Glu Ile Tyr Ser Ser Ser Asn Ala Ser Tyr Ile Val Asn Arg Phe Glu Glu Asn Ser Glu Gin Pro Asp Lys Ala Gly val ser Ser Phe Gin Lys Lys Ser Asn Leu Leu Ser Glu Gly Ile Val Leu Asp Val Ser Ser Lys Thr Arg Leu Gly Glu Ala Ile Glu Lys Glu Asn Pro Ser Leu Arg Glu Val Glu Ile Asp Asn Ser Ser Pro Met Glu Lys Phe Lys Phe Glu Ile Lys Ala Cys Gly Thr Lys Lys Gly Glu Gly Ser Leu Ser Val His Asp Val Thr His Leu Asp Lys Thr Pro Ser Lys Gly Leu Pro Gin Leu Asn Val Thr Glu Lys Val Thr Asp Ala Ser Lys Asp Leu Ser Ser Arg Ser Ser Phe Ala Gin Ser Thr Leu Asn Thr Phe Val Thr Met Gly Lys Arg Lys His Glu Asn Ile Ser Thr Ile Leu Ser Glu Thr Pro Val Leu Arg Asn Gin Thr Ser Ser Tyr Arg Val Glu Lys Ser Lys Phe Glu val Arg Ala Leu Ala Ser Arg Cys Leu val Glu Gly Asp Gin Leu Asp Asp Met Val Ile Ser Lys Glu Asp Met Thr Pro Ser Glu Arg Asp Ser Glu Leu Gly Asn Arg Ile Ser Pro Gly Thr Gin Ala Asp Asn val Glu Arg His Glu Arg Glu His Glu Lys Pro Ile Arg Phe Glu Glu Pro Thr Ser Asp Asn Thr Leu Thr Lys Gly Asp MOR-0444.ST25.txt val Glu Arg Val Ser Glu Asp Asn Pro Arg Cys Ser Gln Pro Leu Arg Ser Val Ala Thr Val Leu Asp Ser Pro Ala Gln Ser Thr Gly Pro Lys Met Phe Ser Thr Leu Glu Phe Ser Phe Gln Asn Leu Arg Thr Arg Arg Leu Glu Arg Leu Ser Arg Leu Gln Ser Thr Gly Tyr Val Ser Lys Cys Met Asn Thr Pro Gln Pro Lys Lys Cys Phe Ala Ala Ala Thr Leu Glu Leu Ser Gln Pro Asp Asp Glu Glu Arg Lys Ala Arg Ala Leu Ala Ala Ala Thr Ser Glu Leu Glu Arg Leu Phe Arg Lys Glu Asp Phe Arg Arg Met Gln Val Leu Gly Gln Phe Asn Leu Gly Phe Ile Ile Ala Lys Leu Glu Arg Asp Leu Phe Ile Val Asp Gln His Ala Ala Asp Glu Lys Phe Asn Phe Glu His Leu Ala Arg Ser Thr Val Leu Asn Gln Gln Pro Leu Leu Gln Pro Leu Asn Leu Glu Leu Ser Pro Glu Glu Glu Val Thr Val Leu Met His Met AS Ile Ile Arg Glu Asn Gly Phe Leu Leu Glu Glu Asn Pro Ser Ala Pro Pro Gly Lys His Phe Arg Leu Arg Ala Ile Pro Tyr Ser Lys Asn Ile Thr Phe Gly Val Glu Asp Leu Lys Asp Leu Ile ser Thr Leu Gly Asp Asn His Gly Glu Cys Ser Val Ala Ser Ser Tyr Lys Thr Ser Lys Thr Asp Ser Ile Cys Pro Ser Arg Val Arg Ala met Leu Ala Ser Arg Ala Cys Arg Ser Ser Val Met Ile Gly Asp Pro Leu MOR-0444.ST25.txt Arg Lys Asn Glu Met Gin Lys Ile Val Glu His Leu Ala Asp Leu Glu Ser Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg His Leu Val Asp Leu Thr Thr Leu Leu Thr Leu Pro Asp Asp Asp Asn Val Asn Asp Asp Asp Asp Asp Asp Ala Thr Ile Ser Leu Ala <210> 37 <211> 399 <212> DNA
<213> Arabidopsis thaliana <400> 37 atgcaaggag attcttctcc gtctccgacg actactagct ctcctttgat aagacctata 60 aacagaaacg taattcacag aatctgttcc ggtcaagtca tcttagacct ctcttcggcc 120 gtcaaggagc ttgtcgagaa tagtctcgac gccggcgcca ccagtataga gattaacctc 180 cgagactacg gcgaagacta ttttcaggtc attgacaatg gttgtggcat ttccccaacc 240 aatttcaagg ttcttgcact taagcatcat acttctaaat tagaggattt cacagatctt 300 ttgaatttga ctacttatgg ttttagagga gaagccttga gctctctctg tgcattggga 360 aatctcactg tggaaacaag aacaaagaat gagccagtt 399 <210> 38 <211> 133 <212> PRT
<213> Arabidopsis thaliana <400> 38 Met Gin Gly Asp Ser Ser Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu Ile Arg Pro Ile Asn Arg Asn Val Ile His Arg Ile Cys Ser Gly Gin val Ile Leu Asp Leu Ser Ser Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly Glu Asp Tyr Phe Gin Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr Asn Phe Lys Val Leu Ala Leu Lys His HiS Thr Ser Lys Leu Glu Asp MOR-0444.ST25.txt Phe Thr Asp Leu Leu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val <210> 39 <211> 2772 <212> DNA
<213> Arabidopsis thaliana <400> 39 atgcaaggag attcttctcc gtctccgacg actactagct ctcctttgat aagacctata 60 aacagaaacg taattcacag aatctgttcc ggtcaagtca tcttagacct ctcttcggcc 120 gtcaaggagc ttgtcgagaa tagtctcgac gccggcgcca ccagtataga gattaacctc 180 cgagactacg gcgaagacta ttttcaggtc attgacaatg gttgtggcat ttccccaacc 240 aatttcaagg ttcttgcact taagcatcat acttctaaat tagaggattt cacagatctt 300 ttgaatttga ctacttatgg ttttagagga gaagccttga gctctctctg tgcattggga 360 aatctcactg tggaaacaag aacaaagaat gagccagttg ctacgctctt gacgtttgat 420 cattctggtt tgcttactgc tgaaaagaag actgctcgcc aaattggtac cactgtcact 480 gttaggaagt tgttctctaa tttacctgta cgaagcaaag agtttaagcg gaatatacgc 540 aaagaatatg ggaagcttgt atctttattg aacgcatatg cgcttattgc gaaaggagtg 600 cggtttgtct gctctaacac gactgggaaa aacccaaagt ctgttgtgct gaacacacaa 660 gggaggggtt cacttaaaga taatatcata acagttttcg gcattagtac ctttacaagt 720 ctacagcctg taagtatatg tgtatcagaa gattgtagag ttgaagggtt tctttccaag 780 cctggacagg gtactggacg caatttagca gatcgacagt atttctttat aaatggtcgg 840 cctgtagata tgccaaaagt cagcaagttg gtgaatgagt tatataaaga tacaagttct 900 cggaaatatc cagttaccat tctggatttt attgtgcctg gtggagcatg tgatttgaat 960 gtcacgcccg ataaaagaaa ggtgttcttt tctgacgaga cttctgttat cggttctttg 1020 agggaaggtc tgaacgagat atattcctcc agtaatgcgt cttatattgt taataggttc 1080 gaggagaatt cggagcaacc agataaggct ggagtttcgt cgtttcagaa gaaatcaaat 1140 cttttgtcag aagggatagt tctggatgtc agttctaaaa caagactagg ggaagctatt 1200 gagaaagaaa atccatcctt aagggaggtt gaaattgata atagttcgcc aatggagaag 1260 tttaagtttg agatcaaggc atgtgggacg aagaaagggg aaggttcttt atcagtccat 1320 gatgtaactc accttgacaa gacacctagc aaaggtttgc ctcagttaaa tgtgactgag 1380 aaagttactg atgcaagtaa agacttgagc agccgctcta gctttgccca gtcaactttg 1440 MOR-0444.ST25.txt aatacttttg ttaccatggg aaaaagaaaa catgaaaaca taagcaccat cctctctgaa 1500 acacctgtcc tcagaaacca aacttctagt tatcgtgtgg agaaaagcaa atttgaagtt 1560 cgtgccttag cttcaaggtg tctcgtggaa ggcgatcaac ttgatgatat ggtcatctca 1620 aaggaagata tgacaccaag cgaaagagat tctgaactag gcaatcggat ttctcctgga 1680 acacaagctg ataatgttga aagacatgag agagaacatg aaaagcctat aaggtttgaa 1740 gaaccaacat cagataacac actcaccaag ggggatgtgg aaagggtttc agaggacaat 1800 ccacggtgca gtcagccact gcgatctgtg gccacagtgc tggattcccc agctcagtca 1860 accggtccta aaatgttttc cacattagaa tttagtttcc aaaacctcag gacaaggagg 1920 ttagagaggc tgtcgagatt gcagtccaca ggttatgtat ctaaatgtat gaatacgcca 1980 cagcctaaaa agtgctttgc cgctgcaaca ttagagttat ctcaaccgga tgatgaagag 2040 cgaaaagcaa gggctttagc tgcagctact tctgagctgg aaaggctttt tcgaaaagag 2100 gatttcagga gaatgcaggt actcgggcaa ttcaatcttg ggttcatcat tgcaaaattg 2160 gagcgagatc tgttcattgt ggatcagcat gcagctgatg agaaattcaa cttcgaacat 2220 ttagcaaggt caactgtcct gaaccagcaa cccttactcc agcctttgaa cttggaactc 2280 tctccagaag aagaagtaac tgtgttaatg cacatggata ttatcaggga aaatggcttt 2340 . cttctagagg agaatccaag tgctcctccc ggaaaacact ttagactacg agccattcct 2400 tatagcaaga atatcacctt tggagtcgaa gatcttaaag acctgatctc aactctagga 2460 gataaccatg gggaatgttc ggttgctagt agctacaaaa ccagcaaaac agattcgatt 2520 tgtccatcac gagtccgtgc aatgctagca tcccgagcat gcagatcatc tgtgatgatc 2580 ggagatccac tcagaaaaaa cgaaatgcag aagatagtag aacacttggc agatctcgaa 2640 tctccttgga attgcccaca cggacgacca acaatgcgtc atcttgtgga cttgacaact 2700 ttactcacat tacctgatga cgacaatgtc aatgatgatg atgatgatga tgcaaccatc 2760 tcattggcat ga 2772 <210> 40 <211> 923 <212> PRT
<213> Arabidopsis thaliana <400> 40 Met Gin Gly Asp Ser Ser Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu Ile Arg Pro Ile Asn Arg Asn Val Ile His Arg Ile Cys Ser Gly Gin Val Ile Leu Asp Leu Ser Ser Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly MOR-0444.ST25.txt Glu Asp Tyr Phe Gin Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr Asn Phe Lys Val Leu Ala Leu Lys His His Thr Ser Lys Leu Glu Asp Phe Thr Asp Leu Leu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val Ala Thr Leu Leu Thr Phe Asp His Ser Gly Leu Leu Thr Ala Glu Lys Lys Thr Ala Arg Gin Ile Gly Thr Thr Val Thr Val Arg Lys Leu Phe Ser Asn Leu Pro Val Arg Ser Lys Glu Phe Lys Arg Asn Ile Arg Lys Glu Tyr Gly Lys Leu Val Ser Leu Leu Asn Ala Tyr Ala Leu Ile Ala Lys Gly Val Arg Phe Val Cys Ser Asn Thr Thr Gly Lys Asn Pro Lys Ser Val Val Leu Asn Thr Gin Gly Arg Gly Ser Leu Lys Asp Asn Ile Ile Thr Val Phe Gly Ile Ser Thr Phe Thr Ser Leu Gin Pro Val Ser Ile Cys Val Ser Glu Asp Cys Arg Val Glu Gly Phe Leu Ser Lys Pro Gly Gin Gly Thr Gly Arg Asn Leu Ala Asp Arg Gin Tyr Phe Phe Ile Asn Gly Arg Pro Val AS Met Pro Lys Val Ser Lys Leu val Asn Glu Leu Tyr Lys Asp Thr Ser Ser Arg Lys Tyr Pro Val Thr Ile Leu Asp Phe Ile Val Pro Gly Gly Ala Cys Asp Leu Asn Val Thr Pro Asp Lys Arg Lys Val Phe Phe Ser Asp Glu Thr Ser Val MOR-0444.ST25.txt Ile Gly Ser Leu Arg Glu Gly Leu Asn Glu Ile Tyr Ser Ser Ser Asn Ala Ser Tyr Ile Val Asn Arg Phe Glu Glu Asn Ser Glu Gin Pro Asp Lys Ala Gly val Ser Ser Phe Gin Lys Lys Ser Asn Leu Leu Ser Glu Gly Ile Val Leu Asp Val Ser Ser Lys Thr Arg Leu Gly Glu Ala Ile Glu Lys Glu Asn Pro Ser Leu Arg Glu val Glu Ile Asp Asn Ser Ser Pro Met Glu Lys Phe Lys Phe Glu Ile Lys Ala Cys Gly Thr Lys Lys Gly Glu Gly Ser Leu Ser Val His Asp Val Thr His Leu Asp Lys Thr Pro Ser Lys Gly Leu Pro Gin Leu Asn Val Thr Glu Lys Val Thr Asp Ala Ser Lys Asp Leu Ser Ser Arg Ser Ser Phe Ala Gin Ser Thr Leu Asn Thr Phe val Thr met Gly Lys Arg Lys His Glu Asn Ile Ser Thr Ile Leu Ser Glu Thr Pro val Leu Arg Asn Gin Thr Ser Ser Tyr Arg Val Glu Lys Ser Lys Phe Glu Val Arg Ala Leu Ala Ser Arg Cys Leu Val Glu Gly Asp Gin Leu Asp AS Met Val Ile Ser Lys Glu Asp Met Thr Pro Ser Glu Arg Asp Ser Glu Leu Gly Asn Arg Ile Ser Pro Gly Thr Gin Ala Asp Asn val Glu Arg His Glu Arg Glu His Glu Lys Pro Ile Arg Phe Glu Glu Pro Thr Ser Asp Asn Thr Leu Thr Lys Gly Asp Val Glu Arg val Ser Glu Asp Asn Pro Arg Cys Ser Gin Pro Leu Arg mOR-0444.ST25.txt Ser val Ala Thr val Leu Asp Ser Pro Ala Gln Ser Thr Gly Pro Lys met Phe Ser Thr Leu Glu Phe Ser Phe Gln Asn Leu Arg Thr Arg Arg Leu Glu Arg Leu Ser Arg Leu Gln Ser Thr Gly Tyr Val Ser Lys Cys Met Asn Thr Pro Gln Pro Lys Lys Cys Phe Ala Ala Ala Thr Leu Glu Leu Ser Gln Pro Asp Asp Glu Glu Arg Lys Ala Arg Ala Leu Ala Ala Ala Thr Ser Glu Leu Glu Arg Leu Phe Arg Lys Glu Asp Phe Arg Arg Met Gln Val Leu Gly Gln Phe Asn Leu Gly Phe Ile Ile Ala Lys Leu Glu Arg Asp Leu Phe Ile Val Asp Gln His Ala Ala Asp Glu Lys Phe Asn Phe Glu His Leu Ala Arg Ser Thr Val Leu Asn Gln Gln Pro Leu Leu Gln Pro Leu Asn Leu Glu Leu Ser Pro Glu Glu Glu Val Thr Val Leu Met His Met Asp Ile Ile Arg Glu Asn Gly Phe Leu Leu Glu Glu Asn Pro Ser Ala Pro Pro Gly Lys His Phe Arg Leu Arg Ala Ile Pro Tyr Ser Lys Asn Ile Thr Phe Gly Val Glu Asp Leu Lys Asp Leu Ile Ser Thr Leu Gly Asp Asn His Gly Glu Cys Ser val Ala Ser Ser Tyr Lys Thr Ser Lys Thr Asp Ser Ile Cys Pro Ser Arg Val Arg Ala Met Leu Ala Ser Arg Ala Cys Arg Ser Ser val met Ile Gly AS Pro Leu Arg Lys Asn Glu met Gln Lys Ile Val Glu His Leu Ala Asp Leu Glu ES a6Pd el.D1D66upp ppeD6pe6p6 puDzela666 pepl6eDe61 eze11616ee 66ePeDD1D1 1466PD61P6 11e116D6p6 614D1D63.D6 161pD1pD61 P64P6D6PD1. PDD1666146 OZET
66111466p6 14DD61.D616 1DP6116111 1D6114p661 p1.6161.DpED 14616pPerD
09Z1 eaD6e661PP PPD4E6P6PP PUIX1061.10 11D1PDD16D D6ap61DD66 666 0OZT 6P6APPAP DPPD1PDD1D PPalP166ED 416P4D6PP6 6BED33.1PP1 UlDPIXE1D6 OVTT l66e6eDD6p EPPAREDDP 61D1pDper6 elD6pD6e6D lep6Dp6611 666 0801 16D1D6p14e 1D6peeeD54 6PD66p61p6 pap66616pp p61D1D1p16 6666p OZOT
DEolpppu666 161.6616p64 ppDpE4pEopp 661DP614D6 p66ppDpD16 6.666 1.P6p1.D6p61 p161.D6p6lp 1114ppe666 616ppe111.3 111136161z pDp661p1p1 PP6a6p6ppl 1616p661ae 1PPAPPREO PDaPD61Da6 1PPPP6PP31 11161p61DD

EDDEZP3PDP laDDE6PEP6 PlEEIDE111D 3331e61e6a 33163e6pp6 plep3361E6 66e3zpe631 311316p6az 3661pE6111 uEuppep66E pEoppelfte 6erpfte6E6 OZL
v661641.166 30zp66v331. 361pepp613 66ppeuppez 33.3p6061.1ez 61.331166pp 1.P66p66ppa 141Dp6apep 64e66p661D 116p6D6pp6 14D6D1D11D 646DuDD46D

61p666popp Drep6PDD16 61.331.16zp6 3661p6E16p 66P1P1P6PP 6P46PplP6P
OtS
11Dep6lD6p 6p6663.6ppl. 116p1144D6 lEDDPPEE'RE E6EEDIOPPP p1661pplee 081' DD116D114e uplaal4p14 61e6lepl.D6 6pp6zpa61P 1.31.31e311E 64ep61D6DD
OZV
4161461p6p p6p6DD6p66 p6p6p663.D1. 1611p6p6Da p61p6Da6pp pp161146Pp zuD64p14e1 peDD1D1161 DDD1.6D14D6 zp6466DD6D Dzppe4D66D D6pp114pb6 1016DD64DD 161631.6D6D D1466pe6p6 6D3PDDlYel P66Dep66e6 P1466p6ap6 OVZ D161D1ppe6 D14161D614 116Dp161D1 I.D6Dp6D66 lD61p666 6666 lpp111p6De DDp66p66D6 up66366666 D66D6p1D61 D61p6D66DD 1146611166 OZT
6pP6Dp1DP6 D56D6E0PDD DPEPPEDD11 D111D1611.1 1P6D1P6E6P DADETAIT

PPEEPDD1ZU PDUD1DI.D1D ZDPDaDaDaD 141.601.PDET ERD1D6PD1D aD11PPEoll TV <00V>
pue[Lpqa s!_sdoppiPJv <m>
vNa <ZTZ>
99VE <TTZ>
TV <OTZ>

PLV nal ...IBS aLI -11-11 LV dsv dsv dsv dsv dsv dsv usv IPA usv dsv dsv dsv aid nal Ju nal nal Jul nal dsv LEA nal spi 6.4v law Jul 0.1d 6.1V A19 Spi 0-Id SAD USV dJ1 0.1d Jas axvszis-14170-110w vs a6Pd 99tE 6D6app PEPP11D1DD 11P11161P6 aDaDalD611 laDa6ppla6 OZVE
DZDETE6101 ElDPDEPZPD P61P1D664P PPI.PPEDDD1 16161DP110 1DDI.PEPDZE

6p6apD661p laD6111161 laDeDe6Dpa Dp61.26pp6D 6611PDDDDD 61PEDPEOPD

DDE016P6D1 DalaP16661 6611pDa6up DaD661Dp6p p6app6aDa6 ppDaDal6p6 OtZE
aDau6ppaD6 p6a6ppDa6p pDalopppp6 666aappDap 6p6pp6leDD 666 166eDapp6p Dupp611661 ETEDDEPPDD PITE664D66 1PD1DED661 666 OZTE
DulD6p6p61 DDa611D6p6 66p6Dpupal 1.6DDp161aD 11646paDDp 6ppDap6.161 16616DppDp pDapaap6aD apfteDappp DaapD6D6a1 D661pDpDpp p6D1.DDED16 16YEDDDPD1 D16D6D11EP 66PPDDEOlD 1DDDEODP11 PDPDPED611 1D1D61p66D

a6appDaa6p pp6p6p1661 DDpDa6Dala 1166Dapple D6lappD6Dp ap66ap6Daa 088?
apPlEipappe 66p6pD666a Dup6Dp6a1D Dappa6papp Daap66pDaD PPDE.I.PEETD

1101a6pDa6 J6 66.D pD6app6P16 6111110Dpa 6p6p6p66pD p6appapp6p ap6aDapD6D 66a1D66ppD pDaaDapapp ap6616DaDD DaDappp6D6 laDa6p6161 OOLZ
6DD616DulD 61D66alopp DDD6aaaDap 116DD661D1 61pDppD6a6 DalDlaDapp ovgz PDapppp66D 6661pDpppD Dp666Dp6aD pla6lapDa6 6DaDDappaa po6pD6616p ossz D6pe6e6pa D66p6D6611 Dpappapap6 app6DD1161 DD6aappDa6 61p6DD6pD6 ozsz al6pD6alap DpapD661.pa DP66PEDDIX
)3666EUPPD PPRP6PD1PP
ovz 6PD1E6PDPI. DETP6PD1UU 6DDD11.11.1X 11.610D66PD 366D66 6106131D1D

16PED6PD6D apeD6aalaD ap6p6aDD16 lE6PlDDEaD EIPPITDDPDE 31.1.EDZEI6P6 OVEZ
101.661UPD1 DUPD6P6PEP 6D1E1.1.1.1.10 UPEID1P11DE 1PEOPD1D1D PPP61D6DEP
08ZZ pp6ap6pDp6 161p6uppop p6pDapaapp ppDalaDe6D esulp6pauDD 6pD6pp6Daa OZZZ ppDaDalaDa laPaa6p6pa D666D6uppp p66p16plaP apapplappp pa6aDaDupp apalaD614a 6p6ap6apap pepapp66pp 6pDpaD1.D66 1161161)1p 6aapp66a6p OOTZ p6pD11666p pu61611PPP D666111eD6 uppal6p6Dp pDpup6aD6a 6666 OtOZ
61DaaDaD61 DD6116161D aDD6pDapDa p6D116D6pa Da6ppDapD6 Dp66D1D61D
0861 p6upp6plaD p6pDpalopp pDpDpaDapa 6pDD661DpD apupD6apa6 PPPEIPD1DRE
0Z61 pD66DpDaap p6pp6aa6pa 61p6a1D66D PEEZPED1UD ETEP6P161U ETETD1DEDD

appD6aDap6 6aapp66ppa apapp6D6pp 166aDuppDa 6p116161Dp uppolaDapa 0081 EPPDPU611) DDE666E011 DD1.61661X6 a61D6papeD pplaapap6p 6aaDapppa6 OtLT
61p6Dp6pDD 666 pDaD161166 66pDPall6p PDDPZUDD11 alapap6666 0891 auD6pplapp 16ap6pp6pa D6 D66 aDa6aDapDa pplappaD6p 6p6611D1D6 a6laDDD61 161p616app 6appeaD166 ap61161D61 666.pp app6aDaaDa 166ppplaaD pap66DppaD app6pappap appp6plaft 661D61D6ap 66666 00ST 6appa6ppop DDP16PDDI.D 6666 666 j.6 1.X1'SZIS'Vti70-210W

MOR-0444.ST25.txt <210> 42 <211> 1109 <212> PRT
<213> Arabidopsis thaliana <400> 42 Met Gin Arg Gin Arg Ser Ile Leu Ser Phe Phe Gin Lys Pro Thr Ala Ala Thr Thr Lys Gly Leu Val Ser Gly Asp Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Pro Arg Phe Asn Val Lys Glu Gly Asp Ala Lys Gly Asp Ala Ser Val Arg Phe Ala Val Ser Lys Ser Val Asp Glu Val Arg Gly Thr Asp Thr Pro Pro Glu Lys Val Pro Arg Arg Val Leu Pro Ser Gly Phe Lys Pro Ala Glu Ser Ala Gly Asp Ala Ser Ser Leu Phe Ser Asn Ile met His Lys Phe Val Lys Val Asp Asp Arg Asp Cys Ser Gly Glu Arg Ser Arg Glu Asp Val Val Pro Leu Asn Asp Ser Ser Leu Cys Met Lys Ala Asn Asp val Ile Pro Gin Phe Arg Ser Asn Asn Gly Lys Thr Gin Glu Arg Asn His Ala Phe Ser Phe Ser Gly Arg Ala Glu Leu Arg Ser Val Glu Asp Ile Gly Val Asp Gly Asp Val Pro Gly Pro Glu Thr Pro Gly met Arg Pro Arg Ala Ser Arg Leu Lys Arg val Leu Glu Asp Glu met Thr Phe Lys Glu Asp Lys Val Pro Val Leu Asp Ser Asn Lys Arg Leu Lys Met Leu Gin Asp Pro Val Cys Gly Glu Lys Lys Glu Val Asn Glu Gly Thr Lys Phe Glu Trp Leu Glu Ser Ser Arg Ile Arg mOR-0444.ST25.txt Asp Ala Asn Arg Arg Arg Pro Asp Asp Pro Leu Tyr Asp Arg Lys Thr Leu His Ile Pro Pro Asp Val Phe Lys Lys Met Ser Ala Ser Gin Lys Gin Tyr Trp Ser Val Lys Ser Glu Tyr Met Asp Ile Val Leu Phe Phe Lys Val Gly Lys Phe Tyr Glu Leu Tyr Glu Leu Asp Ala Glu Leu Gly His Lys Glu Leu Asp Trp Lys Met Thr Met Ser Gly Val Gly Lys Cys Arg Gin Val Gly Ile Ser Glu Ser Gly Ile Asp Glu Ala Val Gin Lys Leu Leu Ala Arg Gly Tyr Lys Val Gly Arg Ile Glu Gln Leu Glu Thr Ser Asp Gin Ala Lys Ala Arg Gly Ala Asn Thr Ile Ile Pro Arg Lys Leu Val Gin Val Leu Thr Pro Ser Thr Ala Ser Glu Gly Asn Ile Gly Pro Asp Ala val His Leu Leu Ala Ile Lys Glu Ile Lys Met Glu Leu Gin Lys Cys Ser Thr Val Tyr Gly Phe Ala Phe Val Asp Cys Ala Ala Leu Arg Phe Trp Val Gly Ser Ile Ser Asp Asp Ala Ser Cys Ala Ala Leu Gly Ala Leu Leu Met Gin Val Ser Pro Lys Glu Val Leu Tyr Asp Ser Lys Gly Leu Ser Arg Glu Ala Gin Lys Ala Leu Arg Lys Tyr Thr Leu Thr Gly Ser Thr Ala val Gin Leu Ala Pro Val Pro Gin Val Met Gly Asp Thr Asp Ala Ala Gly val Arg Asn Ile Ile Glu Ser Asn Gly Tyr Phe Lys Gly Ser Ser Glu Ser Trp Asn Cys Ala Val Asp Gly Leu MOR-0444.ST25.txt Asn Glu Cys Asp Val Ala Leu Ser Ala Leu Gly Glu Leu Ile Asn His Leu Ser Arg Leu Lys Leu Glu Asp Val Leu Lys His Gly Asp Ile Phe Pro Tyr Gin val Tyr Arg Gly Cys Leu Arg Ile Asp Gly Gin Thr met Val Asn Leu Glu Ile Phe Asn Asn Ser Cys Asp Gly Val Leu Gin Gly Pro Leu Asn Lys Tyr Leu Glu Asn Cys Val Ser Pro Thr Gly Lys Arg Leu Leu Arg Asn Trp Ile Cys His Pro Leu Lys Asp Val Glu ser Ile Asn Lys Arg Leu Asp Val Val Glu Glu Phe Thr Ala Asn Ser Glu Ser Met Gin Ile Thr Gly Gin Tyr Leu His Lys Leu Pro Asp Leu Glu Arg Leu Leu Gly Arg Ile Lys Ser Ser Val Arg Ser Ser Ala ser val Leu Pro Ala Leu Leu Gly Lys Lys Val Leu Lys Gin Arg Val Lys Ala Phe Gly Gln Ile Val Lys Gly Phe Arg Ser Gly Ile Asp Leu Leu Leu Ala Leu Gin Lys Glu Ser Asn Met Met Ser Leu Leu Tyr Lys Leu Cys Lys Leu Pro Ile Leu Val Gly Lys Ser Gly Leu Glu Leu Phe Leu Ser Gin Phe Glu Ala Ala Ile Asp Ser Asp Phe Pro Asn Tyr Gin Asn Gin Asp Val Thr Asp Glu Asn Ala Glu Thr Leu Thr Ile Leu Ile Glu Leu Phe Ile Glu Arg Ala Thr Gin Trp Ser Glu val Ile His Thr Ile Ser cys Leu Asp val Leu Arg Ser Phe Ala Ile Ala Ala Ser Leu Ser Ala Gly mOR-0444.ST25.txt Ser met Ala Arg Pro Val Ile Phe Pro Glu Ser Glu Ala Thr Asp Gin Asn Gin Lys Thr Lys Gly Pro Ile Leu Lys Ile Gin Gly Leu Trp His Pro Phe Ala Val Ala Ala Asp Gly Gin Leu Pro Val Pro Asn Asp Ile Leu Leu Gly Glu Ala Arg Arg Ser Ser Gly Ser Ile His Pro Arg Ser Leu Leu Leu Thr Gly Pro Asn met Gly Gly Lys Ser Thr Leu Leu Arg Ala Thr Cys Leu Ala Val Ile Phe Ala Gin Leu Gly Cys Tyr val Pro Cys Glu Ser Cys Glu Ile Ser Leu Val Asp Thr Ile Phe Thr Arg Leu Gly Ala Ser Asp Arg Ile Met Thr Gly Glu Ser Thr Phe Leu Val Glu Cys Thr Glu Thr Ala Ser Val Leu Gin Asn Ala Thr Gin Asp Ser Leu Val Ile Leu Asp Glu Leu Gly Arg Gly Thr Ser Thr Phe Asp Gly Tyr Ala Ile Ala Tyr Ser Val Phe Arg His Leu Val Glu Lys Val Gin Cys Arg Met Leu Phe Ala Thr His Tyr His Pro Leu Thr Lys Glu Phe Ala Ser His Pro Arg Val Thr Ser Lys His Met Ala Cys Ala Phe Lys Ser Arg Ser Asp Tyr Gin Pro Arg Gly Cys Asp Gin Asp Leu Val Phe Leu Tyr Arg Leu Thr Glu Gly Ala Cys Pro Glu Ser Tyr Gly Leu Gin Val Ala Leu Met Ala Gly Ile Pro Asn Gin Val Val Glu Thr Ala Ser Gly Ala Ala Gin Ala Met Lys Arg Ser Ile Gly Glu Asn Phe MOR-0444.ST25.txt Lys Ser Ser Glu Leu Arg Ser Glu Phe Ser Ser Leu His Glu Asp Trp Leu Lys Ser Leu Val Gly Ile Ser Arg Val Ala HIS Asn Asn Ala Pro Ile Gly Glu Asp AS Tyr Asp Thr Leu Phe Cys Leu Trp His Glu Ile Lys Ser Ser Tyr Cys val Pro Lys <210> 43 <211> 5307 <212> DNA
<213> Arabidopsis thaliana <400> 43 aaagataagt tcatacgact tttgtggctc atcaaaggcc atcatcgtcc tctatataca 60 atttagtgct ttatagtaca aaaccttcca cttccctttg tccaaagttt tccaatttaa 120 tttataaaca ggaataatat tatctatata ataaagtgaa aaataactat cattgtccaa 180 ataatttggt cgttgatcat gttactacaa agaaatgaaa tccttagtag aagtatatat 240 atatatatat ttgtaacaca ctcaaaatgg taggtgttgt tacagacaga tgttcgttag 300 cccagtaagc ccaatatgag atttaatggg ccttgatatt ttatagacca aacattgaaa 360 cattgcacgc ctggtctcaa agaacgttaa tacacgcgcc gccggttgcc gccaatccgc 420 tttcccgcca aattcgacac cataaatttc ttctagtcgc tttcgattcc agttccactg 480 aaaaaccacg aaagaagaac atttgcaccg tagttgcaga aggtaggtga aggatttagc 540 tttctctatc ttccaatgga gggtaatttc gaggaacaga acaagcttcc ggagctgaaa 600 ttgggtaatg ttaaacccta gttttttttt tctttctcat tttcgtattc gatttcccaa 660 ttgggtttat gggttttgta aaaggtctga tatttgttat gcattttttt tttaattttt 720 ggaagatgca aagcaagctc aagggtttct ctcgttctac aaaaccctac caaatgtaag 780 ttctcgtttt ctttcgattt ctgggagaag ttagagcttg tacagtgcct ctaattgcaa 840 taaataacac caattctagt cggaaagtag atgctttaaa attagggttt gaagcaattg 900 tagacatttt gttcattggg aagcgaatta ggaaaaaagg cttaagattt tttagcaatt 960 tctcgatctt tgcttatgtg ggttttgatt gttctttgct tcaggatacg agagctgtta 1020 gattctttga tcgcaaggtg agttcattgt tctcaaatgg tctagacttt ggttgtttaa 1080 atgtcgtcat tgatttatgg aaattttttg aatgcatttg caggattatt atacagctca 1140 tggtgaaaat tcagttttca ttgcaaagac ttattatcat acaaccactg ctctacgtca 1200 gctcgggagt ggttcaaatg ctctttcaag cgtaagcatt agtaggaaca tgttcgaaac 1260 gattgctagg gatcttctcc tggagcgtaa tgatcatact gtagaacttt atgaaggaag 1320 cggatcgaat tggagacttg tgaaaacagg ttctcctgga aacattggaa gctttgaaga 1380 06 a6Pd OZVE a66116p6pp 666666 PDITPP611.1 D1464DElll DDD6111611 DPaPPPDPP6 09EE 11DP6allap 3D111611Da 11116111D1 6Dp6p6aDep papepa6616 auebtaaplop NEE Pa6DaDP6PD 6ale6aPPPD DelEDIalUE 6166611.P6P PolD6PP6P1 61601PDpapp OVZE p6pD6pp66p p6papp1611 pap6p661D6 au66Pa611P PraPP1166D pp6p6p6116 081E ZPP1PDP6ZE 1116PPDpal 1-D343.43.46p app6apepp6 131161111P ale6a16peD
OZTE apleeDeaft la66alaDaD Depappp6pD D66.216appa EDDIOR1DDD 6116PDDEIlD
090E 66141p6aD6 laap6ppla6 111161p661 prp6apalaD pa16661D6p lapp66p611 000E 1P1.66paPal 661P1611e1 pa6apapall pp6616apap appppabele Dapeepall6 0b6Z 11661aPp6p DP1a6aDp66 lappoppale aPplaPlaft aza61P166p 61Daplap6p 088Z DDP11.61DP6 e6116P1616 pap6la6pap 6p66uppepa 61D6P66e1P alp6ap66a6 OZ8Z 1.4616ppppD Pa6PDDP6D6 661aPPEPUP P1DETEEDRD peeDeD116e p6a6p66Dp6 09LZ pepo6papp6 p661D616pa pall6pDepe 5656 pp66pDap6p pupD6p6pp6 OOLZ 6pP6Pp6DPD aP66PplaDa 61pD666111 epp6p6PD6p upDp6aapup paapap66pp 0V9Z op6Da66pDa apap6aapep 6pap6p6ppe 6PPPETDED6 laPP6DED11. p6po6eD6p6 08SZ 61D6alep6p pe6eDle6pe e6apaDapp6 61aPpeDDep p6ppappepo alDapap6ap OZSZ ppapp6p661 UPPP6UlDET DDP611.DZP6 P161D1D61.6 U611.66114P 6paPpla6p 09VZ P661apppDa PETPD1P6PD 1P111766P6 3.1DETUPPPEI 1DDP166P6P 6D6PD1PD13 oot"zPD1PD6D11P B6P663D2DP 16eDeep6le 136EDUEUED 1P011.DDDll DCIETP1E4D6 OVEZ PDa6PD6apa pallapappa apaftelave applaPpla6 PP11P1-161P 1.66paPal6p 08ZZ p6apapPeD6 arala6161p ppappa6a1P DallaPp6a1 PPEPDPP116 1PPD1D1Dal OZZZ D61D1PEOla DED6D01113 P166eDleaD apeePalela uppD6eDela 66656Pe 09TZ 6pp6p6p6Da Da6pD6D6a1 lap66p6p61 a6ap6ppapa pp6p6pp6ap app6pDp6Pa OOTZ 101P66pD66 pu116663,D6 up6 61a61 11D616PD11 6P1r4P6Pla P6P6DRETP1 0170Z 1P6P6aPP6a 11P6616D1D DDDP236e26 30661.61E1e DI4D61DP6P 6e16661e6 0861 66PD6Dpea6 lepppefeDpe 6appaDa66a 14611.16ED1 11VP6PEaRe ao6aP6apPp 0Z61 upp6P6e66a P6a6aPP611 6D666p6apa D6pD6apapp 6aapp6p6ap paap66D661 0981 leaP6apap6 P36DD1PPDP Dl1DPE6661 pappeap66e 6apeepaDaD lappp61311 0081 1.16DaDappa 3.pD63.66p3o 1D630D1DRE0 Dollpop611 1.66E031E16 666pa OVLT 16app6p611 papp6666PP 66666 PplapaP6PD lar6ealle6 p6pp66prep 089T 116P6pppup p6pe66p6p6 eDeP4P6ap6 16pD6a6166 p6p661D)Da ap6apa6aDD
0Z91 6eeeD6app 6appopapee D66pDapP61 p6pDplalla PAlEP6PBP ED6D66E1D1 09ST D611.P611.6D 3.1D16v661D ;PVDDVD11D 6DAP1P61E 5.66 aD6Papp66a 00ST aDal6p6D66 papp6apap6 1161paDD66 ap66611pla 6)61p6pD66 le6peollaa 6pepplaapa EDD1D16,116 116E0DEDED p6Eopp6appp 56 1416131461 4x1..szis*vm70-Now mOR-0444.ST25.txt ttcaaatagt aacagggcct aacatgggag ggaagtccac tttcatccgc caggtatgat 3480 gatttcctct agttcagttt tgcttcatag acgtatgact aaagtcggtt tccggccatt 3540 ataaatccca ggttggtgtg attgtgctga tggctcaagt tggttccttt gttccttgtg 3600 ataaagcatc aatttccata agagactgca tctttgcccg tgtaggagca ggcgattgcc 3660 aagtgagttt aagtttagcc ctcaatgaac gaaaaactgc tgatatcctg aacaccctta 3720 ttccaacttt ttttcctttg gtgtgttagc tgcgtggagt gtcaactttt atgcaagaaa 3780 tgcttgaaac cgcatcgata ttgaaaggcg ctactgataa gtcactgata attatcgatg 3840 aacttggtcg tggaacatca acttatgatg gttttggtta gtttctctgc aatttctctt 3900 ctttcatttg gatgttttta gtaagttttc tattatatat tcatttttat ggtcatatgt 3960 gagatttcag tgctcttgac atcatcgtgg tgaatatatc aggtttagct tgggctatat 4020 gtgagcatct ggttcaagtg aaaagagcac caactctgtt tgctactcac ttccatgaac 4080 ttactgcctt ggctcaagca aactctgagg tctctggtaa cactgttggt gtggcaaact 4140 tccatgtcag cgctcacatt gacactgaaa gccgcaaact caccatgctt tacaaggtct 4200 ggtttataaa ttaaaaaatt gctgatctgt tgcagttaaa agtgtctctg tttttatgtt 4260 taatctaaat tacttatttg attttcttac aaagatgaaa ttgaaattaa ttttgtgtgg 4320 tgtgttgttt gtctggttag gttgaaccag gggcctgtga ccagagcttt gggattcatg 4380 tggcggaatt tgccaacttc cctgaaagcg tcgtggccct cgcaagagag aaagctgcag 4440 agctggaaga tttctctccc tcctcgatga taatcaacaa tgaggtcttg attcatttcc 4500 ccctttgttt ttggttgatg atggaatcat tctatcattc acccattctg cagtttatgc 4560 tatattatta taaatctatg tgacaaagat ttaattctcg tattgttgtt tgcaggagag 4620 tgggaagaga aagagcagag aagatgatcc agatgaagta tcaagagggg cagagcgagc 4680 tcacaagttt ctgaaagagt ttgcagcgat gccacttgat aaaatggagc ttaaagattc 4740 acttcaacgg gtacgtgaga tgaaagatga gctagagaaa gatgctgcag actgccactg 4800 gctcaggcag tttctgtgaa gaacccctga cgttttttgg tttttggttt tgtaaatagc 4860 ttaaatcggt tcttgtagtt gtggtcgttg cttgggatga aactaaatga gggcaaaaac 4920 ataattctac attttttgtt agtaaagctc gttaatttac tccctagtgc tatcaattat 4980 tttgcctatt ataattgttg atcaagtact tagagcaacc ccaatggttt ctaaacataa 5040 gtttcttatt ttatagagag aaattttatt ataaaaaaat gtgtgggttt cttgattagt 5100 gaagaaacca tctccaaaat accttatatt cttatataag gtattttgga gagaatttct 5160 aactattcaa gaaacttaca taattaaata ctattatttt tattgtttta atgttaagaa 5220 acttatattt aaaaaccacc aatggaattg ctcttagcta ccatacaaat aattataaaa 5280 atatatcgaa aagtagaaga gccattt 5307 <210> 44 <211> 937 <212> PRT

. .
moR-0444.ST25.txt <213> Arabidopsis thaliana <400> 44 Met Glu Gly Asn Phe Glu Glu Gin Asn Lys Leu Pro Glu Leu Lys Leu Asp Ala Lys Gin Ala Gin Gly Phe Leu Ser Phe Tyr Lys Thr Leu Pro Asn Asp Thr Arg Ala Val Arg Phe Phe Asp Arg Lys Asp Tyr Tyr Thr Ala His Gly Glu Asn Ser Val Phe Ile Ala Lys Thr Tyr Tyr His Thr Thr Thr Ala Leu Arg Gin Leu Gly Ser Gly Ser Asn Ala Leu Ser Ser val Ser Ile Ser Arg Asn Met Phe Glu Thr Ile Ala Arg Asp Leu Leu Leu Glu Arg Asn Asp His Thr Val Glu Leu Tyr Glu Gly Ser Gly Ser Asn Trp Arg Leu Val Lys Thr Gly Ser Pro Gly Asn Ile Gly Ser Phe Glu Asp Val Leu Phe Ala Asn Asn Glu Met Gin Asp Thr Pro Val Val Val Ser Ile Phe Pro Ser Phe His Asp Gly Arg Cys val Ile Gly Met Ala Tyr val Asp Leu Thr Arg Arg Val Leu Gly Leu Ala Glu Phe Leu Asp Asp Ser Arg Phe Thr Asn Leu Glu Ser Ser Leu Ile Ala Leu Gly Ala Lys Glu Cys Ile Phe Pro Ala Glu Ser Gly Lys Ser Asn Glu Cys Lys ser Leu Tyr Asp Ser Leu Glu Arg Cys Ala Val Met Ile Thr Glu Arg Lys Lys His Glu Phe Lys Gly Arg Asp Leu Asp Ser Asp Leu Lys Arg Leu Val Lys Gly Asn Ile Glu Pro Val Arg Asp Leu Val Ser Gly , MOR-0444.ST25.txt Phe Asp Leu Ala Thr Pro Ala Leu Gly Ala Leu Leu Ser Phe Ser Glu Leu Leu Ser Asn Glu Asp Asn Tyr Gly Asn Phe Thr Ile Arg Arg Tyr Asp Ile Gly Gly Phe Met Arg Leu Asp Ser Ala Ala Met Arg Ala Leu Asn Val Met Glu Ser Lys Thr Asp Ala Asn Lys Asn Phe Ser Leu Phe Gly Leu Met Asn Arg Thr Cys Thr Ala Gly Met Gly Lys Arg Leu Leu His Met Trp Leu Lys Gin Pro Leu Val Asp Leu Asn Glu Ile Lys Thr Arg Leu Asp Ile Val Gin Cys Phe Val Glu Glu Ala Gly Leu Arg Gin Asp Leu Arg Gin His Leu Lys Arg Ile Ser Asp Val Glu Arg Leu Leu Arg Ser Leu Glu Arg Arg Arg Gly Gly Leu Gin His Ile Ile Lys Leu Tyr Gln Ser Ala Ile Arg Leu Pro Phe Ile Lys Thr Ala Met Gin Gin Tyr Thr Gly Glu Phe Ala Ser Leu Ile Ser Glu Arg Tyr Leu Lys Lys Leu Glu Ala Leu Ser Asp Gin Asp His Leu Gly Lys Phe Ile Asp Leu Val Glu Cys Ser Val Asp Leu Asp Gin Leu Glu Asn Gly Glu Tyr Met Ile Ser Ser Asn Tyr Asp Thr Lys Leu Ala Ser Leu Lys Asp Gin Lys Glu Leu Leu Glu Gin Gin Ile His Glu Leu His Lys Lys Thr Ala Ile Glu Leu Asp Leu Gin Val Asp Lys Ala Leu Lys Leu Asp Lys Ala Ala Gin Phe Gly His Val Phe Arg Ile Thr Lys Lys Glu Glu Pro Lys Ile MoR-0444.ST25.txt Arg Lys Lys Leu Thr Thr Gin Phe Ile Val Leu Glu Thr Arg Lys Asp Gly val Lys Phe Thr Asn Thr Lys Leu Lys Lys Leu Gly Asp Gin Tyr Gin Ser Val Val Asp Asp Tyr Arg Ser Cys Gin Lys Glu Leu Val Asp Arg Val val Glu Thr Val Thr Ser Phe Ser Glu Val Phe Glu Asp Leu Ala Gly Leu Leu Ser Glu met Asp Val Leu Leu Ser Phe Ala AS Leu Ala Ala Ser Cys Pro Thr Pro Tyr Cys Arg Pro Glu Ile Thr Ser Leu Asp Ala Gly Asp Ile Val Leu Glu Gly Ser Arg His Pro Cys Val Glu Ala Gin Asp Trp Val Asn Phe Ile Pro Asn Asp Cys Arg Leu Met Arg Gly Lys Ser Trp Phe Gin Ile val Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Phe Ile Arg Gin Val Gly Val Ile Val Leu Met Ala Gin Val Gly Ser Phe Val Pro Cys Asp Lys Ala Ser Ile Ser Ile Arg Asp Cys Ile Phe Ala Arg Val Gly Ala Gly Asp Cys Gin Leu Arg Gly Val Ser Thr Phe Met Gin Glu Met Leu Glu Thr Ala Ser Ile Leu Lys Gly Ala Thr Asp Lys Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Cys Glu His Leu Val Gin val Lys Arg Ala Pro Thr Leu Phe Ala Thr His Phe His Glu Leu Thr Ala Leu Ala Gin Ala Asn Ser Glu Val Ser Gly Asn Thr val Gly MOR-0444.ST25.txt Val Ala Asn Phe His Val Ser Ala His Ile Asp Thr Glu Ser Arg Lys Leu Thr met Leu Tyr Lys val Glu Pro Gly Ala Cys Asp Gin Ser Phe Gly Ile His Val Ala Glu Phe Ala Asn Phe Pro Glu Ser Val Val Ala Leu Ala Arg Glu Lys Ala Ala Glu Leu Glu Asp Phe Ser Pro Ser Ser Met Ile Ile Asn Asn Glu Glu Ser Gly Lys Arg Lys Ser Arg Glu Asp Asp Pro Asp Glu Val Ser Arg Gly Ala Glu Arg Ala His Lys Phe Leu Lys Glu Phe Ala Ala Met Pro Leu Asp Lys Met Glu Leu Lys Asp ser Leu Gin Arg Val Arg Glu Met Lys Asp Glu Leu Glu Lys Asp Ala Ala Asp Cys His Trp Leu Arg Gin Phe Leu <210> 45 <211> 3521 <212> DNA
<213> Arabidopsis thaliana <400> 45 ctaagaaagc gcgcgaaaat tggcaaccca agttcgccat agccacgacc acgaccttcc 60 atttctctta aacggaggag attacgaata aagcaattat gggcaagcaa aagcagcaga 120 cgatttctcg tttcttcgct cccaaaccca aatccccgac tcacgaaccg aatccggtag 180 ccgaatcatc aacaccgcca ccgaagatat ccgccactgt atccttctct ccttccaagc 240 gtaagcttct ctccgaccac ctcgccgccg cgtcacccaa aaagcctaaa ctttctcctc 300 acactcaaaa cccagtaccc gatcccaatt tacaccaaag atttctccag agatttctgg 360 aaccctcgcc ggaggaatat gttcccgaaa cgtcatcatc gaggaaatac acaccattgg 420 aacagcaagt ggtggagcta aagagcaagt acccagatgt ggttttgatg gtggaagttg 480 gttacaggta cagattcttc ggagaagacg cggagatcgc agcacgcgtg ttgggtattt 540 acgctcatat ggatcacaat ttcatgacgg cgagtgtgcc aacatttcga ttgaatttcc 600 atgtgagaag actggtgaat gcaggataca agattggtgt agtgaagcag actgaaactg 660 cagccattaa gtcccatggt gcaaaccgga ccggcccttt tttccgggga ctgtcggcgt 720 tgtataccaa agccacgctt gaagcggctg aggatataag tggtggttgt ggtggtgaag 780 96 a6Ed 6166DE6DED PDEDET1DPD 66E6E26611 D6r61E6E4.1 E1E14.64.1D6 D116D1D11.D
09LZ 1.161DDEPET DIXElEDPDE DI6ARP616 PPI4PE6PPE0 P10141DDE1 66661E
OOLZ
D6EDD1E16E DE6ED11D64. 6661E66D1D ED11116166 1E611.3646D E061D6PEDD

6D14ED16D6 EDDE161.41D D116644.66E D4D661Eele DD114.EE114 D6116EED16 08SZ DDlel.E1D61 D6e6EEE66E 6661EDEE1D Dr66DD
1EEEDD611 ElEE6666EE
OZSZ
6ED61ED614. laPEDEDE61 EPPDDDItol. ZDEPIXETPD E11.E4.E1DE6 E6601DE161.D
D4ED16D166 1D16EDE4.ED EEE1E6E611. 6EDDEE6161 DE61E66461 116E6DDD16 00tZ DD161E1DEE 6EEDErE6Ez D1P1D1DPED 111DDD1DPD 611161DE66 1DED613611 OtEZ D4D6EED1.16 DDEII.D66m. 14.1e6EDEDE 4DE1E6E16E D1116e6evo loD14.16E1E

66616D11D6 E6DDEE61.61 lEDD611.D1E DPP61DPEDE0 PaD1D6P1D6 61.E6614D6 OZZZ 61.D6e1.6ear PEETDDDDD1 PD1P1E6Dla P1DE6EP6EP DDRAEZPUP a5PE61666 09TZ 4.1EE61.E1DD D166EEDD11 E6116DDD61. D6E6E4E614. 1PDPDPD1E6 666 66 OOTZ D11D112Pe6 64.11EEE6D1 1E4D6D1.D6E E6EED6D114. eal1D6E1E6 D1DDlle664 OtOZ D6EEEE666E DI.ED16E111 16ED6EEDD6 D1D6EE61.D6 11D6E61DD1 11EEDDE6D6 EDDI.I.DED4E EZDElEDPE0 405/Z10E616 6P6D11.6306 66PP66PPI. EPP1DDAZD

1D1D11.DEEE E66DD61EED E611661611. 64DDDD1ED1. 4.1E1161D11. 4.E611EvEr6 0981 P611.14D1DP 1D16D61.61 DePAZDITP DEt1P16P66P 61EEE61D1D r6EED6EEE1 0081 ED6611.D66D 66 ee6666D611 DE1111E3.D6 ee661E116E DEaleD11.6P
OLT
6PDEDAPEE 1D61DE66D1 ED111D1EE6 EEDEE1EE66 16DEED14.E1. E61D1ED1E6 E1D1.61E1D6 EDE641D16E D1.DD1D1D61 6D1D1E1111 6E61DDED1E 1.611EED6E6 P6u61D1166 EE6EE61466 146E6l6PD6 ED1D6PDDDI. 10116P1PD1. D1E6661ED6 09ST 14.D61D11zE 6E61D11161 D6 6D66 D4D61D1E1E 6411EEE6E1 E6D61E1D1D
00ST DlED4DE6a6 661DEDE6E1 1D11D66EDD 11661E1E16 POUlaDeDED ED1EE61E1E
Ott' ElEDD14.E11 DD1.D661D1E E66D1E661e 6ED1.1EE1EE EEE61614.66 E66116EDEE

D61D1DElru DD6EDI.D1D1 DE64.E6E6ED EDEE16EED1 61.14.D4.D6D1 11ED1DD666 OZET 6PeDDEllap D1P66PEU61 11E661416P DPEPDaDlPD D614.116DEE 1DDADIODD
09ZT ETPD1163.DE 61D1PDPDA 1PDPE61P41 PEDPIXD3.16 EDE611a611 D1.61E66EE
0OZT EuE64D61D6 6e664D6EE6 leeefteele 61e6ue6E11 DEE166ED6D 6EDZERPPPP
OKI 6162PaluD1 11E11.66u61 E6E4.6ED64.E E1661EED6E D111611E66 DD616 DEE6616E6D 116DEEED1D DE1DDE661D 61.EDED6616 666 PEI1DUPPDPP
OZOT DE014.1D1DD 6EDD6611D6 11.64D6E64D ETDDPD1611 D66 P6 1E6646EE6E 61ED41.4EE1 E64rED43.6r 6EE61E1116 116EE6166e DE6D111EEE

61.3.6a66116 1161.66316e 6E1161E64.1 116E64EEE6 14.E166161.D 66ElaEDE6E
5565 E5e6pEap66 1.611.616111 6611D111Ev 1.6e6pDvpqa 6666ep lxvszis*V17170-tiow MOR-0444.ST25.txt tagccattgc ctatgcaaca ttacagcatc tcctagcaga aaagagatgt ttggttcttt 2880 ttgtcacgca ttaccctgaa atagctgaga tcagtaacgg attcccaggt tctgttggga 2940 cataccatgt ctcgtatctg acattgcaga aggataaagg cagttatgat catgatgatg 3000 tgacctacct atataagctt gtgcgtggtc tttgcagcag gagctttggt tttaaggttg 3060 ctcagcttgc ccagatacct ccatcatgta tacgtcgagc catttcaatg gctgcaaaat 3120 tggaagctga ggtacgtgca agagagagaa atacacgcat gggagaacca gaaggacatg 3180 aagaaccgag aggcgcagaa gaatctattt cggctctagg tgacttgttt gcagacctga 3240 aatttgctct ctctgaagag gacccttgga aagcattcga gtttttaaag catgcttgga 3300 agattgctgg caaaatcaga ctaaaaccaa cttgttcatt ttgatttaat cttaacatta 3360 tagcaactgc aaggtcttga tcatctgtta gttgcgtact aacttatgtg tattagtata 3420 acaagaaaag agaattagag agatggattc taatccggtg ttgcagtaca tcttttctcc 3480 acccgcataa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 3521 <210> 46 <211> 1081 <212> PRT
<213> Arabidopsis thaliana <400> 46 Met Gly Lys Gin Lys Gin Gin Thr Ile Ser Arg Phe Phe Ala Pro Lys Pro Lys Ser Pro Thr His Glu Pro Asn Pro Val Ala Glu Ser Ser Thr Pro Pro Pro Lys Ile Ser Ala Thr Val Ser Phe Ser Pro Ser Lys Arg Lys Leu Leu Ser Asp His Leu Ala Ala Ala Ser Pro Lys Lys Pro Lys Leu Ser Pro His Thr Gin Asn Pro Val Pro Asp Pro Asn Leu His Gin Arg Phe Leu Gin Arg Phe Leu Glu Pro Ser Pro Glu Glu Tyr Val Pro Glu Thr Ser Ser Ser Arg Lys Tyr Thr Pro Leu Glu Gln Gin Val val Glu Leu Lys Ser Lys Tyr Pro AS val val Leu Met Val Glu Val Gly Tyr Arg Tyr Arg Phe Phe Gly Glu Asp Ala Glu Ile Ala Ala Arg Val MOR-0444.ST25.txt Leu Gly Ile Tyr Ala His met Asp His Asn Phe Met Thr Ala Ser val Pro Thr Phe Arg Leu Asn Phe His val Arg Arg Leu val Asn Ala Gly Tyr Lys Ile Gly val Val Lys Gin Thr Glu Thr Ala Ala Ile Lys Ser His Gly Ala Asn Arg Thr Gly Pro Phe Phe Arg Gly Leu Ser Ala Leu Tyr Thr Lys Ala Thr Leu Glu Ala Ala Glu Asp Ile Ser Gly Gly Cys Gly Gly Glu Glu Gly Phe Gly Ser Gin Ser Asn Phe Leu Val cys Val val Asp Glu Arg val Lys Ser Glu Thr Leu Gly Cys Gly Ile Glu Met ser Phe Asp Val Arg Val Gly Val val Gly Val Glu Ile Ser Thr Gly Glu val Val Tyr Glu Glu Phe Asn Asp Asn Phe Met Arg Ser Gly Leu Glu Ala val Ile Leu Ser Leu Ser Pro Ala Glu Leu Leu Leu Gly Girt Pro Leu Ser Gin Gln Thr Glu Lys Phe Leu Val Ala His Ala Gly Pro Thr Ser Asn Val Arg Val Glu Arg Ala Ser Leu Asp Cys Phe Ser Asn Gly Asn Ala Val Asp Glu val Ile Ser Leu Cys Glu Lys Ile Ser Ala Gly Asn Leu Glu Asp Asp Lys Glu met Lys Leu Glu Ala Ala Glu Lys Gly Met ser Cys Leu Thr val His Thr Ile met Asn met Pro His Leu Thr Val Gin Ala Leu Ala Leu Thr Phe Cys His Leu Lys Gin Phe Gly Phe Glu Arg Ile Leu Tyr Gin Gly Ala Ser Phe Arg Ser Leu Ser ser MOR-0444.ST25.txt Asn Thr Glu met Thr Leu Ser Ala Asn Thr Leu Gin Gin Leu Glu val Val Lys Asn Asn Ser Asp Gly Ser Glu Ser Gly Ser Leu Phe His Asn Met Asn His Thr Leu Thr Val Tyr Gly Ser Arg Leu Leu Arg His Trp Val Thr His Pro Leu Cys Asp Arg Asn Leu Ile Ser Ala Arg Leu Asp Ala Val Ser Glu Ile Ser Ala Cys Met Gly Ser His Ser Ser Ser Gin Leu Ser Ser Glu Leu val Glu Glu Gly Ser Glu Arg Ala Ile Val Ser Pro Glu Phe Tyr Leu Val Leu Ser Ser Val Leu Thr Ala Met Ser Arg Ser Ser Asp Ile Gin Arg Gly Ile Thr Arg Ile Phe His Arg Thr Ala Lys Ala Thr Glu Phe Ile Ala Val met Glu Ala Ile Leu Leu Ala Gly Lys Gin Ile Gin Arg Leu Gly Ile Lys Gin Asp Ser Glu met Arg Ser met Gin Ser Ala Thr Val Arg Ser Thr Leu Leu Arg Lys Leu Ile Ser val Ile Ser Ser Pro Val Val Val Asp Asn Ala Gly Lys Leu Leu Ser Ala Leu Asn Lys Glu Ala Ala Val Arg Gly Asp Leu Leu Asp Ile Leu Ile Thr Ser Ser Asp Gin Phe Pro Glu Leu Ala Glu Ala Arg Gin Ala val Leu val Ile Arg Glu Lys Leu Asp Ser Ser Ile Ala Ser Phe Arg Lys Lys Leu Ala Ile Arg Asn Leu Glu Phe Leu Gin Val Ser Gly Ile Thr His Leu Ile Glu Leu Pro Val Asp Ser Lys Val Pro Met Asn Trp , MOR-0444.ST25.txt Val Lys Val Asn Ser Thr Lys Lys Thr Ile Arg Tyr His Pro Pro Glu Ile Val Ala Gly Leu Asp Glu Leu Ala Leu Ala Thr Glu His Leu Ala Ile val Asn Arg Ala Ser Trp Asp Ser Phe Leu Lys Ser Phe Ser Arg .

Tyr Tyr Thr Asp Phe Lys Ala Ala Val Gin Ala Leu Ala Ala Leu Asp Cys Leu His Ser Leu Ser Thr Leu Ser Arg Asn Lys Asn Tyr Val Arg Pro Glu Phe Val Asp Asp Cys Glu Pro Val Glu Ile Asn Ile Gin Ser Gly Arg His Pro Val Leu Glu Thr Ile Leu Gin Asp Asn Phe Val Pro Asn Asp Thr Ile Leu His Ala Glu Gly Glu Tyr Cys Gin Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Cys Tyr Ile Arg Gin Val Ala Leu Ile Ser Ile met Ala Gin Val Gly Ser Phe Val Pro Ala Ser Phe Ala Lys Leu His Val Leu Asp Gly Val Phe Thr Arg Met Gly Ala Ser Asp Ser Ile Gin His Gly Arg Ser Thr Phe Leu Glu Glu Leu Ser Glu Ala Ser His Ile Ile Arg Thr Cys Ser Ser Arg Ser Leu Val Ile Leu Asp Glu Leu Gly Arg Gly Thr Ser Thr His Asp Gly Val Ala Ile Ala Tyr Ala Thr Leu Gin His Leu Leu Ala Glu Lys Arg Cys Leu val Leu Phe Val Thr His Tyr Pro Glu Ile Ala Glu Ile Ser Asn Gly Phe Pro Gly Ser Val Gly Thr Tyr His Val Ser Tyr Leu Thr Leu Gin Lys Asp Lys MOR-0444.ST25.txt Gly Ser Tyr Asp His Asp Asp Val Thr Tyr Leu Tyr Lys Leu Val Arg Gly Leu Cys Ser Arg Ser Phe Gly Phe Lys Val Ala Gin Leu Ala Girt Ile Pro Pro Ser Cys Ile Arg Arg Ala Ile Ser Met Ala Ala Lys Leu Glu Ala Glu Val Arg Ala Arg Glu Arg Asn Thr Arg Met Gly Glu Pro Glu Gly His Glu Glu Pro Arg Gly Ala Glu Glu Ser Ile Ser Ala Leu Gly Asp Leu Phe Ala AS Leu Lys Phe Ala Leu Ser Glu Glu Asp Pro Trp Lys Ala Phe Glu Phe Leu Lys His Ala Trp Lys Ile Ala Gly Lys Ile Arg Leu Lys Pro Thr Cys Ser Phe <210> 47 <211> 7080 <212> DNA
<213> Arabidopsis thaliana <400> 47 ctcttcgccg actgtttcac tccccttctc tctcactctc tgtgcgcttt attccactct 60 ccgatggctc cgtctcgccg acagatcagc ggaagatctc cgttggtgaa ccagcagcgt 120 caaatcacct ccttctttgg gaaatctgct tcatcatctt cttctccgtc tccatctcct 180 tcaccatctc tctccaataa gaaaaccccc aaatctaaca accctaaccc taaatctccg 240 tctccgtcac catctccgcc taagaaaacc cccaaattga accctaaccc tagttctaat 300 cttcctgctc gtagtcctag ccctggtcct gatactcctt ctcctgtaca gtccaagttt 360 aagaagcccc ttctcgtcat cggacagaca ccttcgcctc ctcaatcggt ggtaattact 420 tacggtgacg aggtggtggg gaagcaagtt agggtttatt ggcctttgga taaaaaatgg 480 tatgatggga gcgtgacgtt ttatgataag ggtgagggta agcatgtggt tgagtatgaa 540 gatggggaag aagagtcttt ggatttggga aaggagaaga ctgagtgggt ggttggggaa 600 aaatcaggag ataggtttaa tcgattgaaa cgaggcgctt cggctttgag aaaagttgtg 660 acggatagtg atgatgatgt ggagatgggt aatgtggaag aagataaaag tgacggtgat 720 gattctagcg atgaggattg gggaaagaat gttgggaagg aggtttgtga gagtgaagaa 780 gatgatgtgg agttggttga tgagaatgaa atggatgaag aagagttggt ggaagagaaa 840 gatgaagaaa cttctaaagt taatagagta tccaaaactg actctagaaa gcggaagact 900 ZOT DElud Ot6Z plzu666341 lee61.31113 eop11631D1 Eul.puel6e4 100D1PPP6P D3PEEDE6EZ
088z 16elpepee6 e6p6eDuvD6 lell6e613.6 166m.36po Depelzell.E pp66166DD6 OZ8Z 666.D
1p1D61DD61. 1.D1PlaPp63. 6eD613.e6ee DI.e61v66ep 1.116vDpel6 plaup1.13.1.1. 1.11.1m663.E. 611.Elp461.6 6DI.D111-eDe pl6Dulellp e3611661D3 OOLZ
v1.661pp6au 611.6u1.31.1.6 e61.66poD66 61Dplpelp6 PP6P3PDP6D 6666 Ot9Z 61-1451x461. 66lIalREDE D6P6PAPDP IODZUE1DEZ 1.11.6pre6u6 6p66E.E.61Dp 08SZ 6113D661.pe 61.u66Dpapp lopell.v1D6 ze6r66661.r 6EDP61D6Dp OZSZ
D66eueepez 3.61.1.6pD61.6 zel6er6D63 62v61611.66 plp111.1161 DI4DEP6PDE
09tz 6a6ezpal.D6 66666e6 1.elaueeee6 el.61.p1DD61 u61666111D pD1161.11.up 00tZ
peaDppp61.1. )1.3613461z leP316RED1 1.6upzulauD 6611.6E414p vloaep6Tel.
OtEZ
DpDDDI.61.el 6ppplp66pe Da11.66upu6 e6eue6DD6 DeeD6p661D 6ED1e61.Dpp 08ZZ DUPP6PDPEP DRP6D1613.6 P111.1666D1. p1D666tope 66lee6llee 666EE
OZZZ
EzPeE6E166 61.11.Del.63.D 61.ppEo661.E. DZIXDUZ1PP PETET16160 p1.1.61.1D111.

eaDze61.6D6 PED1.16PDDI. DRUDEIEZPIX uP6PD71.146 11.1.66PEP6P 1.1.6r1.1pvp6 001?
P613.uprepl. 61.D341.1.1.pp 6pe6e663Dz 1.1.u661.614p DIODEPDET6 U666P151.14 OVOZ
lelaleD6Ze ZeDP61.DDDD ETEDP414D3. DZDZE161.EP 1D4D1.61.1p1. 613DTD1.1.1r 086T PPDP11P3P4 1616614eD1 341D14666e penD6zEll. PeseDZED661. 14SEPP6DDP

Peepull.61p pppp4164D4 epufleu61.11 e363.e63.66e 1DEPIXD3OP EllE1461116 0981 lorr166ET6 atorzuPpra el.P661Dpp6 6ppaD6p66D 16TeDepole 661e6e6111 11D6p6leaD 1.1peel.6661. r6eD6ETD66 1.61.11.1.Dalp 1.661repl.p6 6e66 OtL1 661.e61.1E1.3. 1P11.DPPZEZ 16D11.1Plap Ezl.m.r6Dp p6p1.66ppp1. 1D1.1p161.16 PEPDE661x3. PDEIPEPAPP P1.116E6661. 6666 PD111.111.03 661.61.111e6 ZP1P1.1DeD1 3.1DPEDZEEZ D6ev61.6611. 11.1.P66ppla 661D1v2P66 1pD6D6E6pp 6eD3.66vDD6 6e661peell eeeepe61.63. 111E6loplp DDZDDE1D10 PDPEoft6D)1 PE01P11Pp6e 61x63.ppl.DD 363u6e66ee eu1361E666 upoppeouffe 3ee61661.1.1 Ott' 1D1.1.6r1.46P P1DET4D11P 1D 6i 61)66P16n le1.61e6leD

1301Plazap 1141.66131.D im6r66eD6 6aDE.I.D1D1.1 3P611elpto 6e61.6 OZET
111PPel6lp p6v6e1.6p66 p3.1.661.663p 1D611.1.P3.D6 6p6e1.1.31.to DalPPPPPPP

PpeD3.1.1D1.6 un61.6E666 1111.1D6D31 lvpu6v6zpl. 1r616)1D61. 661.11r6plo 614D1.361.u6 6662p66116 1.6DMP5PP 66141.666PP )1661666P1. p6a36EDPI.D
OKI P4103.61611 363.1663461. 163.P3.616e1 p6231.34641 zapP6zDvD6 laD1D161.E.1. 661.61416n 41a61.1ED61 1.1e1613.D61 ezepzee161 1.6z1D1.D1.6p OZOT ETEEIDDEPE1 66PPDP6641 PETP6PPE06 4DAP661.61 1.61D13066P ez1.1.166ree 614Dze1.pp) 66PDPIX61D PETPAPPPP 6PPET61661 66pDapee63 pe3.6ee616e 1.xl..szts'ttt0-110w MOR-0444.ST25.txt ggagaagacc atatatgaag ttggaattat ctacaagcga atcaattgtc aaccgtcttc 3000 tgcttattct agtgagggaa agattctagg tgatggttca agctttcttc caaaaatgtt 3060 gtctgaatta gcaactgaag ataagaatgg tagcctggca ctctctgctc ttggtggtgc 3120 catttactac ctgcgacaag cattcttgga tgagagtctg cttagatttg caaagtttga 3180 atccctgcct tactgtgatt tcagcaacgt taatgagaag cagcacatgg ttcttgatgc 3240 tgctgctctt gaaaaccttg agatatttga aaacagtaga aatggaggct attcagggta 3300 aagtttctct atcttaccat gtattattaa acataattga tgtgttctaa atctagagtg 3360 ttgtcttttg aagaacgctg tatgctcaac tgaatcaatg tatcactgca tctgggaaac 3420 ggttactgaa aacatggctg gcaagacctt tatataatac ggaactgatc aaggaacgac 3480 aagatgctgt agcaattctg cgggtgagtc tttcaacaag ttgtttgact ttgctgctgt 3540 catttctctg tctctcaact agacaataac ttggcatctt ggtttcacat ttgatcattt 3600 ttcatgtctg tttcgctatc catggatctc tcctcagaat tacactattt ccccattatg 3660 ggtgttcaag accatttttg ccactgtttc actggcaaag atgatgtttt cctatgcgtt 3720 caactaacca tctatttcta gaacttattc cctaagatta taaaacttac tctgcttctt 3780 cagcatgtca aggctttcgt ttacactatc catctgacaa tgtattatgg tactgtccct 3840 tccctcaggg tgaaaatctt ccgtactcac tggaattccg gaagtcgttg tccagacttc 3900 cagacatgga acggttgatt gcacgtatgt tttctagcat gtaagggatt agctagattg 3960 agatgttaat tcttacatta tatgtttata ccaaagactt actaaacata tttgttaaac 4020 ttgtgttacg tgttatagtg aagctagtgg aagaaatggc gataaagtgg tgctatatga 4080 agatacagct aagaagcagg tacaggaatt catatcaact ctacgtggtt gtgaaacaat 4140 ggcagaagca tgctcttctc tccgtgctat cttgaagcat gatacatcca ggcggctgct 4200 tcatttacta actcctggta taatcaattt gctccatatt cacattctta tactggcaaa 4260 ttgcacagca tctcatatca tttctctgcc aggtcaaagt cttccaaata tatcatcctc 4320 cataaagtat ttcaaggatg cttttgactg ggtagaagct cacaattctg gacgtgtaat 4380 accccatgaa ggagcagatg aagagtatga ttgtgcctgc aaaacagtag aagaatttga 4440 gtccagtttg aaaaaacatc tgaaagagca acggaaatta ctcggagatg catcagtgag 4500 aattacttca ctattttttt ttactcctta aatggctaat caaccgaggg ttttctgatc 4560 agatctttgg tgctcttttg tcttcttatc cagataaact atgttacagt tggaaaagat 4620 gaatacctct tggaagttcc tgaaagttta agtgggagtg ttcctcatga ttatgaatta 4680 tgctcatcga aaaaggtaaa agttgtacca agtttcacat tctaaagaaa ttggcatttc 4740 gctttcgtca taacaagtcg atagtcttct cgtaattgct gtctgctgat atatttacta 4800 tatagagacc cttaatttta aacatgagat tttcttactt tttactctct ttcagggtgt 4860 ctctcgatat tggactccta ccataaagaa attattaaaa gagctatcac aagcaaaatc 4920 tgaaaaagag tcggccctga agagcatttc acagagattg attggacgtt tctgcgagca 4980 mOR-0444.ST25.txt tcaagaaaaa tggagacaat tggtttctgc aacagctggt atggacaagt tcatgtttta 5040 aaaaaaaaaa attgtttaag gaattttcag catcttcctt cagaatatgt atcttgctta 5100 tccaattcct gttaattact gtcacccagt gttagctttg tgggtcgtcg cttggaccct 5160 tttcgttgtg aacatttgtt gagctagtta gaattgagtt tgatcccaca ctttatagat 5220 tgagttagaa gtaggcatgc agaagaaaat gaatcttagg cagacgtata gttcaatcac 5280 atcttataag caagaggttt cttgggtgga agattgtttt atagaattag gcatgcaaac 5340 aactttgcac ttagaccttt atgtggatac atttttgaca tgaattcttt ctattgcaga 5400 gctggacgtg ttgatcagcc tcgcttttgc aagtgattct tatgaaggag taagatgccg 5460 cccagtaata tctggttcta catctgatgg tgttccacac ttgtctgcca ctggtctagg 5520 gcatccagtt ctaaggggtg attcgttagg cagaggctct tttgtaccaa ataatgtaaa 5580 gataggtggt gctgagaaag ccagtttcat cctcctcaca ggccctaata tgggtggaaa 5640 atcaaccctt cttcgccaag tttgcttggc tgtaatcttg gctcaggtaa gctatcattt 5700 gaaaaaactt tgtaggcaat gggctttgac ccgtttaatt ttgatgaaag aaactcaagc 5760 aatgatgatc ttttcacaga ttggagcaga tgtcccagca gaaacctttg aggtttcgcc 5820 tgttgacaaa atttgtgtcc ggatgggtgc aaaagatcat atcatggcag gacaaagcac 5880 gtttttaaca gaactttcag aaactgcggt aatgttggta agtaatgttc attctgtttg 5940 tcaaattgat tacatgaagc tttctaagat aaatgtgaaa cttgccacag tggttaccct 6000 tttgagagtt ggtcacaggc tttgttaaac tatgcgaatg ccaacaaacg cactgataga 6060 atgttttata ttaataatat gcagacatca gccacccgaa actcgctggt ggtgctagat 6120 gagcttggac gaggaacagc cacatcagat gggcaagcca ttgcgtatgt tgaatcaatt 6180 attgcgtatc atgttttttg ggacttactg ttattgttca ctttatctaa aatatcttaa 6240 ctatttacag ggaatccgta cttgagcact tcatagaaaa ggtgcagtgt agaggattct 6300 tctctactca ttatcatcgt ctctctgtgg attatcaaac caatccaaag gtattgtgaa 6360 aagtgtctgc ttcagtttct gggtttgaaa gacttgagaa ctatcaataa taatctgatt 6420 gtttgtgtac attctgaaac ttgtcaaaaa ccgatcagtc ttgaatattt gtttggatag 6480 gtctcacttt gccatatggc atgtcaaata ggagaaggaa tcggtggagt agaagaagtt 6540 acatttctct atagattgac tcctggtgca tgtcctaaaa gttatggagt taacgttgct 6600 cggttagctg gtaagaacac tgaattctct actccatcac ctctactcag ttaaacagaa 6660 gcagtcactc atcaaattgt tttggtttta atctccatag gtcttccaga ttacgtactc 6720 cagagagccg tgataaaatc ccaagaattc gaggctttgt acggtaaaaa ccatagaaaa 6780 accgatcata aattagcagc aatgataaag cagatcatca gcagtgttgc atcagattct 6840 gattactcag cttcaaagga ctcattgtgt gagctacact ccatggccaa tacatttctc 6900 cggttaacca actaatttaa cagctctacg cctttccggt ttgtcgttct tcttgtaact 6960 ctttaaccaa ggtcaatcca cgagcttcgt cgtgtcaaat actaaaacct gagtcagcct 7020 MOR-0444.ST25.txt gaaactaaac tcctgagtag agactcagtt ttgaggtgtg ggtttagctt ctgagtcttt 7080 <210> 48 <211> 1324 <212> PRT
<213> Arabidopsis thaliana <400> 48 Met Ala Pro Ser Arg Arg Gin Ile Ser Gly Arg Ser Pro Leu Val Asn Gin Gin Arg Gin Ile Thr Ser Phe Phe Gly Lys Ser Ala Ser Ser Ser Ser Ser Pro Ser Pro Ser Pro Ser Pro Ser Leu Ser Asn Lys Lys Thr Pro Lys Ser Asn Asn Pro Asn Pro Lys Ser Pro Ser Pro Ser Pro Ser Pro Pro Lys Lys Thr Pro Lys Leu Asn Pro Asn Pro Ser Ser Asn Leu Pro Ala Arg Ser Pro Ser Pro Gly Pro Asp Thr Pro Ser Pro Val Gin Ser Lys Phe Lys Lys Pro Leu Leu Val Ile Gly Gin Thr Pro Ser Pro Pro Gin Ser Val Val Ile Thr Tyr Gly Asp Glu Val Val Gly Lys Gin Val Arg Val Tyr Trp Pro Leu Asp Lys Lys Trp Tyr Asp Gly Ser Val Thr Phe Tyr Asp Lys Gly Glu Gly Lys His Val Val Glu Tyr Glu Asp Gly Glu Glu Glu Ser Leu Asp Leu Gly Lys Glu Lys Thr Glu Trp val val Gly Glu Lys Ser Gly Asp Arg Phe Asn Arg Leu Lys Arg Gly Ala Ser Ala Leu Arg Lys val Val Thr Asp Ser Asp Asp Asp Val Glu Met Gly Asn Val Glu Glu Asp Lys Ser Asp Gly Asp Asp Ser Ser Asp Glu Asp Trp Gly Lys Asn Val Gly Lys Glu Val Cys Glu Ser Glu Glu Asp MOR-0444.ST25.txt Asp val Glu Leu val Asp Glu Asn Glu met Asp Glu Glu Glu Leu Val Glu Glu Lys Asp Glu Glu Thr Ser Lys Val Asn Arg Val Ser Lys Thr Asp Ser Arg Lys Arg Lys Thr Ser Glu Val Thr Lys Ser Gly Gly Glu Lys Lys Ser Lys Thr Asp Thr Gly Thr Ile Leu Lys Gly Phe Lys Ala Ser Val Val Glu Pro Ala Lys Lys Ile Gly Gin Ala Asp Arg Val Val Lys Gly Leu Glu Asp Asn Val Leu Asp Gly Asp Ala Leu Ala Arg Phe Gly Ala Arg Asp Ser Glu Lys Phe Arg Phe Leu Gly Val Asp Arg Arg Asp Ala Lys Arg Arg Arg Pro Thr Asp Glu Asn Tyr Asp Pro Arg Thr Leu Tyr Leu Pro Pro Asp Phe Val Lys Lys Leu Thr Gly Gly Gin Arg Gin Trp Trp Glu Phe Lys Ala Lys His Met Asp Lys Val Val Phe Phe Lys Met Gly Lys Phe Tyr Glu Leu Phe Glu Met Asp Ala His Val Gly Ala Lys Glu Leu Asp Ile Gin Tyr Met Lys Gly Glu Gin Pro His Cys Gly Phe Pro Glu Lys Asn Phe Ser Val Asn Ile Glu Lys Leu val Arg Lys Gly Tyr Arg Val Leu Val Val Glu Gin Thr Glu Thr Pro Asp Gin Leu Glu Gin Arg Arg Lys Glu Thr Gly Ser Lys Asp Lys Val Val Lys Arg Glu Val Cys Ala Val Val Thr Lys Gly Thr Leu Thr Asp Gly Glu Met Leu Leu Thr Asn Pro Asp Ala Ser Tyr Leu Met Ala Leu Thr Glu . .
MoR-0444.ST25.txt Gly Gly Glu Ser Leu Thr Asn Pro Thr Ala Glu His Asn Phe Gly val Cys Leu val Asp Val Ala Thr Gin Lys Ile Ile Leu Gly Gin Phe Lys Asp Asp Gin Asp Cys Ser Ala Leu Ser Cys Leu Leu Ser Glu met Arg Pro Val Glu Ile Ile Lys Pro Ala Lys Val Leu Ser Tyr Ala Thr Glu Arg Thr Ile Val Arg Gin Thr Arg Asn Pro Leu Val Asn Asn Leu Val Pro Leu Ser Glu Phe Trp Asp Ser Glu Lys Thr Ile Tyr Glu val Gly Ile Ile Tyr Lys Arg Ile Asn Cys Gin Pro Ser Ser Ala Tyr ser Ser Glu Gly Lys Ile Leu Gly Asp Gly Ser Ser Phe Leu Pro Lys met Leu Ser Glu Leu Ala Thr Glu Asp Lys Asn Gly Ser Leu Ala Leu ser Ala Leu Gly Gly Ala Ile Tyr Tyr Leu Arg Gin Ala Phe Leu Asp Glu Ser Leu Leu Arg Phe Ala Lys Phe Glu ser Leu Pro Tyr cys Asp Phe Ser Asn Val Asn Glu Lys Gin His Met Val Leu Asp Ala Ala Ala Leu Glu Asn Leu Glu Ile Phe Glu Asn Ser Arg Asn Gly Gly Tyr Ser Gly Thr Leu Tyr Ala Gin Leu Asn Gin Cys Ile Thr Ala Ser Gly Lys Arg Leu Leu Lys Thr Trp Leu Ala Arg Pro Leu Tyr Asn Thr Glu Leu Ile Lys Glu Arg Gin Asp Ala Val Ala Ile Leu Arg Gly Glu Asn Leu Pro Tyr ser Leu Glu Phe Arg Lys Ser Leu Ser Arg Leu Pro Asp Met Glu Arg moR-0444.sT25.txt Leu Ile Ala Arg met Phe Ser Ser Ile Glu Ala Ser Gly Arg Asn Gly Asp Lys Val Val Leu Tyr Glu Asp Thr Ala Lys Lys Gln Val Gin Glu Phe Ile Ser Thr Leu Arg Gly Cys Glu Thr Met Ala Glu Ala Cys Ser Ser Leu Arg Ala Ile Leu Lys His Asp Thr Ser Arg Arg Leu Leu His Leu Leu Thr Pro Gly Gin Ser Leu Pro Asn Ile Ser Ser ser Ile Lys Tyr Phe Lys Asp Ala Phe Asp Trp val Glu Ala His Asn Ser Gly Arg Val Ile Pro His Glu Gly Ala Asp Glu Glu Tyr Asp Cys Ala Cys Lys Thr Val Glu Glu Phe Glu Ser Ser Leu Lys Lys His Leu Lys Glu Gin Arg Lys Leu Leu Gly AS Ala Ser Ile Asn Tyr Val Thr Val Gly Lys Asp Glu Tyr Leu Leu Glu Val Pro Glu Ser Leu Ser Gly Ser val Pro His Asp Tyr Glu Leu Cys Ser Ser Lys Lys Gly Val Ser Arg Tyr Trp Thr Pro Thr Ile Lys Lys Leu Leu Lys Glu Leu Ser Gin Ala Lys Ser Glu Lys Glu Ser Ala Leu Lys Ser Ile Ser Gin Arg Leu Ile Gly Arg Phe Cys Glu His Gin Glu Lys Trp Arg Gin Leu Val Ser Ala Thr Ala Glu Leu Asp Val Leu Ile Ser Leu Ala Phe Ala Ser Asp Ser Tyr Glu Gly val Arg Cys Arg Pro Val Ile Ser Gly Ser Thr Ser Asp Gly Val Pro His Leu Ser Ala Thr Gly Leu Gly His Pro Val Leu MOR-0444.ST25.txt Arg Gly Asp Ser Leu Gly Arg Gly Ser Phe Val Pro Asn Asn val Lys Ile Gly Gly Ala Glu Lys Ala Ser Phe Ile Leu Leu Thr Gly Pro Asn Met Gly Gly Lys ser Thr Leu Leu Arg Gin Val Cys Leu Ala Val Ile Leu Ala Gin Ile Gly Ala Asp Val Pro Ala Glu Thr Phe Glu Val Ser Pro val Asp Lys Ile Cys Val Arg Met Gly Ala Lys Asp His Ile met Ala Gly Gin Ser Thr Phe Leu Thr Glu Leu Ser Glu Thr Ala Val met Leu Thr Ser Ala Thr Arg Asn Ser Leu Val Val Leu Asp Glu Leu Gly Arg Gly Thr Ala Thr Ser Asp Gly Gin Ala Ile Ala Glu Ser val Leu Glu His Phe Ile Glu Lys Val Gin Cys Arg Gly Phe Phe Ser Thr His Tyr His Arg Leu Ser Val Asp Tyr Gin Thr Asn pro Lys Val Ser Leu Cys His Met Ala Cys Gin Ile Gly Glu Gly Ile Gly Gly Val Glu Glu Val Thr Phe Leu Tyr Arg Leu Thr Pro Gly Ala Cys Pro Lys Ser Tyr Gly Val Asn val Ala Arg Leu Ala Gly Leu Pro Asp Tyr Val Leu Gin Arg Ala val Ile Lys Ser Gin Glu Phe Glu Ala Leu Tyr Gly Lys Asn His Arg Lys Thr Asp His Lys Leu Ala Ala Met Ile Lys Gin Ile Ile Ser Ser Val Ala Ser Asp Ser Asp Tyr Ser Ala Ser Lys Asp Ser MOR-0444.ST25.txt Leu Cys Glu Leu His Ser Met Ala Asn Thr Phe Leu Arg Leu Thr Asn <210> 49 <211> 2501 <212> DNA
<213> Oryza sativa <400> 49 cggcacgaga ttttgcagtc tcctctcctc ctccgctcga gcgagtgagt cccgaccacg 60 tcgctgccct cgcctcaccg ccggccaacc gccgtgacga gagatcgagc agggcggggc 120 atggacgagc cttcgccgcg cggaggtggg tgcgccgggg agccgccccg catccggagg 180 ttggaggagt cggtggtgaa ccgcatcgcg gcgggggagg tgatccagcg gccgtcgtcg 240 gcggtgaagg agctcatcga gaacagcctc gacgctggcg cctccagcgt ctccgttgcg 300 gtgaaggacg gtggcctcaa gctcatccag gtctccgatg acggccatgg catcaggttt 360 gaggatttgg caatattgtg cgaaaggcat actacctcaa agttatctgc atacgaggat 420 ctgcagacca taaaatcgat ggggttcaga ggggaggctt tggctagtat gacttatgtt 480 ggccatgtta ccgtgacaac gataacagaa ggccaattgc acggctacag ggtttcttac 540 agagatggtg taatggagaa tgagcctaag ccttgcgctg cggtgaaagg aactcaagtc 600 atggttgaaa atctatttta caacatggta gcccgcaaga aaacattgca gaactccaat 660 gatgactacc ccaagatcgt agacttcatc agtcggtttg cagtccatca catcaacgtt 720 accttctctt gcagaaagca tggagccaat agagcagatg ttcatagtgc aagtacatcc 780 tcaaggttag atgctatcag gagtgtctat ggggcttctg tcgttcgtga tctcatagaa 840 ataaaggttt catatgagga tgctgcagat tcaatcttca agatggatgg ttacatctca 900 aatgcaaatt atgtggcaaa gaagattaca atgattcttt tcataaatga taggcttgta 960 gactgtactg ctttgaaaag agctattgaa tttgtgtact ctgcaacatt gcctcaagca 1020 tccaaacctt tcatatacat gtccatacat cttccatcag aacacgtgga tgttaatata 1080 cacccaacca agaaagaggt tagccttttg aatcaagagc gtattattga aacaataaga 1140 aatgctattg aggaaaaact gatgaattct aatacaacca ggatattcca aactcaggca 1200 ttaaacttat cagggattgc tcaagctaac ccacaaaagg ataaggtttc tgaggccagt 1260 atgggttctg gaacaaaatc tcaaaaaatt cctgtgagcc aaatggtcag aacagatcca 1320 cgcaatccat ctggaagatt gcacacctac tggcacgggc aatcttcaaa tcttgaaaag 1380 aaatttgatc ttgtatctgt aagaaatgtt gtaagatcaa ggagaaacca aaaagatgct 1440 ggtgatttgt caagccgtca tgagctcctt gtggaaatag attctagctt ccatcctggc 1500 cttttggaca ttgtcaagaa ctgcacatat gttggacttg ccgatgaagc ctttgctttg 1560 , MOR-0444.ST25.txt atacaacaca atacccgctt ataccttgta aatgtggtaa atattagtaa agaacttatg 1620 taccagcaag ctttgtgccg ttttgggaac ttcaatgcta ttcagctcag tgaaccagct 1680 ccacttcagg agttgctggt gatggcactg aaagacgatg aattgatgag tgatgaaaag 1740 gatgatgaga aactggagat tgcagaagta aacactgaga tactaaaaga aaatgctgag 1800 atgattaatg agtacttttc tattcacatt gatcaagatg gcaaattgac aagacttcct 1860 gttgtactgg accagtacac ccctgatatg gaccgtcttc cagaatttgt gttggcttta 1920 ggaaatgatg ttacttggga tgacgagaaa gagtgcttca gaacagtagc ttctgctgta 1980 ggaaacttct atgcacttca tcccccaatc cttccaaatc catctgggaa tggcattcat 2040 ttatacaaga aaaatagaga ttcaatggct gatgaacatg ctgagaatga tctaatatca 2100 gatgaaaatg acgttgatca agaacttctt gcggaagcag aagcagcatg ggcccaacgt 2160 gagtggacca ttcagcatgt cttgtttcca tccatgcgac ttttcctcaa gcccccgaag 2220 tcaatggcaa cagatggaac gtttgtgcag gttgcttcct tggagaaact ctacaagatt 2280 tttgaaaggt gttagctcat aagtgagaaa atgaaggcag agtaagatca tgattcatgg 2340 agtgtttttg aaaatgtgta taatttcacc gtattatgta ctttgatagt gtctgtagaa 2400 actgaagaaa gaaagatggc tttacttctg aattgaaagt taacgatgcc agcaattgta 2460 tattctgatc aaccaaaaaa aaaaaaaaaa aaaaaaaaaa a 2501 <210> 50 <211> 724 <212> PRT
<213> Oryza sativa <400> 50 Met Asp Glu Pro Ser Pro Arg Gly Gly Gly Cys Ala Gly Glu Pro Pro Arg Ile Arg Arg Leu Glu Glu Ser Val Val Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ser Ser Ala Val Lys Glu Leu Ile Glu Asn Ser Leu Asp Ala Gly Ala Ser Ser Val Ser Val Ala val Lys Asp Gly Gly Leu Lys Leu Ile Gin Val Ser Asp Asp Gly His Gly Ile Arg Phe Glu Asp Leu Ala Ile Leu Cys Glu Arg His Thr Thr Ser Lys Leu Ser Ala Tyr Glu Asp Leu Gin Thr Ile Lys Ser met Gly Phe Arg Gly Glu , MOR-0444.ST25.txt Ala Leu Ala Ser met Thr Tyr Val Gly His val Thr val Thr Thr Ile Thr Glu Gly Gin Leu His Gly Tyr Arg Val Ser Tyr Arg Asp Gly Val Met Glu Asn Glu Pro Lys Pro Cys Ala Ala Val Lys Gly Thr Gin Val Met Val Glu Asn Leu Phe Tyr Asn Met Val Ala Arg Lys Lys Thr Leu Gin Asn Ser Asn Asp Asp Tyr Pro Lys Ile Val Asp Phe Ile Ser Arg Phe Ala Val His His Ile Asn Val Thr Phe Ser Cys Arg Lys His Gly Ala Asn Arg Ala Asp val HiS Ser Ala Ser Thr Ser Ser Arg Leu Asp Ala Ile Arg Ser val Tyr Gly Ala Ser Val val Arg Asp Leu Ile Glu Ile Lys Val Ser Tyr Glu Asp Ala Ala Asp Ser Ile Phe Lys Met Asp Gly Tyr Ile ser Asn Ala Asn Tyr Val Ala Lys Lys Ile Thr Met Ile Leu Phe Ile Asn Asp Arg Leu Val Asp Cys Thr Ala Leu Lys Arg Ala Ile Glu Phe Val Tyr Ser Ala Thr Leu Pro Gin Ala Ser Lys Pro Phe Ile Tyr Met Ser Ile His Leu Pro Ser Glu His Val Asp Val Asn Ile His Pro Thr Lys Lys Glu Val Ser Leu Leu Asn Gin Glu Arg Ile Ile Glu Thr Ile Arg Asn Ala Ile Glu Glu Lys Leu Met Asn Ser Asn Thr Thr Arg Ile Phe Gin Thr Gin Ala Leu Asn Leu Ser Gly Ile Ala Gin Ala Asn Pro Gin Lys Asp Lys Val Ser Glu Ala Ser Met Gly Ser Gly mOR-0444.ST25.txt Thr Lys Ser Gin Lys Ile Pro val Ser Gin met val Arg Thr Asp Pro Arg Asn Pro Ser Gly Arg Leu His Thr Tyr Trp His Gly Gin Ser Ser Asn Leu Glu Lys Lys Phe Asp Leu Val Ser val Arg Asn val val Arg Ser Arg Arg Asn Gin Lys Asp Ala Gly Asp Leu Ser Ser Arg His Glu Leu Leu val Glu Ile Asp Ser Ser Phe His Pro Gly Leu Leu AS Ile Val Lys Asn Cys Thr Tyr val Gly Leu Ala Asp Glu Ala Phe Ala Leu Ile Gin His Asn Thr Arg Leu Tyr Leu val Asn val val Asn Ile Ser Lys Glu Leu Met Tyr Gin Gin Ala Leu Cys Arg Phe Gly Asn Phe Asn Ala Ile Gin Leu Ser Glu Pro Ala Pro Leu Gin Glu Leu Leu val met Ala Leu Lys AS Asp Glu Leu met ser Asp Glu Lys Asp AS Glu Lys Leu Glu Ile Ala Glu val Asn Thr Glu Ile Leu Lys Glu Asn Ala Glu met Ile Asn Glu Tyr Phe ser Ile His Ile Asp Gin Asp Gly Lys Leu Thr Arg Leu Pro val val Leu Asp Gin Tyr Thr Pro Asp met AS Arg Leu Pro Glu Phe val Leu Ala Leu Gly Asn Asp val Thr Trp Asp Asp Glu Lys Glu Cys Phe Arg Thr val Ala Ser Ala val Gly Asn Phe Tyr Ala Leu His Pro Pro Ile Leu Pro Asn Pro Ser Gly Asn Gly Ile His Leu Tyr Lys Lys Asn Arg AS Ser met Ala ASp Glu HiS Ala Glu Asn mOR-0444.ST25.txt Asp Leu Ile Ser Asp Glu Asn Asp Val Asp Gin Glu Leu Leu Ala Glu Ala Glu Ala Ala Trp Ala Gin Arg Glu Trp Thr Ile Gln His Val Leu Phe Pro Ser met Arg Leu Phe Leu Lys Pro Pro Lys Ser met Ala Thr Asp Gly Thr Phe Val Gin Val Ala Ser Leu Glu Lys Leu Tyr Lys Ile Phe Glu Arg Cys

Claims

What is claimed is:

1. A method for producing hybridoma cells producing high-affinity antibodies from in vitro immunized immunoglobulin producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein said parental hybridoma cells express a dominant negative allele of a mismatch repair gene operably linked to a promoter;
c) incubating said parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
d) screening antibodies produced from said hypermutated hybridoma cells for binding to said antigen; and e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
thereby producing hybridoma cells producing high-affinity antibodies

2. A method for producing hybridoma cells that produce high titers of antibodies from in vitro immunized immunoglobulin producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein said parental hybridoma cells express a dominant negative allele of a mismatch repair gene operably linked to a promoter;
c) incubating said parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells, d) performing a screen of said hypermutated hybridoma cells for antibodies produced in higher titers than that produced by said parental hybridoma cells;
and e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by said parental hybridoma cells;
thereby producing hybridoma cells that produce high titers of antibodies.

3. A method as claimed in claim 1 or claim 2, further comprising inactivation of said dominant negative allele of said mismatch repair gene, thereby stabilizing the genome of said hypermutated hybridoma.

4. A method as claimed in claim 3, wherein the step of inactivating said dominant negative allele of a mismatch repair gene is carried out by knocking out said dominant negative allele or removing an inducer of said dominant negative allele.

5. A method for producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells, wherein said parental hybridoma cells express a dominant negative allele of a mismatch repair gene operably linked to a promoter;
c) incubating said parental hybridoma cells to allow for mutagenesis, thereby forming hypermutated hybridoma cells;
d) screening antibodies to antigen for antibodies produced from said hypermutated hybridoma cells for binding to said antigen;
e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
f) cloning immunoglobulin genes from selected hypermutated hybridoma cells into a mammalian expression cell;
thereby producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

6. A method as claimed in any one of claims 1 to 5, wherein the dominant negative mismatch repair gene is introduced into said parental hybridoma cells after the fusion of said myeloma cells with said immunoglobulin-producing cells.

7. A method as claimed in any one of claims 1 to 5, wherein said myeloma cells express a dominant negative mismatch repair gene which is also expressed in said parental hybridoma cells.

8. A method for producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;
c) performing a screen for binding of antibodies produced from said parental hybridoma cells to antigen;
d) cloning immunoglobulin genes from parental hybridoma cells that produce antibodies that bind antigen into a mammalian expression cell, wherein said mammalian expression cell expresses a dominant negative allele of a mismatch repair gene;
e) performing a screen for mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental hybridoma cells;
thereby producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized human immunoglobulin-producing cells.

9. A method for producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;

b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;
c) performing a screen for binding of antibodies produced from said parental hybridoma cells to antigen;
d) cloning immunoglobulin genes from parental hybridoma cells that produce antibodies that bind antigen into a parental mammalian expression cell, wherein said parental mammalian expression cell expresses a dominant negative allele of a mismatch repair gene operably linked to a promoter;
e) incubating said parental mammalian expression cell to allow for mutagenesis, thereby forming hypermutated mammalian expression cells;
performing a screen of hypermutated mammalian expression cells for cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental hybridoma cells; and 9) performing a screen of said hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental hybridoma cells for cells that produce higher titers of antibodies than said parental mammalian expression cells;
thereby producing mammalian expression cells that produce high titers of high-affinity antibodies from in vitro immunized immunoglobulin-producing cells.

10. A method as claimed in claim 8 or claim 9, wherein said dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell prior to introduction of said immunoglobulin genes.

11. A method as claimed in claim 8 or claim 9, wherein said dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell after introduction of said immunoglobulin genes.

12. A method as claimed in claim 8 or claim 9, wherein said dominant negative allele of a mismatch repair gene is introduced into said mammalian expression cell simultaneously with said immunoglobulin genes.

13. A method as claimed in any one of claims 1 to 12, wherein said dominant negative allele of a mismatch repair gene comprises a dominant negative allele of a gene selected from PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3, MSH1, or homologs of PMSR genes.

14. A method as claimed in claim 13, wherein said dominant negative allele of a mismatch repair gene comprises a dominant negative allele of a PMS2 gene.

15. A method for producing hybridoma cells producing high-affinity antibodies from in vitro immunized immunoglobulin producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;
c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells;
d) screening antibodies produced from said hypermutated hybridoma cells for binding to said antigen; and e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
thereby producing hybridoma cells producing high-affinity antibodies.

16. A method for producing hybridoma cells that produce high titers of antibodies from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;

c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated hybridoma cells;
d) performing a screen of said hypermutated hybridoma cells for antigen-specific antibodies produced in higher titers than that produced by said parental hybridoma cells; and e) selecting hypermutated hybridoma cells that produce higher titers of antibodies than that produced by said parental hybridoma cells;
thereby producing hybridoma cells that produce high titers of antibodies, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula;
wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O- alkyl, S-alkyl, N- alkyl, O- alkenyl, S- alkenyl, N- alkenyl, O- alkynyl, S- alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups;
wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups.

17. A method for producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;
c) performing a screen for binding of antibodies produced from said parental hybridoma cells to antigen;
d) cloning immunoglobulin genes from parental hybridoma cells that produce antibodies that bind antigen into a mammalian expression cell, thereby forming parental mammalian expression cells;
e) incubating said parental mammalian expression cells in the presence of at least one chemical inhibitor of mismatch repair, thereby forming hypermutated mammalian expression cells;
f) performing a screen for hypermutated mammalian expression cells that secrete antibodies with higher affinity for antigen as compared to antibodies produced from said parental hybridoma cells;
thereby producing mammalian expression cells that produce high affinity antibodies from in vitro immunized immunoglobulin-producing cells.

18. A method as claimed in claim 17 further comprising screening, prior to collection of said antibodies from said hypermutated mammalian expression cells, for hypermutated mammalian expression cells that also produce antibodies in higher titers than said parental hybridoma cells.

19. A method for producing mammalian expression cells that produce high affinity antibodies to a selected antigen from in vitro immunized immunoglobulin-producing cells comprising:
a) combining donor cells comprising immunoglobulin-producing cells with an immunogenic antigen in vitro;
b) fusing said immunoglobulin-producing cells with myeloma cells to form parental hybridoma cells;
c) incubating said parental hybridoma cells in the presence of at least one chemical inhibitor of mismatch repair to form hypermutated hybridoma cells;
d) performing a screen for binding of antigen for antibodies produced from said hypermutated hybridoma cells;
e) selecting hypermutated hybridoma cells that produce antibodies with greater affinity for said antigen than antibodies produced by said parental hybridoma cells;
f) cloning immunoglobulin genes from said hypermutated hybridoma cells into a mammalian expression cell, thereby forming parental mammalian expression cells;
thereby producing mammalian expression cells that produce high-affinity antibodies from in vitro immunized immunoglobulin-producing cells, wherein said chemical inhibitor of mismatch repair is an anthracene having the formula:
wherein R1-R10 are independently hydrogen, hydroxyl, amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O- alkyl, S-alkyl, N- alkyl, O- alkenyl, S- alkenyl, N- alkenyl, O- alkynyl, S- alkynyl, N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO2, an aldehyde group, an ester, an ether, a crown ether, a ketone, an organosulfur compound, an organometallic group, a carboxylic acid, an organosilicon or a carbohydrate that optionally contains one or more alkylated hydroxyl groups;
wherein said heteroalkyl, heteroaryl, and substituted heteroaryl contain at least one heteroatom that is oxygen, sulfur, a metal atom, phosphorus, silicon or nitrogen; and wherein said substituents of said substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heteroaryl are halogen, CN, NO2, lower alkyl, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino; and wherein said amino groups are optionally substituted with an acyl group, or 1 to 3 aryl or lower alkyl groups.

20. A method as claimed in claim 1 or claim 15, further comprising a screen for hypermutated hybridomas that also produce antibodies in higher titres than said parental hybridomas.

21. A method as claimed in claim 15, 16, 19, or 20, further comprising the step of removing said chemical inhibitor following hypermutation, thereby stabilising the genome of said hypermutated hybridoma.

22. A method as claimed in claim 1, or 2, further comprising incubating said parental hybridoma cells with a chemical mutagen.

23. A method as claimed in claim 19, further comprising the steps of:
incubating said mammalian expression cell in the presence of at least one chemical inhibitor of mismatch repair, thereby forming a hypermutated mammalian expression cell; and screening for hypermutated mammalian expression cells that produce higher titres of antibodies than said parental mammalian expression cells.

24. A method as claimed in claim 17, 18, or 23, further comprising removing said chemical inhibitor, thereby stabilising the genome of said hypermutated mammalian expression cells.

25. A method as claimed in claim 2, 9, 16, 20, or 23, wherein said higher titres of said antibodies is at least 1.5-8 fold greater than the titre produced by said parental hybridoma cell.

26. A method as claimed in claim 15, 16, 17, 19, or 23, wherein R1-R10 are independently hydrogen, hydroxyl, methyl ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl, or hydroxybutyl.

27. A method as claimed in claim 16, 19, or 23, wherein the chemical inhibitor is 1,2-dimethylanthracene, 9,10-dimethylanthracene, 7,8-dimethylanthracene, 9,10-diphenylanthracene, 9,10-dihydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, dimethylanthracene-1,2-diol, 9-hydroxymethyl-10-methylanthracene-1,2-diol, 9-hydroxymethyl-10-methylanthracene-3,4-diol, or 9,10-di-m-tolylanthracene.