CA2352532C - Compositions and methods for increasing bone mineralization - Google Patents

Abstract

A novel class or family of TGF-.beta. binding proteins is disclosed. Also disclosed are assays for selecting molecules for increasing bone mineralization and methods for utilizing such molecules.

Description

COMPOSITIONS AND METHODS FOR INCREASING BONE MINERALIZATION
TECHNICAL FIELD
The present invention relates generally to pharmaceutical products and methods and, more specifically, to methods and compositions suitable for increasing the mineral content of bone. Such compositions and methods may be utilized to treat a wide variety of conditions, including for example, osteopenia, osteoporosis, fractures and other disorders in which low bone mineral density are a hallmark of the disease.
BACKGROUND OF THE INVENTION
Two or three distinct phases of changes to bone mass occur over the life of an individual (see Riggs, West .1 Med. /54:63-77, 1991). The first phase occurs in both men and women, and proceeds to attainment of a peak bone mass. This first phase is achieved through linear growth of the endochondral growth plates, and radial growth due to a rate of periosteal apposition. The second phase begins around age 30 for trabecular bone (flat bones such as the vertebrae and pelvis) and about age 40 for cortical bone (e.g., long bones found in the limbs) and continues to old age.
This phase is characterized by slow bone loss, and occurs in both men and women. In women, a third phase of bone loss also occurs, most likely due to postmenopausal estrogen deficiencies. During this phase alone, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular compartment (see Riggs, supra).
Loss of bone mineral content can be caused by a wide variety of conditions, and may result in significant medical problems. For example, osteoporosis is a debilitating disease in humans characterized by marked decreases in skeletal bone mass and mineral density, structural deterioration of bone including degradation of bone microarchitecture and corresponding increases in bone fragility and susceptibility to fracture in afflicted individuals. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone), a condition found in approximately 25 million people in the United States. Another 7-8 million patients in the United States have been diagnosed with clinical osteoporosis (defined as bone mineral content greater than 2.5 standard deviations below that of mature young adult bone). Osteoporosis is one of the most expensive diseases for the health care system, costing tens of billions of dollars annually in the United States. In addition to health care-related costs, long-term residential care and lost working days add to the financial and social costs of this disease. Worldwide approximately 75 million people are at risk for osteoporosis.
The frequency of osteoporosis in the human population increases with age, and among Caucasians is predominant in women (who comprise 80% of the osteoporosis patient pool in the United States). The increased fragility and susceptibility to fracture of skeletal bone in the aged is aggravated by the greater risk of accidental falls in this population. More than 1.5 million osteoporosis-related bone fractures are reported in the United States each year. Fractured hips, wrists, and vertebrae are among the most common injuries associated with osteoporosis. Hip fractures in particular are extremely uncomfortable and expensive for the patient, and for women correlate with high rates of mortality and morbidity.
Although osteoporosis has been defined as an increase in the risk of fracture due to decreased bone mass, none of the presently available treatments for skeletal disorders can substantially increase the bone density of adults.
There is a strong perception among all physicians that drugs are needed which could increase bone density in adults, particularly in the bones of the wrist, spinal column and hip that are at risk in osteopenia and osteoporosis.
Current strategies for the prevention of osteoporosis may offer some benefit to individuals but cannot ensure resolution of the disease. These strategies include moderating physical activity (particularly in weight-bearing activities) with the onset of advanced age, including adequate calcium in the diet, and avoiding consumption of products containing alcohol or tobacco. For patients presenting with clinical osteopenia or osteoporosis, all current therapeutic drugs and strategies are directed to reducing further loss of bone mass by inhibiting the process of bone absorption, a natural component of the bone remodeling process that occurs constitutively.
For example, estrogen is now being prescribed to retard bone loss.
There is, however, some controversy over whether there is any long term benefit to patients and whether there is any effect at all on patients over 75 years old.
Moreover, use of estrogen is believed to increase the risk of breast and endometrial cancer.
High doses of dietary calcium, with or without vitamin D has also been suggested for postmenopausal women. However, high doses of calcium can often have unpleasant gastrointestinal side effects, and serum and urinary calcium levels must be continuously monitored (see Khosla and Rigss, Mayo Clin. Proc. 70:978-982, 1995).

Other therapeutics which have been suggested include calcitonin, bisphosphonates, anabolic steroids and sodium fluoride. Such therapeutics however, have undesirable side effects (e.g., calcitonin and steroids may cause nausea and provoke an immune reaction, bisphosphonates and sodium fluoride may inhibit repair of fractures, even though bone density increases modestly) that may prevent their usage (see Khosla and Rigss, supra).
No currently practiced therapeutic strategy involves a drug that stimulates or enhances the growth of new bone mass. The present invention provides compositions and methods which can be utilized to increase bone mineralization, and thus may be utilized to treat a wide variety of conditions where it is desired to increase bone mass. Further, the present invention provides other, related advantages.
SUMMARY OF THE INVENTION
As noted above, the present invention provides a novel class or family of TGF-beta binding-proteins, as well as assays for selecting compounds which increase bone mineral content and bone mineral density, compounds which increase bone mineral content and bone mineral density and methods for utilizing such compounds in the treatment or prevention of a wide variety of conditions.
Within one aspect of the present invention, isolated nucleic acid = molecules are provided, wherein said nucleic acid molecules are selected from the group consisting of: (a) an isolated nucleic acid molecule comprising sequence ID Nos.
1, 5, 7, 9, 11, 13, or, 15, or complementary sequence thereof; (b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecule of (a) under conditions of high stringency; and (c) an isolated nucleic acid that encodes a TGF-beta binding-protein according to (a) or (b). Within related aspects of the present invention, isolated nucleic acid molecules are provided based upon hybridization to only a portion of one of the above-identified sequences (e.g., for (a) hybridization may be to a probe of at least 20, 25, 50, or 100 nucleotides selected from nucleotides 156 to 539 or 555 to 687 of Sequence ID No. 1). As should be readily evident, the necessary stringency to be utilized for hybridization may vary based upon the size of the probe. For example, for a 25-mer probe high stringency conditions could include: 60 mM Tris pH
8.0, 2 mM EDTA, 5x Denhardt's, 6x SSC, 0.1% (w/v) N-laurylsarcosine, 0.5% (w/v) NP-40 (nonidet P-40) overnight at 45 degrees C, followed by two washes with with 0.2x SSC
/ 0.1% SDS at 45-50 degrees. For a 100-mer probe under low stringency conditions, suitable conditions might include the following: 5x SSPE, 5x Denhardt's, and 0.5%
SDS overnight at 42-50 degrees, followed by two washes with 2x SSPE (or 2x SSC) /0.1% SDS at 42-50 degrees.
Within related aspects of the present invention, isolated nucleic acid molecules are provided which have homology to Sequence ID Nos. 1, 5, 7, 9, 11, 13, or 15, at a 50%, 60%, 75%, 80%, 90%, 95%, or 98% level of homology utilizing a Wilbur-Lipman algorithm. Representative examples of such isolated molecules include, for example, nucleic acid molecules which encode a protein comprising Sequence ID NOs. 2, 6, 10, 12, 14, or 16, or have homology to these sequences at a level of 50%, 60%, 75%, 80%, 90%, 95%, or 98% level of homology utilizing a Lipman-Pearson algorithm.
Isolated nucleic acid molecules are typically less than 100kb in size, and, within certain embodiments, less than 50kb, 25kb, 10kb, or even 5kb in size.
Further, isolated nucleic acid molecules, within other embodiments, do not exist in a "library"
of other unrelated nucleic acid molecules (e.g., a subclone BAC such as described in GenBank Accession No. AC003098 and EM13 No. AQ171546). However, isolated nucleic acid molecules can be found in libraries of related molecules (e.g., for shuffling, such as is described in U.S. Patent Nos. 5,837,458; 5,830,721; and 5,811,238). Finally, isolated nucleic acid molecules as described herein do not include nucleic acid molecules which encode Dan, Cerberus, Gremlin, or SCGF (U.S.
Patent No. 5,780,263).
Also provided by the present invention are cloning vectors which contain the above-noted nucleic acid molecules, and expression vectors which comprise a promoter (e.g., a regulatory sequence) operably linked to one of the above-noted nucleic acid molecules. Representative examples of suitable promoters include tissue-specific promoters, and viral -- based promoters (e.g., CMV-based promoters such as CMV I-E, SV40 early promoter, and MuLV LTR). Expression vectors may also be based upon, or derived from viruses (e.g., a "viral vector"). Representative examples of viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors and retroviral vectors. Also provided are host cells containing or comprising any of above-noted vectors (including for example, host cells of human, monkey, dog, rat, or mouse origin).
Within other aspects of the present invention, methods of producing TGF-beta binding-proteins are provided, comprising the step of culturing the aforementioned host cell containing vector under conditions and for a time sufficient to produce the TGF-beta binding protein. Within further embodiments, the protein produced by this method may be further purified (e.g., by column chromatography, affinity purification, and the like). Hence, isolated proteins which are encoded by the above-noted nucleic acid molecules (e.g., Sequence ID NOs. 2, 4, 6, 8, 10, 12, 14, or 16) may be readily produced given the disclosure of the subject application.
It should also be noted that the aforementioned proteins, or fragments thereof, may be produced as fusion proteins. For example, within one aspect fusion 5 proteins are provided comprising a first polypeptide segment comprising a TGF-beta binding-protein encoded by a nucleic acid molecule as described above, or a portion thereof of at least 10, 20, 30, 50, or 100 amino acids in length, and a second polypeptide segment comprising a non-TGF-beta binding-protein. Within certain embodiments, the second polypeptide may be a tag suitable for purification or recognition (e.g., a polypeptide comprising multiple anionic amino acid residues - see U.S. Patent No. 4,851,341), a marker (e.g., green fluorescent protein, or alkaline phosphatase), or a toxic molecule (e.g., ricin).
Within another aspect of the present invention, antibodies are provided which are capable of specifically binding the above-described class of TGF-beta binding proteins (e.g., human BEER). Within various embodiments, the antibody may be a polyclonal antibody, or a monoclonal antibody (e.g, of human or murine origin).
Within further embodiments, the antibody is a fragment of an antibody which retains the binding characteristics of a whole antibody (e.g., an F(ab')2, F(ab),, Fab', Fab, or Fv fragment, or even a CDR). Also provided are hybridomas and other cells which are capable of producing or expressing the aforementioned antibodies.
Within related aspects of the invention, methods are provided detecting a TGF-beta binding protein, comprising the steps of incubating an antibody as described above under conditions and for a time sufficient to permit said antibody to bind to a TGF-beta binding protein, and detecting the binding. Within various embodiments the antibody may be bound to a solid support to facilitate washing or separation, and/or labeled. (e.g., with a marker selected from the group consisting of enzymes, fluorescent proteins, and radioisotopes).
Within other aspects of the present invention, isolated oligonucleotides are provided which hybridize to a nucleic acid molecule according to Sequence ID
NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, or 18 or the complement thereto, under conditions of high stringency. Within further embodiments, the oligonucleotide may be found in the sequence which encodes Sequence ID Nos. 2, 4, 6, 8, 10, 12, 14, or 16. Within certain embodiments, the oligonucleotide is at least 15, 20, 30, 50, or 100 nucleotides in length. Within further embodiments, the oligonucleotide is labeled with another molecule (e.g., an enzyme, fluorescent molecule, or radioisotope). Also provided are primers which are capable of specifically amplifying all or a portion of the above-_WO 00/32773 PCT/US99/27990 mentioned nucleic acid molecules which encode TGF-beta binding-proteins. As utilized herein, the term "specifically amplifying" should be understood to refer to primers which amplify the aforementioned TGF-beta binding-proteins, and not other TGF-beta binding proteins such as Dan, Cerberus, Gremlin, or SCGF (U.S. Patent No.
5,780,263).
Within related aspects of the present invention, methods are provided for detecting a nucleic acid molecule which encodes a TGF-beta binding protein, comprising the steps of incubating an oligonucleotide as described above under conditions of high stringency, and detecting hybridization of said oligonucleotide.
Within certain embodiments, the oligonucleotide may be labeled and/or bound to a solid support.
Within other aspects of the present invention, ribozymes are provided which are capable of cleaving RNA which encodes one of the above-mentioned TGF-beta binding-proteins (e.g., Sequence ID NOs, 2, 6, 8, 10, 12, 14, or 16).
Such ribozymes may be composed of DNA, RNA (including 2'-0-methyl ribonucleic acids), nucleic acid analogs (e.g., nucleic acids having phosphorothioate linkages) or mixtures thereof. Also provided are nucleic acid molecules (e.g., DNA or cDNA) which encode these ribozymes, and vectors which are capable of expressing or producing the ribozymes. Representative examples of vectors include plasmids, retrotransposons, cosmids, and viral-based vectors (e.g., viral vectors generated at least in part from a retrovirus, adenovirus, or, adeno-associated virus). Also provided are host cells (e.g., human, dog, rat, or mouse cells) which contain these vectors. In certain embodiments, the host cell may be stably transformed with the vector.
Within further aspects of the invention, methods are provided for producing ribozymes either synthetically, or by in vitro or in vivo transcription. Within further embodiments, the ribozymes so produced may be further purified and /
or formulated into pharmaceutical compositions (e.g., the ribozyme or nucleic acid molecule encoding the ribozyme along with a pharmaceutically acceptable carrier or diluent). Similarly, the antisense oligonucleotides and antibodies or other selected molecules described herein may be formulated into pharmaceutical compositions.
Within other aspects of the present invention, antisense oligonucleotides are provided comprising a nucleic acid molecule which hybridizes to a nucleic acid molecule according to Sequence ID NOs. 1, 3, 5, 7, 9, 11, 13, or 15, or the complement thereto, and wherein said oligonucleotide inhibits the expression of TGF-beta binding protein as described herein (e.g., human BEER). Within various embodiments, the oligonucleotide is 15, 20, 25, 30, 35, 40, or 50 nucleotides in length.
Preferably, the oligonucleotide is less than 100, 75, or 60 nucleotides in length. As should be readily evident, the oligonucleotide may be comprised of one or more nucleic acid analogs, ribonucleic acids, or deoxyribonucleic acids. Further, the oligonucleotide may be modified by one or more linkages, including for example, covalent linkage such as a phosphorothioate linkage, a phosphotriester linkage, a methyl phosphonate linkage, a methylene(methylimino) linkage, a morpholino linkage, an amide linkage, a polyamide linkage, a short chain alkyl intersugar linkage, a cycloalkyl intersugar linkage, a short chain heteroatomic intersugar linkage and a heterocyclic intersugar linkage.
One representative example of a chimeric. oligonucleotide is provied in U.S.
Patent No.
5,989,912.
Within yet another aspect of the present invention, methods are provided for increasing bone mineralization, comprising introducing into a warm-blooded animal an effective amount of the ribozyme as described above. Within related aspects, such methods comprise the step of introducing into a patient an effective amount of the nucleic acid molecule or vector as described herein which is capable of producing the desired ribozyme, under conditions favoring transcription of the nucleic acid molecule to produce the ribozyme.
Within other aspects of the invention transgenic, non-human animals are provided. Within one embodiment a transgenic animal is provided whose germ cells and somatic cells contain a nucleic acid molecule encoding a TGF-beta binding-protein as described above which is operably linked to a promoter effective for the expression of the gene, the gene being introduced into the animal, or an ancestor of the animal, at an embryonic stage, with the proviso that said animal is not a human. Within other embodiments, transgenic knockout animals are provided, comprising an animal whose germ cells and somatic cells comprise a disruption of at least one allele of an endogenous nucleic acid molecule which hybridizes to a nucleic acid molecule which encodes a TGF-binding protein as described herein, wherein the disruption prevents transcription of messenger RNA from said allele as compared to an animal without the disruption, with the proviso that the animal is not a human. Within various embodiments, the disruption is a nucleic acid deletion, substitution, or, insertion.
Within other embodiments the transgenic animal is a mouse, rat, sheep, pig, or dog.
Within further aspects of the invention, kits are provided for the detection of TGF-beta binding-protein gene expression, comprising a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15; (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of (a); (c) a nucleic acid molecule that is a fragment of (a) or (b) of at least 15, 2030, 50, 75, or, 100 nucleotides in length. Also provided are kits for the detection of a TGF-beta binding-protein which comprise a container that comprise one of the TGF-beta binding protein antibodies described herein.
For example, within one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) mixing one or more candidate molecules with TGF-beta-binding-protein encoded by the nucleic acid molecule according to claim 1 and a selected member of the TGF-beta family of proteins (e.g., BlVIP
5 or 6), (b) determining whether the candidate molecule alters the signaling of the TGF-beta family member, or alters the binding of the TGF-beta binding-protein to the TGF-beta family member. Within certain embodiments, the molecule alters the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation.
Within this aspect of the present invention, the candidate molecule(s) may alter signaling or binding by, for example, either decreasing (e.g., inhibiting), or increasing (e.g., enhancing) signaling or binding.
Within yet another aspect, methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the step of determining whether a selected molecule inhibits the binding of TGF-beta binding-protein to bone, or an analogue thereof. Representative examples of bone or analogues thereof include hydroxyapatite and primary human bone samples obtained via biopsy.
Within certain embodiments of the above-recited methods, the selected molecule is contained within a mixture of molecules and the methods may further comprise the step of isolating one or more molecules which are functional within the assay. Within yet other embodiments, TGF-beta family of proteins is bound to a solid support and the binding of TGF-beta binding-protein is measured or TGF-beta binding-protein are bound to a solid support and the binding of TGF-beta proteins are measured.
Utilizing methods such as those described above, a wide variety of molecules may be assayed for their ability to increase bone mineral content by inhibiting the binding of the TGF-beta binding-protein to the TGF-beta family of proteins. Representative examples of such molecules include proteins or peptides, organic molecules, and nucleic acid molecules.
Within other related aspects of the invention, methods are provided for increasing bone mineral content in a warm-blooded animal, comprising the step of administering to a warm-blooded animal a therapeutically effective amount of a molecule identified from the assays recited herein. Within another aspect, methods are provided for increasing bone mineral content in a warm-blooded animal, comprising the step of administering to a warm-blooded animal a therapeutically effective amount of a molecule which inhibits the binding of the TGF-beta binding-protein to the TGF-beta super-family of proteins, including bone morphogenic proteins (BMPs).
Representative examples of suitable molecules include antisense molecules, ribozymes, ribozyme genes, and antibodies (e.g., a humanized antibody) which specifically recognize and alter the activity of the TGF-beta binding-protein.
Within another aspect of the present invention, methods are provided for increasing bone mineral content in a warm-blooded animal, comprising the steps of (a) introducing into cells which home to the bone a vector which directs the expression of a molecule which inhibits the binding of the TGF-beta binding-protein to the TGF-beta family of proteins and bone morphogenic proteins (BIWPs), and (b) administering the vector-containing cells to a warm-blooded animal. As utilized herein, it should be understood that cells "home to bone" if they localize within the bone matrix after peripheral administration. Within one embodiment, such methods further comprise, prior to the step of introducing, isolating cells from the marrow of bone which home to the bone. Within a further embodiment, the cells which home to bone are selected from the group consisting of CD34+ cells and osteoblasts.
Within other aspects of the present invention, molecules are provided (preferably isolated) which inhibit the binding of the TGF-beta binding-protein to the TGF-beta super-family of proteins.
Within further embodiments, the molecules may be provided as a composition, and can further comprise an inhibitor of bone resorption.
Representative examples of such inhibitors include calcitonin, estrogen, a bisphosphonate, a growth factor having anti-resorptive activity and tamoxifen.
Representative examples of molecules which may be utilized in the afore-mentioned therapeutic contexts include, e.g., ribozymes, ribozyme genes, antisense molecules, and/or antibodies (e.g., humanized antibodies). Such molecules may depending upon their selection, used to alter, antagonize, or agonize the signalling or binding of a TGF-beta binding-protein family member as described herein Within various embodiments of the invention, the above-described molecules and methods of treatment or prevention may be utilized on conditions such as osteoporosis, osteomalasia, periodontal disease, scurvy, Cushing's Disease, bone fracture and conditions due to limb immobilization and steroid usage.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic. illustration comparing the amino acid sequence of Human Dan; Human Gremlin; Human Cerberus and Human Beer. Arrows indicate the Cysteine backbone.
ia Figure 2 summarizes the results obtained from surveying a variety of human tissues for the expression of a TGF-beta binding-protein gene, specifically, the Human Beer gene. A semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) procedure was used to amplify a portion of the gene from first-strand cDNA synthesized from total RNA (described in more detail in EXAMPLE
2A).
Figure 3 summarizes the results obtained from RNA in situ hybridization of mouse embryo sections, using a cRNA probe that is complementary to the mouse Beer transcript (described in more detail in EXAMPLE 2B). Panel A is a transverse section of 10.5 dpc embryo. Panel B is a sagittal section of 12.5 dpc embryo and panels C and D are sagittal sections of 15.5 dpc embryos.
Figure 4 illustrates, by western blot analysis, the specificity of three-different polyclonal antibodies for their respective antigens (described in more detail in EXAMPLE 4). Figure 4A shows specific reactivity of an anti-H. Beer antibody for H.
Beer antigen, but not H. Dan or H. Gremlin. Figure 4B shows reactivity of an anti-H.
Gremlin antibody for H. Gremlin antigen, but not H. Beer or H. Dan. Figure 4C
shows .
reactivity of an anti-H. Dan antibody for H. Dan, but not H. Beer or H.
Gremlin.
Figure 5 illustrates, by western blot analysis, the selectivity of the TGF-beta binding-protein, Beer, for BMP-5 and BMP-6, but not BMP-4 (described in more detail in EXAMPLE 5).
Figure 6 demonstrates that the ionic interaction between the TGF-beta =
binding-protein, Beer, and BM13-5 has a dissociation constant in the 15-30 TIM
range.

DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Prior to setting forth the invention in detail, it may be helpful to an understanding thereof to set forth definitions of certain terms and to list and to define .. the abbreviations that will be used hereinafter.
"Molecule" should be understood to include proteins or peptides (e.g., antibodies, recombinant binding partners, peptides with a desired binding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acid molecules, and nucleic acid analogues such as PNA); and organic or inorganic compounds.
"TGF-beta" should be understood to include any known or novel member of the TGF-beta super-family, which also includes bone morphogenic proteins (BMPs).
"TGF-beta receptor" should be understood to refer to the receptor specific for a particular member of the TGF-beta super-family (including bone .. morphogenic proteins (BMPs)).
"TGF-beta binding-protein" should be understood to refer to a protein with specific binding affinity for a particular member or subset of members of the TGF-beta super-family (including bone morphogenic proteins (BMPs)). Specific examples of TGF-beta binding-proteins include proteins encoded by Sequence ID
Nos.
.. 1, 5, 7, 9, 11, 13, and 15.
Inhibiting the "binding of the TGF-beta binding-protein to the TGF-beta family of proteins and bone morphogenic proteins (BMPs)" should be understood to refer to molecules which allow the activation of TGF-beta or bone morphogenic proteins (BMPs), or allow the binding of TGF-beta family members including bone .. morphogenic proteins (BMPs) to their respective receptors, by removing or preventing TGF-beta from binding to TGF-binding-protein. Such inhibition may be accomplished, for example, by molecules which inhibit the binding of the TGF-beta binding-protein to specific members of theTGF-beta super-family.
"Vector" refers to an assembly which is capable of directing the expression of desired protein. The vector must include transcriptional promoter elements which are operably linked to the gene(s) of interest. The vector may be composed of either deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (e.g., a DNA-RNA chimeric). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may also be incorporated into the vectors described herein.
An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a TGF-binding protein that has been separated from the genomic DNA of a eukaryotic cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. The isolated nucleic acid molecule may be genomic DNA, cDNA, RNA, or composed at least in part of nucleic acid analogs.
An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Within certain embodiments, a particular protein preparation contains an isolated polypeptide if it appears nominally as a single band on SDS-PAGE gel with Coomassie Blue staining. "Isolated" when referring to organic molecules means that the compounds are greater than 90 percent pure utilizing methods which are well known in the art (e.g., NMR., melting point).
"Sclerosteosis" Sclerosteosis is a term that was applied by Hansen (1967) (Hansen, H. G., Sklerosteose.ln: Opitz, H.; Schmid, F., Handbuch der Kinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351-355) to a disorder similar to van Buchem hyperostosis corticalis generalisata but possibly differing in radiologic appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases. The jaw has an unusually square appearance in this condition.
"Humanized antibodies" are recombinant proteins in which murine complementary determining regions of monoclonal antibodies have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
As used herein, an "antibody fragment" is a portion of an antibody such as F(ab'),, F(ab)2, Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-TGF-beta binding-protein monoclonal antibody fragment binds with an epitope of TGF-beta binding-protein.
The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the õ

light chain variable region, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("sPv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervari ab le region.
A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, enzymes, and other marker moieties.
As used herein, an "immunoconjugate" is a molecule comprising an anti-TGF-beta binding-protein antibody, or an antibody fragment, and a detectable label. An immunoconjugate has roughly the same, or only slightly reduced, ability to bind TGF-beta binding-protein after conjugation as before conjugation.
Abbreviations: TGF-beta ¨ "Transforming Growth Factor-beta", TGF-¨ "Transforming Growth Factor-beta binding-protein" (one representative TGF-bBP is designated "H. Beer"); BMP ¨ "bone morphogenic protein"; PCR ¨
"polymerase chain reaction"; RT-PCR PCR process in which RNA is first transcribed into DNA at the first step using reverse transcriptase (RT); cDNA - any DNA
made by copying an RNA sequence into DNA form.
As noted above, the present invention provides a novel class of TGF-beta binding-proteins, as well as methods and compositions for increasing bone mineral content in warm-blooded animals. Briefly, the present inventions are based upon the unexpected discovery that a mutation in the gene which encodes a novel member of the TGF-beta binding-protein family results in a rare condition (sclerosteosis) characterized by bone mineral contents which are one- to four-fold higher than in normal individuals.
Thus, as discussed in more detail below this discovery has led to the development of assays which may be utilized to select molecules which inhibit the binding of the TGF-beta binding-protein to the TGF-beta family of proteins and bone morphogenic proteins (BMPs), and methods of utilizing such molecules for increasing the bone mineral content of warm-blooded animals (including for example, humans).
DISCUSSION OF THE DISEASE KNOWN AS SCLEROSTEOSIS
Sclerosteosis is a term that was applied by Hansen (1967) (Hansen, H.
G., Sklerosteose.In: Opitz, H.; Schmid, F., Handbuch der Kinderheilkunde.
Berlin:
Springer (pub.) 6 1967. Pp. 351-355) to a disorder similar to van Buchem hyperostosis corticalis generalisata but possibly differing in radiologic appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases.
Sclerosteosis is now known to be an autosomal semi-dominant disorder which is characterized by widely disseminated sclerotic lesions of the bone in the adult.
The condition is progressive. Sclerosteosis also has a developmental aspect which is associated with syndactyly (two or more fingers are fused together). The Sclerosteosis Syndrome is associated with large stature and many affected individuals attain a height of six feet or more. The bone mineral content of homozygotes can be 1 to 6 fold over normal individuals and bone mineral density can be 1 to 4 fold above normal values (e.g., from unaffected siblings).
The Sclerosteosis Syndrome occurs primarily in Afrikaaners of Dutch descent in South Africa. Approximately 1/140 individuals in the Afrikaaner population are carriers of the mutated gene (heterozygotes). The mutation shows 100%
penetrance. There are anecdotal reports of increased of bone mineral density in heterozygotes with no associated pathologies (syndactyly or skull overgrowth).
It appears at the present time that there is no abnormality of the pituitary-hypothalamus axis in Sclerosteosis. In particular, there appears to be no over-production of growth hormone and cortisone. In addition, sex hormone levels are normal in affected individuals. However, bone turnover markers (osteoblast specific alkaline phosphatase, osteocalcin, type 1 procollagen C' propeptide (PICP), and total alkaline phosphatase; (see Cornier, C., Curr. Opin. in Rheu. 7:243, 1995) indicate that there is hyperosteoblastic activity associated with the disease but that there is normal to slightly decreased osteoclast activity as measured by markers of bone resorption (pyridinoline, deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant acid phosphatases and galactosyl hydroxylysine (see Cornier, supra)).
Sclerosteosis is characterized by the continual deposition of bone throughout the skeleton during the lifetime of the affected individuals. In homozygotes the continual deposition of bone mineral leads to an overgrowth of bone in areas of the skeleton where there is an absence of mechanoreceptors (skull, jaw, cranium).
In homozygotes with Sclerosteosis, the overgrowth of the bones of the skull leads to cranial compression and eventually to death due to excessive hydrostatic pressure on the brain stern. In all other parts of the skeleton there is a generalized and diffuse sclerosis. Cortical areas of the long bones are greatly thickened resulting in a substantial increase in bone strength. Trabecular connections are increased in thickness which in turn increases the strength of the trabecular bone. Sclerotic bones appear unusually opaque to x-rays.
As described in more detail in Example 1, the rare genetic mutation that is responsible for the Sclerosteosis syndrome has been localized to the region of human 5 chromosome 17 that encodes a novel member of the TGF-beta binding-protein family (one representative example of which is designated "H. Beer"). As described in more detail below, based upon this discovery, the mechanism of bone mineralization is more fully understood, allowing the development of assays for molecules which increase bone mineralization, and use of such molecules to increase bone mineral content, and in 10 the treatment or prevention of a wide number of diseases.
TGF-BETA SUPER-FAMILY
The Transforming Growth Factor-beta (TGF:beta) super-family contains a variety of growth factors that share common sequence elements and structural motifs (at both the secondary and tertiary levels). This protein family is known to exert a wide 15 spectrum of biological responses on a large variety of cell types. Many of them have important functions during the embryonal development in pattern formation and tissue specification; in adults they are involved, e.g., in wound healing and bone repair and bone remodeling, and in the modulation of the immune system. In addition to the three TGF-beta's, the super-family includes the Bone Morphogenic Proteins (BMPs), Activins, Inhibins, Growth and Differentiation Factors (GDFs), and Glial-Derived Neurotrophic Factors (GDNFs). Primary classification is established through general sequence features that bin a specific protein into a general sub-family.
Additional stratification within the sub-family is possible due to stricter sequence conservation between members of the smaller group. In certain instances, such as with BMP-5, BMP-6 and BMP-7, this can be as high as 75 percent amino acid homology between members of the smaller group. This level of identity enables a single representative sequence to illustrate the key biochemical elements of the sub-group that separates it from other members of the larger family.
TGF-beta signals by inducing the formation of hetero-oligomeric complexes of type I and type II receptors. The crystal structure of TGF-beta2 has been determined. The general fold of the TGF-beta2 monomer contains a stable, compact, cysteine knotlike structure formed by three disulphide bridges. Dimerization, stabilized by one disulphide bridge, is antiparallel.
TGF-beta family members initiate their cellular action by binding to receptors with intrinsic serine/threonine kinase activity. This receptor family consists _ _ of two subfamilies, denoted type I and type II receptors. Each member of the TGF-beta family binds to a characteristic combination of type I and type II receptors, both of which are needed for signaling. In the current model for TGF-beta receptor activation, TGF-beta first binds to the type II receptor (TbR-II), which occurs in the cell membrane in an oligomeric form with activated kinase. Thereafter, the type I
receptor (TbR-I), which can not bind ligand in the absence of TbR-II, is recruited into the complex. TbR-II then phosphorylates TbR-I predominantly in a domain rich in glycine and serine residues (GS domain) in the juxtamembrane region, and thereby activates TbR-I.
Thus far seven type I receptors and five type II receptors have been identified.
BONE MORPHOGENIC PROTEINS (BMPs) ARE KEY REGULATORY PROTEINS IN
DETERMINING BONE MINERAL DENSITY IN HUMANS
A major advance in the understanding of bone formation was the identification of the bone morphogenic proteins (BMPs), also known as osteogenic proteins (OPs), which regulate cartilage and bone differentiation in vivo.
BMPs/OPs induce endochondral bone differentiation through a cascade of events which include formation of cartilage, hypertrophy and calcification of the cartilage, vascular invasion, differentiation of osteoblasts, and formation of bone. As described above, the BMPs/OPs (BIVFP 2-14, and osteogenic protein 1 and -2, OP-1 and OP-2) are members of the TGF-beta super-family. The striking evolutionary conservation between members the BMP/OP sub-family suggests that they are critical in the normal development and function of animals. Moreover, the presence of multiple forms of BMPs/OPs raises an important question about the biological relevance of this apparent redundancy. In addition to postfetal chondrogenesis and osteogenesis, the BMPs/OPs play multiple roles in skeletogenesis (including the development of craniofacial and dental tissues) and in embryonic development and organogenesis of parenchymatous organs, including the kidney. It is now understood that nature relies on common (and few) molecular mechanisms tailored to provide the emergence of specialized tissues and organs. The BMP/OP super-family is an elegant example of nature parsimony in programming multiple specialized functions deploying molecular isoforms with minor variation in amino acid motifs within highly conserved carboxy-terminal regions.
BMP ANTAGONISM
The BMP and Activin sub-families are subject to significant post-_ translational regulation. An intricate extracellular control system exists, whereby a high affinity antagonist is synthesized and exported, and subsequently complexes selectively with BMPs or activins to disrupt their biological activity (W.C.
Smith (1999) TIG 15(1) 3-6). A number of these natural antagonists have been identified, and 3 based on sequence divergence appear to have evolved independently due to the lack of primary sequence conservation. There has been no structural work to date on this class of proteins. Studies of these antagonists has highlighted a distinct preference for interacting and neutralizing BMP-2 and BMP-4. Furthermore, the mechanism of inhibition seems to differ for the different antagonists (S. lemura et al.
(1998) Proc Nail Acad Sci USA 95 9337-9342).
NOVEL TGF -BETA BENDING-PROTEINS
1. Background re: TGF-beta binding-proteins As noted above, the present invention provides a novel class of TGF-beta binding-proteins that possess a nearly identical cysteine (disulfide) scaffold when compared to Human DAN, Human Gremlin, and Human Cerberus, and SCGF (U.S.
Patent No. 5,780,263) but almost no homology at the nucleotide level (for background information, see generally Hsu, D.R., Economides, A.N., Wang, X., Eimon, P.M., Harland, R.M., "The Xenopus Dorsalizing Factor Gremlin Identifies a Novel Family of Secreted Proteins that Antagonize BM? Activities," Molecular Cell 1:673-683, 1998).
One representative example of the novel class of TGF-beta binding-proteins is disclosed in Sequence ID Nos. 1, 5, 9, 11, 13, and 15.
Representative members of this class of binding proteins should also be understood to include variants of the TGF-beta binding-protein (e.g., Sequence ID Nos. 5 and 7). As utilized herein, a "TGF-beta binding-protein variant gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID
Nos: 2, 10, 12, 14 or 16. Such variants include naturally-occurring polymorphisms or allelic variants of TGF-beta binding-protein genes, as well as synthetic genes that contain conservative amino acid substitutions of these amino acid sequences.
Additional variant forms of a TGF-beta binding-protein gene are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein.
TGF-beta binding-protein variant genes can be identified by determining whether the genes hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ
ID Nos:
1, 5, 7, 9, 11, 13, or 15 under stringent conditions. In addition, TGF-beta binding-protein variant genes should encode a protein having a cysteine backbone.
¨ ¨

As an alternative, TGF-beta binding-protein variant genes can be identified by sequence comparison. As used herein, two amino acid sequences have "100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing two nucleotide or amino acid sequences by determining optimal alignment are well-known to those of skill in the art (see, for example, Peruski and Peruski, The Internet and the New Biology: Tools for Genornic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins," in Methods in Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.), Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc. 1998)).
A variant TGF-beta binding-protein should have at least a 50% amino acid sequence identity to SEQ ID NOs: 2, 6, 10, 12, 14 or 16 and preferably, greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity. Alternatively, TGF-beta binding-protein variants can be identified by having at least a 70% nucleotide sequence identity to= SEQ ID NOs: 1, 5, 9, 11, 13 or 15. Moreover, the present invention contemplates TGF-beta binding-protein gene variants having greater than 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1. Regardless of the particular method used to identify a TGF-beta binding-protein variant gene or variant TGF-beta binding-protein, a variant TGF-beta binding-protein or a polypeptide encoded by a variant TGF-beta binding-protein gene can be functionally characterized by, for example, its ability to bind to and/or inhibit the signaling of a selected member of the TGF-beta family of proteins, or by its ability to bind specifically to an anti-TGF-beta binding-protein antibody.
The present invention includes functional fragments of TGF-beta binding-protein genes. Within the context of this invention, a "functional fragment" of a TGF-beta binding-protein gene refers to a nucleic acid molecule that encodes a portion of a TGF-beta binding-protein polypeptide which either (1) possesses the above-noted function activity, or (2) specifically binds with an anti-TGF-beta binding-protein antibody. For example, a functional fragment of a TGF-beta binding-protein gene described herein comprises a portion of the nucleotide sequence of SEQ ID
Nos:

1, 5, 9, 11, 13, or 15.
2. Isolation of the TGF-beta binding-protein gene DNA molecules encoding a binding-protein gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon, for example, SEQ ID NO:1.
For example, the first step in the preparation of a cDNA library is to isolate RNA using methods well-known to those of skill in the art. In general, RNA
isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants. For example, total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and Separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)1; Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)"]).
Alternatively, total RNA can be isolated by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Ausubel (1995) at pages 4-I to 4-6; Wu (1997) at pages 33-41).
In order to construct a cDNA library, poly(A)+ RNA must be isolated from a total RNA preparation. Poly(A)+ RNA can be isolated from total RNA by using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Ausubel (1995) at pages 4-11 to 4-12).
Double-stranded cDNA molecules are synthesized from poly(A) + RNA
using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double-stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, Maryland), CLONTECH Laboratories, Inc. (Palo Alto, California), Promega Corporation (Madison, Wisconsin) and Stratagene Cloning Systems (La Jolla, California).
The basic approach for obtaining TGF-beta binding-protein cDNA clones can be modified by constructing a subtracted cDNA library which is enriched in TGF-binding-protein-specific cDNA molecules. Techniques for constructing subtracted libraries are well-known to those of skill in the art (see, for example, Sargent, "Isolation of Differentially Expressed Genes," in Meth. bizyinol. 152:423, 1987, and Wu et al. (eds.), "Construction and Screening of Subtracted and Complete Expression cDNA
Libraries," in Methods in Gene Biotechnology, pages 29-65 (CRC Press, Inc. 1997)).
Various cloning vectors are appropriate for the construction of a cDNA
5 library. For example, a cDNA library can be prepared in a vector derived from bacteriophage, such as a Xgt10 vector (see, for example, Huynh et al., "Constructing and Screening cDNA Libraries in Xgt10 and Xgtl 1," in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52).
Alternatively, double-stranded cDNA molecules can be inserted into a 10 plasmid vector, such as a pBluescript vector (Stratagene Cloning Systems;
La Jolla, California), a LambdaGEM-4 (Promega Corp.; Madison, Wisconsin) or other commercially available vectors. Suitable cloning vectors also can be obtained from the American Type Culture Collection (Rockville, Maryland).
In order to amplify the cloned cDNA molecules, the cDNA library is 15 inserted into a prokaryotic host, using standard techniques. For example, a cDNA library can be introduced into competent E. coil DH5 cells, which can be obtained from Life Technologies, Inc. (Gaithersburg, Maryland).
A human genomic DNA library can be prepared by means well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).
20 Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases. Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).
Nucleic acid molecules that encode a TGF-beta binding-protein gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the human TGF-beta binding-protein gene, as described herein. General methods for screening libraries with PCR are provided by, for example, Yu et al., "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover, techniques for using PCR to isolate related genes are described by, for example, Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 317-337 (Humana Press, Inc.
1993).
Alternatively, human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Rockville, Maryland).
A library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ID NO:1, using standard methods (see, fir example, Ausubel (1995) at pages 6-1 to 6-11).
Anti-TGF-beta binding-protein antibodies, produced as described below, can also be used to isolate DNA sequences that encode TGF-beta binding-protein genes from cDNA libraries. For example, the antibodies can be used to screen Xgtl 1 expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al., "Screening X expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), pages 1-14 (Oxford University Press 1995)).
The sequence of a TGF-beta binding-protein cDNA or TGF-beta binding-protein genomic fragment can be determined using standard methods.
Moreover, the identification of genomic fragments containing a TGF-beta binding-protein promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see, generally, Ausubel (1995)).
As an alternative, a TGF-beta binding-protein gene can be obtained by synthesizing DNA molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al., Plant Malec. Biol. 21:1131, 1993; Bambot et al., PCR Methods and Applications 2:266, 1993; Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods' and Applications, White (ed.), pages 263-268, (Humana Press, Inc.
1993); Holowachuk et al., PCR Methods Appl. 4:299, 1995).

3. Production of TGF-beta binding-protein genes Nucleic acid molecules encoding variant TGF-beta binding-protein genes can be obtained by screening various cDNA or genomic libraries with polynucleotide probes having nucleotide sequences based upon SEQ ID NO:1, 5, 9, 11, 13, or 15, using procedures described above. TGF-beta binding-protein gene variants can also be constructed synthetically. For example, a nucleic acid molecule can be devised that encodes a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, or 16. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID
NOs: 2, 6, 8, 10, 12, 14 or 16, in which an alkyl amino acid is substituted for an alkyl amino acid in a TGF-beta binding-protein amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a TGF-beta binding-protein amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid in a TGF-beta binding-protein amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in a TGF-beta binding-protein amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a TGF-beta binding-protein amino acid sequence, a basic amino acid is substituted for a basic amino acid in a TGF-beta binding-protein amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in a TGF-beta binding-protein amino acid sequence.
Among the common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. In making such substitutions, it is important to, where possible, maintain the cysteine backbone outlined in Figure 1.
Conservative amino acid changes in a TGF-beta binding-protein gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID
NO:1 . Such "conservative amino acid" variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed II/biogenesis: A Practical Approach (IRL Press 1991)).
The functional ability of such variants can be determined using a standard method, such as the assay described herein. Alternatively, a variant TGF-beta binding-protein polypeptide can be identified by the ability to specifically bind anti-TGF-beta binding-- ¨

_WO 00/32773 protein antibodies.
Routine deletion analyses of nucleic acid molecules can be performed to obtain "functional fragments" of a nucleic acid molecule that encodes a TGF-beta binding-protein polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1 can be digested with BaI31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for activity, or for the ability to bind anti-TGF-beta binding-protein antibodies. One alternative to exonuclease digestion is to use oligenucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
Alternatively, particular fragments of a TGF-beta binding-protein gene can be synthesized using the polymerase chain reaction.
Standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molex. Gen, Genet. 240:113, 1993; Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems, Proceedings of ISIR-TNO
Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987);
Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. I, Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem.
2'70:29270, 1995; Fukunaga et al., J. Biol. Chem. 270:25291, 1995; Yamaguchi et al., Biochem. Pharmacol. 50:1295, 1995; and Meisel et al., Plan/Mo/cc. Biol. 30:1, 1996.
The present invention also contemplates functional fragments of a TGF-beta binding-protein gene that have conservative amino acid changes.
A TGF-beta binding-protein variant gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ID NOs: 1, 5, 9, 11, 13, or, 15 and 2, 6, 10, 12, 14, or 16, as discussed above. An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant TGF-beta binding-protein gene can hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID Nos: 1, 5, 9, 11, 13, or, 15, or a portion thereof of at least 15 or 20 nucleotides in length. As an illustration of stringent hybridization conditions, a nucleic acid molecule having a variant TGF-beta binding-protein sequence can bind with a fragment of a nucleic acid molecule having a sequence from SEQ ID NO:1 in a buffer containing, for example, 5xSSPE (1xSSPE
=
180 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA (pH 7.7), 5xDenhardt's solution (100xDenhardt's = 2% (w/v) bovine serum albumin, 2%
(w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone) and 0.5% SDS incubated overnight at 55-60 C.
Post-hybridization washes at high stringency are typically performed in 0.5xSSC
(1xSSC = 150 mM sodium chloride, 15 mM trisodium citrate) or in 0.5xSSPE at 55-'C.
Regardless of the particular nucleotide sequence of a variant TGF-beta binding-protein gene, the gene encodes a polypeptide that can be characterized by its functional activity, or by the ability to bind specifically to an anti-TGF-beta binding-protein antibody. More specifically, variant TGF-beta binding-protein genes encode polypeptides which exhibit at least 50%, and preferably, greater than 60, 70, 80 or 90%, of the activity of polypeptides encoded by the human TGF-beta binding-protein gene described herein.
4. Production of TGF-beta binding-protein in Cultured Cells To express a TGF-beta binding-protein gene, a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
TGF-beta binding-proteins of the present invention are preferably expressed in mammalian cells. Examples of mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK;
ATCC
CRL 1573), baby hamster kidney cells (BHK-21; ATCC CRL 8544), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K I; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL
1650) and murine embryonic cells (1\11H-3T3; ATCC CRL 1658).
For a mammalian host, the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene . _ which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
Transcriptional regulatory sequences include a promoter region sufficient to 5 direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene [Hamer et al., .I. Molec. Appl. Genet.
1:273, 1982], the TK promoter of Herpes virus [McKnight, Cell 31:355, 1982], the Sr-/U early promoter [Benoist et al., Nature 290:304, 19811, the Rous sarcoma virus promoter [Gorman et al., Proc. Na!'! Acacl Sc). USA 9:6777,-1982], the cytomegalovirus promoter [Foecking et al., io Gene 45:101, 1980], and the mouse mammary tumor virus promoter (see, generally, = Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 163-181 (John Wiley &
Sons, Inc. 1996)).
Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA
15 polymerase promoter, can be used to control TGF-beta binding-protein gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mot C'ell. Biol. /0:4529, 1990; Kaufman et al., Nucl. Acids Res. /9:4485, 1991).
TGF-beta binding-protein genes may also be expressed in bacterial, yeast, insect, or plant cells. Suitable promoters that can be used to express TGF-beta binding-20 protein polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the PR
and PL promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. col', promoters of B. subtilis, the promoters of the bacteriophages of Bacillus, Streptonlyces promoters, the int promoter of bacterio-25 phage lambda, the bla promoter of pBR322, and the CAT promoter of the chloram-phenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Glick, Ind Microhiol. /:277, 1987, Watson et al., Molecular Biology of the Gene, 4th Ed (Benjamin Cummins 1987), and by Ausubel et al. (1995).
Preferred prokaryotic hosts include E. coli and Bacillus subtilus.
Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DHIOB/p3, DH1 1 S, C600, HB101, 1M101, JM105, 1M109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA
Cloning: A Practical Approach, Glover (Ed.) (IRL Press 1985)).

Methods for expressing proteins in prokaryotic hosts are well-known to those of skill in the art (see, for example, Williams et al., "Expression of foreign proteins in E. coil using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), page 15 (Oxford University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc. 1995); and Georgiou, "Expression of Proteins in Bacteria,"
in Protein Engineering: Principles and Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc. 1996)).
to The baculovirus system provides an efficient means to introduce cloned TGF-beta binding-protein genes into insect cells. Suitable expression vectors are based upon the Autographa califbrnica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa californica nuclear polyhedrosis virus immediate-early gene promoter (le-I) and the delayed early 39K promoter, baculovirus p10 promoter, and the Drosophila metallothionein promoter. Suitable insect host cells include cell lines derived from IPLB-Sf:21, a Spodoptera frugiperda pupal ovarian cell line, such as SP
(ATCC CRL 1711), Sf21AE, and 5f21 (Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2 cells.
Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al., "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel et al., "The baculovirus expression system," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
Promoters for expression in yeast include promoters from GALI
(galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOXI
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include YIp-based vectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp vectors, such as YCp19. One skilled in the art will appreciate that there are a wide variety of suitable vectors for expression in yeast cells.
Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells. General methods of culturing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into Plants,"
in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like. Preferably, the transfected cells are selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991). Methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are also provided by Ausubel (1995).
General methods for expressing and recovering foreign protein produced by a mammalian cell system is provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Grisshammer et al., "Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from a baculovirus system are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc., 1995).
More generally, TGF-beta binding-protein can be isolated by standard techniques, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, HPLC and the like. Additional variations in TGF-beta binding-protein isolation and purification can be devised by those of skill in the art.
For example, anti-TGF-beta binding-protein antibodies, obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
5. Production of Antibodies to TGF-beta binding-proteins Antibodies to TGF-beta binding-protein can be obtained, for example, using the product of an expression vector as an antigen. Particularly useful anti-TGF-beta binding-protein antibodies "bind specifically" with TGF-beta binding-protein of Sequence ID Nos. 2, 6, 10, 12, 14, or 16, but not to other TGF-beta binding-proteisn such as Dan, Cerberus, SCGF, or Gremlin. Antibodies of the present invention (including fragments and derivatives thereof) may be a polyclonal or, especially a monoclonal antibody. The antibody may belong to any immunoglobulin class, and may be for example an IgG, for example IgGI, IgG2, IgG3, IgG4; IgE; IgM; or IgA
antibody. It may be of animal, for example mammalian origin, and may be for example a murine, rat, human or other primate antibody. Where desired the antibody may be an internalising antibody.
Polyclonal antibodies to recombinant TGF-beta binding-protein can be prepared using methods well-known to those of skill in the art (see, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995)). Although polyclonal antibodies are typically raised in animals such as rats, mice, rabbits, goats, or sheep, an anti-TGF-beta binding-protein antibody of the present invention may also be derived from a subhuman primate antibody.

General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/11465 (1991), and in Losman et al., Mt. Cancer 46:310, 1990.
The antibody should comprise at least a variable region domain. The variable region domain may be of any size or amino acid composition and will generally comprise at least one hypervariable amino acid sequence responsible for antigen binding embedded in a framework sequence. In general terms the variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) chain variable domains. Thus for example the V region domain may be monomeric and be a VH
or VL domain where these are capable of independently binding antigen with acceptable affinity. Alternatively the V region domain may be dimeric and contain VH-VH, VH-VL, or VL-VL, dimers in which the VH and VL chains are non-covalently associated (abbreviated hereinafter as Fv). Where desired, however, the chains may be covalently coupled either directly, for example via a disulphide bond between the two variable domains, or through a linker, for example a peptide linker, to form a single chain domain (abbreviated hereinafter as scFv).
The variable region domain may be any naturally occuring variable domain or an engineered version thereof. By engineered version is meant a variable region domain which has been created using recombinant DNA engineering techniques. Such engineered versions include those created for example from natural antibody variable regions by insertions, deletions or changes in or to the amino acid sequences of the natural antibodies.
Particular examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from one antibody and the remainder of the variable region domain from a second antibody.
The variable region domain may be covalently attached at a C-terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example where a VH domain is present in the variable region domain this may be linked to an immunoglobulin CHI domain or a fragment thereof. Similarly a VL domain may be linked to a CK domain or a fragment thereof. In this way for example the antibody may be a Fab fragment wherein the antigen binding domain contains associated VH and VL
domains covalently linked at their C-termini to a CH1 and CK domain respectively. The CHI
domain may be extended with further amino acids, for example to provide a hinge region domain as found in a Fab' fragment, or to provide further domains, such as antibody CH2 and CH3 domains.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al.
(eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies:
Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).
Antibodies for use in the invention may in general be monoclonal (prepared by conventional immunisation and cell fusion procedures) or in the case of fragments, derived therefrom using any suitable standard chemical e.g. reduction or enzymatic cleavage and/or digestion techniques, for example by treatment with pepsin.
More specifically, monoclonal anti-TGF-beta binding-protein antibodies can be generated utilizing a variety of techniques. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495, 1975; and Coligan et al. (eds.), Current Protocols in Immunology, /:2.5.1-2.6.7 (John Wiley 8z Sons 1991) ["Coligan"];
Picksley et al., "Production of monoclonal antibodies against proteins expressed in E.
_ _WO 00/32773 co/i," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), page 93 (Oxford University Press 1995)).
Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a TGF-beta binding-protein gene product, verifying the 5 presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
10 In addition, an anti-TGF-beta binding-protein antibody of the present invention may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from 15 embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 20 1994; and Taylor et al., Int. 11711711111. 6:579, 1994.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and 25 pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G
(IgG)," in Methods' in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).
For particular uses, it may be desirable to prepare fragments of anti-TGF-beta binding-protein antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be 30 obtained by pepsin or papain digestion of whole antibodies by conventional methods.
As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab'),. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab =WO 00/32773 .31 fragments and an Fe fragment directly. These methods are described, for example, by Goldenberg, U. S . patent No. 4,331,647, Nisonoff et al . , Arch Biochem.
Biophys.
89:230, 1960, Porter, Biochem. .1. 73:119, 1959, Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
to Alternatively, the antibody may be a recombinant or engineered antibody obtained by the use of recombinant DNA techniques involving the manipulation and re-expression of DNA encoding antibody variable and/or constant regions. Such DNA
is known and/or is readily available from DNA libraries including for example phage-antibody libraries (see Chiswell, D J and McCafferty, J. Tibtech. 10 80-84 (1992)) or where is desired can be synthesised. Standard molecular biology and/or chemistry procedures may be used to sequence and manipulate the DNA, for example, to introduce codons to create cysteine residues, to modify, add or delete other amino acids or domains as desired.
From here, one or more replicable expression vectors containing the DNA
may be prepared and used to transform an appropriate cell line, e.g. a non-producing 20 myeloma cell line, such as a mouse NSO line or a bacterial, e.g. E.coli line, in which production of the antibody will occur. In order to obtain efficient transcription and translation, the DNA sequence in= each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operably linked to the variable domain sequence. Particular methods for producing antibodies in this way are generally 25 well known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al (Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1989); DNA sequencing can be performed as described in Sanger et al (PNAS 74, 5463, (1977)) and the Amersham International plc sequencing handbook; and site directed mutagenesis can be carried out according to the method of Kramer et al (Nucl.
Acids Res.
30 12 9441, (1984)) and the Anglian Biotechnology Ltd handbook.
Additionally, there are numerous publications, detailing techniques suitable for the preparation of antibodies by manipulation of DNA, creation of expression vectors and transformation of appropriate cells, for example as reviewed by Mountain A and Adair, J R in Biotechnology and Genetic Engineering Reviews (ed. Tombs, M P. 10, Chapter 1, 1992, Intercept, Andover, UK) and 35 in International Patent Specification No. WO 91/09967.
Where desired, the antibody according to the invention may have one or effector or reporter molecules attached to it and the invention extends to such modified proteins. The effector or reporter molecules may be attached to the antibody through any available amino acid side-chain, terminal amino acid or, where present carbohydrate functional group located in the antibody, always provided of course that this does not adversely affect the binding properties and eventual usefulness of the molecule.
Particular functional groups include, for example any free amino, imino, thiol, hydroxyl, carboxyl or aldehyde group. Attachment of the antibody and the effector and/or reporter molecule(s) may be achieved via such groups and an appropriate functional group in the effector or reporter molecules. The linkage may be direct or indirect, through spacing or bridging groups.
Effector molecules include, for example, antineoplastic agents, toxins (such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. ricin and fragments thereof) biologically active proteins, for example enzymes, nucleic acids and fragments thereof, e.g. DNA, RNA and fragments thereof, naturally occurring and synthetic polymers e.g. polysaccharides and polyalkylene polymers such as poly(ethylene glycol) and derivatives thereof, radionuclides, particularly radioiodide, and chelated metals. Suitable reporter groups include chelated metals, fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
Particular antineoplastic agents include cytotoxic and cytostatic agents, for example alkylating agents, such as nitrogen mustards (e.g. chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracil mustard) and derivatives thereof, triethylenephosphoramide, triethylenethiophosphor-amide, busulphan, or cisplatin;
antimetabolites, such as methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid, antibiotics, such as bleomycins (e.g.
bleomycin sulphate), doxorubicin, daunorubicin, mitomycins (e.g. mitomycin C), actinomycins (e.g. dactinomycin) plicamycin, calichaemicin and derivatives thereof, or esperamicin and derivatives thereof; mitotic inhibitors, such as etoposide, vincristine or vinblastine and derivatives thereof; alkaloids, such as ellipticine; polyols such as taxicin-I or taxicin-II; hormones, such as androgens (e.g. dromostanolone or testolactone), progestins (e.g. megestrol acetate or medroxyprogesterone acetate), estrogens (e.g.
dimethylstilbestrol diphosphate, polyestradiol phosphate or estramustine phosphate) or antiestrogens (e.g.
tamoxifen); anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea;
hydrazines, such as procarbazine; or imidazoles, such as dacarbazine.
Particularly useful effector groups are calichaemicin and derivatives thereof (see for example South African Patent Specifications Nos. 85/8794, 88/8127 and 90/2839).
Chelated metals include chelates of di-or tripositive metals having a _ coordination number from 2 to 8 inclusive. Particular examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). In general the metal is preferably a radionuclide. Particular radionuclides 99m 186 188 58 60 67 195 199 110 203 206 207 include Tc, Re, Re, Co, Co, Cu, Au, Au, Ag, Pb, Bi, Bi, In, Ga, Ga, Y, Y, Tb, Gd and 47Sc.
The chelated metal may be for example one of the above types of metal chelated with any suitable polydentate chelating agent, for example acyclic or cyclic polyamines, polyethers, (e.g. crown ethers and derivatives thereof);
polyamides; porphyrins;
and carbocyclic derivatives.
In general, the type of chelating agent will depend on the metal in use. One particularly useful group of chelating agents in conjugates according to the invention, however, are acyclic and cyclic polyamines, especially polyaminocarboxylic acids, for example diethylenetriaminepentaacetic acid and derivatives thereof, and macrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives (for example as described in International Patent Specification No. WO 92/22583); and polyamides, especially desferrioxamine and derivatives thereof.
Thus for example when it is desired to use a thiol group in the antibody as the point of attachment this may be achieved through reaction with a thiol reactive group present in the effector or reporter molecule. Examples of such groups include an a-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone, or a disulphide. These and other suitable linking procedures are generally and more particularly described in International Patent Specifications Nos. WO
93/06231, WO
92/22583, WO 90/091195 and WO 89/01476.
ASSAYS FOR SELECTING MOLECULES WHICH INCREASE BONE DENSITY
As discussed above, the present invention provides methods for selecting and/or isolating compounds which are capable of increasing bone density. For example, within one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) mixing a selected molecule with TGF-beta binding protein and a selected member of the TGF-beta family of proteins, (b) determining whether the selected molecule stimulates signaling by the TGF-beta family of proteins, or inhibits the binding of the TGF-beta binding protein to the TGF-beta family of proteins.
Within certain embodiments, the molecule enhances the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation.

.3 4 Within other aspects of the invention, methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) exposing a selected molecule to cells which express TGF-beta binding-protein and (b) determining whether the expression (or activity) of TGF-beta binding-protein from said exposed cells decreases, and therefrom determining whether the compound is capable of increasing bone mineral content. Within one embodiment, the cells are selected from the group consisting of the spontaneously transformed or untransformed normal human bone from bone biopsies and rat parietal bone osteoblasts. Such methods may be accomplished in a wide variety of assay formats including, for example, Countercurrent Immuno-Electrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition or Competition assays, and sandwich assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also Antibodies: A
Laboratory Manual, supra).
Representative embodiments of such assays are provided below in Examples 5 and 6. Briefly, a family member of the TGF-beta super-family or a TGF-beta binding protein is first bound to a solid phase, followed by addition of a candidate molecule. The labeled family member of the TGF-beta super-family or a TGF-beta binding protein is then added to the assay, the solid phase washed, and the quantity of bound or labeled TGF-beta super-family member or TGF-beta binding protein on the solid support determined. Molecules which are suitable for use in increasing bone mineral content as described herein are those molecules which decrease the binding of TGF-beta binding protein to a member or members of the TGF-beta super-family in a statistically significant manner. Obviously, assays suitable for use within the present invention should not be limited to the embodiments described within Examples 2 and 3.
In particular, numerous parameters may be altered, such as by binding TGF-beta to a solid phase, or by elimination of a solid phase entirely.
Within other aspects of the invention, methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) exposing a selected molecule to cells which express TGF-beta and (b) determining whether the activity of TGF-beta from said exposed cells is altered, and therefrom determining whether the compound is capable of increasing bone mineral content. Similar to the above described methods, a wide variety of methods may be utilized to assess the changes of TGF-beta binding-protein expression due to a selected test compound.

For example, within one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) mixing a selected molecule with TGF-beta-binding-protein and a selected member of the TGF-beta family of proteins, 5 (b) determining whether the selected molecule up-regulates the signaling of the TGF-beta family of proteins, or inhibits the binding of the TGF-beta binding-protein to the TGF-beta family of proteins. Within certain embodiments, the molecule enhances the ability of TGF-beta to function as a positive regulator of mechemchymal cell differentiation.
10 Similar to the above described methods, a wide variety of methods may be utilized to assess stimulation of TGF-beta due to a selected test compound.
One such representative method is provided below in Example 6 (see also Durham et al., Endo. 136:1374-1380.
Within yet other aspects of the present invention, methods are provided 15 for determining whether a selected molecule is capable of increasing bone mineral content, comprising the step of determining whether a selected molecule inhibits the binding of TGF-beta binding-protein to bone, or an analogue thereof. As utilized herein, it should be understood that bone or analogues thereof refers to hydroxyapatite, or a surface composed of a powdered form of bone, crushed bone or intact bone.
20 Similar to the above described methods, a wide variety of methods may be utilized to assess the inhibition of TGF-beta binding-protein localization to bone matrix.
One such representative method is provided below in Example 7.
It should be noted that while the methods recited herein may refer to the analysis of an individual test molecule, that the present invention should not be so limited. In particular, the selected molecule may be contained within a mixture of compounds. Hence, the recited methods may further comprise the step of isolating a molecule which inhibits the binding of TGF-beta binding-protein to a TGF-beta family member.
CANDIDATE MOLECULES
30 A wide variety of molecules may be assayed for their ability to inhibit the binding of TGF-beta binding-protein to a TGF-beta family member.
Representative examples which are discussed in more detail below include organic molecules, proteins or peptides, and nucleic acid molecules. Although it should be evident from the discussion below that the candidate molecules described herein may be utilized in the assays described herein, it should also be readily apparent that such molecules can also be utilized in a variety of diagnostic and therapeutic settins.
1. Organic Molecules Numerous organic molecules may be assayed for their ability to inhibit the binding of TGF-beta binding-protein to a TGF-beta family member.
For example, within one embodiment of the invention suitable organic molecules may be selected from either a chemical library, wherein chemicals are assayed individually, or from combinatorial chemical libraries where multiple compounds are assayed at once, then deconvoluted to determine and isolate the most active compounds.
Representative examples of such combinatorial chemical libraries include those described by Agrafiotis et al., "System and method of automatically generating chemical compounds with desired properties," U.S. Patent No.
5,463,564;
Armstrong, R.W., "Synthesis of combinatorial arrays of organic compounds through the use of multiple component combinatorial array syntheses," WO 95/02566;
Baldwin, J.J. et al., "Sulfonamide derivatives and their use," WO 95/24186; Baldwin, J.J. et al., "Combinatorial dihydrobenzopyran library," WO 95/30642; Brenner, S., "New kit for preparing combinatorial libraries," WO 95/16918; Chenera, B. et al., "Preparation of library of resin-bound aromatic carbocyclic compounds," WO 95/16712; Ellman, J.A., "Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support," U.S. Patent No. 5,288,514; Felder, E. et al., "Novel combinatorial compound libraries," WO 95/16209; Lerner, R. et al., "Encoded combinatorial chemical libraries,"
WO 93/20242; Pavia, M.R. et al., "A method for preparing and selecting pharmaceutically useful non-peptide compounds from a structurally diverse universal library," WO 95/04277; Summerton, J.E. and D.D. Weller, "Morpholino-subunit combinatorial library and method," U.S. Patent No. 5,506,337; Holmes, C., "Methods for the Solid Phase Synthesis of Thiazolidinones, Metathiazanones, and Derivatives thereof," WO 96/00148; Phillips, G.B. and G.P. Wei, "Solid-phase Synthesis of Benzimidazoles," Tel. Letters 37:4887-90, 1996; Ruhland, B. et al., "Solid-supported Combinatorial Synthesis of Structurally Diverse 13-Lactams," J. Amer. Chem.
Soc.
111:253-4, 1996; Look, G.C. et al., "The Indentification of Cyclooxygenase-1 Inhibitors from 4-Thiazolidinone Combinatorial Libraries," Bioorg and Med.
Chem.
Letters 6:707-12, 1996.

11.

2. Proteins and Peptides A wide range of proteins and peptides may likewise be utilized as candidate molecules for inhibitors of the binding of TGF-beta binding-protein to a TGF-beta family member.
a. Combinatorial Peptide Libraries Peptide molecules which are putative inhibitors of the binding of TGF-beta binding-protein to a TGF-beta family member may be obtained through the screening of combinatorial peptide libraries. Such libraries may either be prepared by one of skill in the art (see e.g., U.S. Patent Nos. 4,528,266 and 4,359,535, and Patent I() Cooperation Treaty Publication Nos. WO 92/15679, WO 92/15677, WO
90/07862, WO
90/02809, or purchased from commercially available sources (e.g., New England Biolabs Ph.D.Tm Phage Display Peptide Library Kit).
b. Antibodies Antibodies which inhibit the binding of TGF-beta binding-protein to a TGF-beta family member may readily be prepared given the disclosure provided herein. Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F(ab')2, Fv variable regions, or complementarity determining regions). As discussed above, antibodies are understood to be specific against TGF-beta binding-protein , or against a specific TGF-beta family member, if they bind with a Ka of greater than or equal to107m-', preferably.greater than or equal to 108M-1, and do not bind to other TGF;beta binding-proteins, or, bind with a Ka of less than or equal to 106M-'. Furthermore, antibodies of the present invention should block or inhibit the binding of TGF-beta binding-protein to a TGF-beta family member.
The affinity of a monoclonal antibody or binding partner, as well as inhibition of binding can be readily determined by one of ordinary skill in the art (see Scatchard, Ann. NY. Acad ScL 51:660-672, 1949).
Briefly, polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Typically, the TGF-beta binding-protein or unique peptide thereof of 13-20 amino acids (preferably conjugated to keyhole limpet hemocyanin by cross-linking with glutaraldehyde) is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, along with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to the protein or peptide. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993, = see also Monoclonal Antibodies, Hybrid.omas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference).
Briefly, within one embodiment a subject animal such as a rat or mouse is immunized with TGF-beta binding-protein or portion thereof as described above.
The protein may be admixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response. Between one and three weeks after the initial immunization the animal may be reimmunized with another booster immunization, and tested for reactivity to the protein utilizing assays described above. Once the animal has reached a plateau in its reactivity to the injected protein, it is sacrificed, and organs which contain large numbers of B cells such as the spleen and lymph nodes are harvested.
Cells which are obtained from the immunized animal may be immortalized by infection with a virus such as the Epstein-Barr virus (EBV) (see Glasky and Reading, Hyhridoma 8(4):377-389, 1989). Alternatively, within a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibody. Suitable myeloma lines include, for example, NS-1 (ATCC
No.
TIB 18), and P3X63 - Ag 8.653 (ATCC No. CRL 1580).
Following the fusion, the cells may be placed into culture plates containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as fetal bovine serum (FBS, e., from Hyclone, Logan, Utah, or JRH
Biosciences). Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells such as HAT
(hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After about seven days, the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which are reactive against TGF-beta binding-protein (depending on the aptigen used), and which block or inhibit the binding of TGF-beta binding-protein to a TGF-beta family member.
A wide variety of assays may be utilized to determine the presence of antibodies which are reactive against the proteins of the present invention, including for example countercurrent immuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, western blots, immunoprecipitation, inhibition or competition assays, and sandwich assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also Antibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Following several clonal dilutions and reassays, a hybridoma producing antibodies reactive against the desired protein may be isolated.
Other techniques may also be utilized to construct monoclonal antibodies (see William D. Huse et at., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies:
Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. S'ci.
USA
86:5728-5732, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990). These references describe a commercial system available from Stratagene (La Jolla, California) which enables the production of antibodies through recombinant techniques. Briefly, mRNA is isolated from a B
cell population, and utilized to create heavy and light chain immunoglobulin cDNA
expression libraries in the XImmunoZap(H) and XImmunoZap(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E. coll.
Similarly, portions or fragments, such as Fab and Fv fragments, of antibodies may also be constructed utilizing conventional enzymatic digestion or recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody. Within one embodiment, the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers _ _ may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. Stratagene (La Jolla, California) sells primers for mouse and human variable regions including, among others, primers for VHa, VHb, CHI, VL and CL regions. These primers may be utilized to amplify heavy VHc, VHd, 5 or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTM H or ImmunoZAPTM L (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression.
Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced (see Bird et al., Science 242:423-426, 10 1988). In addition, such techniques may be utilized to change a "murine"
antibody to a "human" antibody, without altering the binding specificity of the antibody.
Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratoty Manual, Harlow and Lane (eds.), Cold Spring Harbor 15 Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.
C; Mutant TGF-beta binding-proteins As described herein and below in the Examples (e.g., Examples 8 and 20 9), altered versions of TGF-beta binding-protein which compete with native TGF-beta binding-protein's ability to block the activity of a particular TGF-beta family member should lead to increased bone density. Thus, mutants of TGF-beta binding-protein which bind to the TGF-beta family member but do not inhibit the function of the TGF-beta family member would meet the criteria. The mutant versions must effectively 25 compete with the endogenous inhibitory functions of TGF-beta binding-protein.
d. Production of proteins Although various genes (or portions thereof) have been provided herein, it should be understood that within the context of the present invention, reference to one or more of these genes includes derivatives of the genes that are substantially 30 similar to the genes (and, where appropriate, the proteins (including peptides and polypeptides) that are encoded by the genes and their derivatives). As used herein, a nucleotide sequence is deemed to be "substantially similar" if: (a) the nucleotide sequence is derived from the coding region of the above-described genes and includes, for example, portions of the sequence or allelic variations of the sequences discussed _ _ _ above, or alternatively, encodes a molecule which inhibits the binding of TGF-beta binding-protein to a member of the TGF-beta family, (b) the nucleotide sequence is capable of hybridization to nucleotide sequences of the present invention under moderate, high or very high stringency (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) the DNA sequences are degenerate as a result of the genetic code to the DNA
sequences defined in (a) or (b). Further, the nucleic acid molecule disclosed herein includes both complementary and non-complementary sequences, provided the sequences otherwise meet the criteria set forth herein. Within the context of the present invention, high stringency means standard hybridization conditions (e.g., 5XSSPE, 0.5% SDS at 65 C, or the equivalent).
The structure of the proteins encoded by the nucleic acid molecules described herein may be predicted from the primary translation products using the hydrophobicity plot function of, for example, P/C Gene or 1ntelligenetics Suite (Intelligenetics, Mountain View, California), or according to the methods described by Kyte and Doolittle (11V1ol. Biol. 157:105-132, 1982).
Proteins of the present invention may be prepared in the form of acidic or basic salts, or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction. Furthermore, various substitutions, deletions, or additions may be made to the amino acid or nucleic acid sequences, the net effect of which is to retain or further enhance or decrease the biological activity of the mutant or wild-type protein. Moreover, due to degeneracy in the genetic code, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence.
Other derivatives of the proteins disclosed herein include conjugates of the proteins along with other proteins or polypeptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins which may be added to facilitate purification or identification of proteins (see U.S.
Patent No.
4,851,341, see also, Hopp et al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteins such as Flag/TGF-beta binding-protein be constructed in order to assist in the identification, expression, and analysis of the protein.
Proteins of the present invention may be constructed using a wide variety of techniques described herein. Further, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence.
Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncation derivatives of proteins (e.g., a soluble extracellular portion) may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion.
Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).
Mutations which are made in the nucleic acid molecules of the present invention preferably preserve the reading frame of the coding sequences.
Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect translation of the mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed mutants screened for indicative biological activity. Alternatively, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence.
Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion.
Nucleic acid molecules which encode proteins of the present invention may also be constructed utilizing techniques of PCR mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402-3406, 1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene 88:107-111, 1990), or by use of randomly mutagenized oligonucleotides (Horwitz et al., Genome 3:112-117, 1989).
The present invention also provides for the manipulation and expression of the above described genes by culturing host cells containing a vector capable of expressing the above-described genes. Such vectors or vector constructs include either synthetic or cDNA-derived nucleic acid molecules encoding the desired protein, which are operably linked to suitable transcriptional or translational regulatory elements.
Suitable regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect, or plant genes. Selection of appropriate regulatory elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of regulatory elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a transcriptional terminator, and a ribosomal binding sequence, including a translation initiation signal.
Nucleic acid molecules that encode any of the proteins described above may be readily expressed by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, insect, or plant cells.
Methods for transforming or transfecting such cells to express foreign DNA are well known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S.
Patent No. 4,784,950; Axel et al., U.S. Patent No, 4,399,216; Goeddel et al., U.S.
Patent No. 4,766,075; and Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol. 104:1067-1071, 1994; and Paszkowski et al., Biotech. 24:387-392, 1992).
Bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.
Representative examples of bacterial host cells include DH5a. (Stratagene, LaJolla, California).
Bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the ¾-lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the T7 RNA
polymerase promoter (Studier et al., Meth. Enzymol. /85:60-89, 1990), the lambda promoter (Elvin et al., Gene 87:123-126, 1990), the tip promoter (Nichols and Yanofsky, Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20:231, 1982). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977), the pUC plasmids pUC18, _ pUC19, pUC118, pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, California).
Yeast and fungi host cells suitable for carrying out the present invention include, among others, Saccharomyces pornhe, Saccharomyces cerevisiae, the genera Pichia or Kinyveromyces and various species of the genus A.spergilhts (McKnight et al., U.S. Patent No. 4,935,349). Suitable expression vectors for yeast and fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl. Acad.
Sci.
USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8:121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof.
Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980; Alber and Kawasaki, .1. Mot Appl. Genet. 1:419-434, 1982) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms firr Chemicals, Hollaender etal. (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples of useful promoters for fungi vectors include those derived from A.spergillus nidulans glycolytic genes, such as the ad,h3 promoter (McKnight et al., EMBO .1. 4:2093-2099, 1985). The expression units may also include a transcriptional terminator. An example of a suitable terminator is the adh3 terminator (McKnight et al., ibid., 1985).
As with bacterial vectors, the yeast vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include leu2 (Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al., ihid).
Another suitable selectable marker is the cat gene, which confers chloramphenicol resistance on yeast cells.
Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc.
Natl. Acad Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA
81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983). The genotype of the host cell may contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.
. _ Protocols for the transformation of yeast are also well known to those of ordinary skill in the art. For example, transformation may be readily accomplished either by preparation of spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA
75:1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., .1.
5 Bacteriology 153:163, 1983). Transformation of fungi may also be carried out using polyethylene glycol as described by Cullen et al. (Bio/Technology 5:369, 1987).
Viral vectors include those which comprise a promoter that directs the expression of an isolated nucleic acid molecule that encodes a desired protein as described above. A wide variety of promoters may be utilized within the context of the 10 present invention, including for example, promoters such as MolVILV LTR, RSV LTR, Friend MuLV LTR, adenoviral promoter (Ohno et al., Science 265:781-784, 1994), neomycin phosphotransferase promoter/enhancer, late parvovirus promoter (Koering et al., Hum. Gene Therap. 5:457-463, 1994), Herpes TK promoter, SV40 promoter, metallothionein Ha gene enhancer/promoter, cytomegalovirus immediate early 15 promoter, and the cytomegalovirus immediate late promoter. Within particularly preferred embodiments of the invention, the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731; and WO 90/07936). Representative examples of suitable tissue specific promoters include neural specific enolase promoter, platelet derived growth factor beta promoter, bone morphogenic protein promoter, human 20 alphal-chimaerin promoter, synapsin I promoter and synapsin II promoter.
In addition to the above-noted promoters, other viral-specific promoters (e.g., retroviral promoters (including those noted above, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal or parasitic (e.g., malarial) -specific promoters may be utilized in order to target a specific cell or tissue which is infected with a virus, 25 bacteria, fungus or parasite.
Mammalian cells suitable for carrying out the present invention include, among others COS, CHO, Sa0S, osteosarcomas, KS483, MG-63, primary osteoblasts, and human or mammalian bone marrow stroma. Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the 30 transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Bone specific promoters include the bone sialo-protein and the promoter for osteocalcin. Viral promoters include the cytomegalovirus immediate early promoter (Boshart et al., Cell 41:521-530, 1985), cytomegalovirus immediate late promoter, SV40 promoter (Subramani et al., !viol Cell. Biol. /:854-864, 1981), MMTV
35 LTR, RSV LTR, metallothionein-1, adenovirus El a. Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a , -----mouse VK promoter (Bergman et al., Proc. Natl. Acad ScL USA 8/:704I-7045, 1983;
Grant et al., Nucl. Acids Res. /5:5496, 1987) and a mouse VH promoter (Loh et al, Cell 33:85-93, 1983). The choice of promoter will depend, at least in part, upon the level of expression desired or the recipient cell line to be transfected.
Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the peptide or protein of interest. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest.
Suitable polyadenylation signals include the early or late polyadenylation signals from (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res.
9:3719-3730, 1981). The expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer. Expression vectors may also include sequences encoding the adenovirus VA RNAs. Suitable expression vectors can be obtained from commercial sources (e.g., Stratagene, La Jolla, California).
Vector constructs comprising cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell /4:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., Elv1B0 .1. /:841-845, 1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987). To identify cells that have stably integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene.
Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Massachusetts, Mammalian cells containing a suitable vector are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest.
Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable, selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level.
Cells that satisfy these criteria can then be cloned and scaled up for production.
Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods include calcium phosphate mediated transfection, electroporation, lipofection, retroviral, adenoviral and protoplast fusion-mediated transfection (see Sambrook et al., supra). Naked vector constructs can also be taken up by muscular cells or other suitable cells subsequent to injection into the muscle of a mammal (or other animals).
Numerous insect host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of baculoviruses as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-224,1990).
Numerous plant host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of Agrobacteriutn rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (.1 Biosci. (Bangalore)11:47-58, 1987).
Within related aspects of the present invention, proteins of the present invention may be expressed in a transgenic animal whose germ cells and somatic cells contain a gene which encodes the desired protein and which is operably linked to a promoter effective for the expression of the gene. Alternatively, in a similar manner transgenic animals may be prepared that lack the desired gene (e.g., "knock-out" mice).
Such transgenics may be prepared in a variety of non-human animals, including mice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al., Nature 3/5:680-683, 1985, Palmiter et al., Science 222:809-814, 1983, Brinster et al., Proc. Nall.
Acad Sci.
USA 82:4438-4442, 1985, Palmiter and Brinster, Cell 41:343-345, 1985, and U.S.

Patent Nos. 5,175,383, 5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384). Briefly, an expression vector, including a nucleic acid molecule to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, for example, by microinjection.
Integration of the injected DNA is detected by blot analysis of DNA from tissue samples.
It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny. Tissue-specific expression may be achieved through the use of a tissue-specific promoter, or through the use of an _ inducible promoter, such as the metallothionein gene promoter (Palmiter et al., 1983, Mid), which allows regulated expression of the transgene.
Proteins can be isolated by, among other methods, culturing suitable host and vector systems to produce the recombinant translation products of the present invention. Supernatants from such cell lines, or protein inclusions or whole cells where the protein is not excreted into the supernatant, can then be treated by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant may be first concentrated using commercially available protein concentration filters, such as an Arnicon or Millipore Pellicon ultrafiltration unit.
Following concentration, the concentrate may be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support.
Alternatively, anion or cation exchange resins may be employed in order to purify the protein. As a further alternative, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps may be employed to further purify the protein.
Other methods of isolating the proteins of the present invention are well known in the skill of the art.
A protein is deemed to be "isolated" within the context of the present invention if no other (undesired) protein is detected pursuant to SDS-PAGE
analysis followed by Coomassie blue staining. Within other embodiments, the desired protein can be isolated such that no other (undesired) protein is detected pursuant to SDS-PAGE analysis followed by silver staining.
3. Nucleic Acid Molecules Within other aspects of the invention, nucleic acid molecules are provided which are capable of inhibiting TGF-beta binding-protein binding to a member of the TGF-beta family. For example, within one embodiment antisense oligonucleotide molecules are provided which specifically inhibit expression of TGF-beta binding-protein nucleic acid sequences (see generally, Hirashima et al.
in Molecular Biology of RNA: New Perspectives (M. Inouye and B. S. Dudock, eds., Academic Press, San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of Gene Expression (IS. Cohen, ed., 1989 MacMillan Press, London); Stein and Cheng, Science 261:1004-1012, 1993; WO 95/10607; U.S. Patent No. 5,359,051; WO 92/06693; and EP-A2-612844). Briefly, such molecules are constructed such that they are complementary to, and able to form Watson-Crick base pairs with, a region of transcribed TGF-beta binding-protein mRNA sequence. The resultant double-stranded nucleic acid interferes with subsequent processing of the mRNA, thereby preventing protein synthesis (see Example 10).
Within other aspects of the invention, ribozymes are provided which are capable of inhibiting the TGF-beta binding-protein binding to a member of the TGF-beta family. As used herein, "ribozymes" are intended to include RNA molecules that contain anti-sense sequences for specific recognition, and an RNA-cleaving enzymatic activity. The catalytic strand cleaves a specific site in a target RNA at greater than stoichiometric concentration. A wide variety of ribozymes may be utilized within the context of the present invention, including for example, the hammerhead ribozyme (for example, as described by Forster and Symons, Cell 48:211-220, 1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988;
Haseloff and Gerlach, Nature 334:585, 1988); the hairpin ribozyme (for example, as described by Haseloff et al., U.S. Patent No. 5,254,678, issued October 19, 1993 and Hempel et at., European Patent Publication No. 0 360 257, published March 26, 1990);
and Tetrahymena ribosomal RNA-based ribozymes (see Cech et al., U.S. Patent No.
4,987,071). Ribozymes of the present invention typically consist of RNA, but may also be composed of DNA, nucleic acid analogs (e.g., phosphorothioates), or chimerics thereof (e.g., DNA/RNA/RNA).
4. Labels The gene product or any of the candidate molecules described above and below, may be labeled with a variety of compounds, including for example, fluorescent molecules, toxins, and radionuclides.
Representative examples of fluorescent molecules include fluorescein, Phycohili proteins, such as phycoerythrin, rhodamine, Texas red and luciferase. Representative examples of toxins include ricin, abrin diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, 1-123, 1-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. In addition, the antibodies described above may also be labeled or conjugated to one partner of a ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein.
Methods for conjugating or labeling the molecules described herein with the representative labels set forth above may be readily accomplished by one of ordinary skill in the art (see Trichothecene Antibody Conjugate, U.S. Patent No.
4,744,981; Antibody Conjugate, U.S. Patent No. 5,106,951; Fluorogenic Materials and Labeling Techniques, U.S. Patent No. 4,018,884; Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Patent No. 4,897,255; and Metal Radionuclide Chelating Compounds for Improved Chelation Kinetics, U.S. Patent No.
4,988,496; see also Inman, Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme 5 Purification: Part B, Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; see also Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem. 171:1-32, 1988).
PHARMACEUTICAL COMPOSITIONS
As noted above, the present invention also provides a variety of 10 pharmaceutical compositions, comprising one of the above-described molecules which inhibits the TGF-beta binding-protein binding to a member of the TGF-beta family along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails 15 combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.
20 In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible 25 expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.
METHODS OF TREATMENT
The present invention also provides methods for increasing the mineral 30 content and mineral density of bone. Briefly, numerous conditions result in the loss of bone mineral content, including for example, disease, genetic predisposition, accidents which result in the lack of use of bone (e.g., due to fracture), therapeutics which effect bone resorption, or which kill bone forming cells and normal aging. Through use of the molecules described herein which inhibit the TGF-beta binding-protein binding to a _ _ TGF-beta family member such conditions may be treated or prevented. As utilized herein, it should be understood that bone mineral content has been increased, if bone mineral content has been increased in a statistically significant manner (e.g., greater than one-half standard deviation), at a selected site.
A wide variety of conditions which result in loss of bone mineral content may be treated with the molecules described herein. Patients with such conditions may be identified through clinical diagnosis utilizing well known techniques (see, e.g., Harrison's Principles of Internal Medicine, McGraw-Hill, Inc.).
Representative examples of diseases that may be treated included dysplasias, wherein there is abnormal growth or development of bone. Representative examples of such conditions include achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's, hypophosphatemic rickets, Marfan's, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, fractures, periodontal disease, pseudoarthrosis and pyogenic osteomyelitis.
Other conditions which may be treated or prevented include a wide variety of causes of osteopenia (i.e., a condition that causes greater than one standard deviation of bone mineral content or density below peak skeletal mineral content at youth). Representative examples of such conditions include anemic states, conditions caused steroids, conditions caused by heparin, bone marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, senility, postmenopausal state, oligomenorrhea , amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, transient regional osteoporosis and osteomalacia.
Within one aspect of the present invention, bone mineral content or density may be increased by administering to a warm-blooded animal a therapeutically effective amount of a molecule which inhibits the TGF-beta binding-protein binding to a TGF-beta family member. Examples of warm-blooded animals that may be treated include both vertebrates and mammals, including for example horses, cows, pigs, sheep, dogs, cats, rats and mice. Representative examples of therapeutic molecules include ribozymes, ribozyme genes, antisense oligonucleotides and antibodies (e.g, humanized antibodies).
Within other aspects of the present invention, methods are provided for increasing bone density, comprising the step of introducing into cells which home to bone a vector which directs the expression of a molecule which inhibits the TGF-beta binding-protein binding to a member of the TGF-beta family, and administering the , vector containing cells to a warm-blooded animal. Briefly, cells which home to bone may be obtained directly from the bone of patients (e.g., cells obtained from the bone marrow such as CD34+, osteoblasts, osteocytes, and the like), from peripheral blood, or from cultures.
A vector which directs the expression of a molecule that inhibits the TGF-beta binding-protein binding to a member of the TGF-beta family is introduced into the cells. Representative examples of suitable vectors include viral vectors such as herpes viral vectors (e.g., U.S. Patent No. 5,288,641), adenoviral vectors (e.g., WO
94/26914, WO 93/9191; Kolls et al., .PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993;
Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. .1 Neurosci.
5(10:1287-1291, 1993; Vincent et al., Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat.
Genet. 1(5):372-378, 1992; and Levrero et al., Gene 101(2):195-202, 1991), adeno-associated viral vectors (WO 95/13365; Flotte etal.., PNAS 90(22):10613-10617, 1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap.
5:457-463, 1994), pox virus vectors (Panicali and Paoletti, PNAS '9:4927-4931, 1982; and Ozaki et al., Biochem. Biophys. Res. (OMM. 193(2):653-660, 1993), and retroviruses (e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698;
WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 93/10218). Viral vectors may likewise be constructed which contain a mixture of different elements (e.g., promoters, envelope sequences and the like) from different viruses, or non-viral sources. Within various embodiments, either the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.
Within other embodiments of the invention, nucleic acid molecules which encode a molecule which inhibits the TGF-beta binding-protein binding to a member of the TGF-beta family themselves may be administered by a variety of techniques, including, for example, administration of asialoosomucoid (ASOR) conjugated with poly-L-lysine DNA complexes (Cristano et al., PNAS 92122-92126, 1993), DNA linked to killed adenovirus (Curie' et al., Hum. Gene Ther.
3(2):147-154, 1992), cytofectin-mediated introduction (DMRIE-DOPE, Vical, California), direct DNA injection (Acsadi et al., Nature 352:815-818, 1991); DNA ligand (Wu et al., I of Biol. Chem. 264:16985-16987, 1989); lipofection (Feigner et al., Proc. Nall.
Acad. Sci.
USA 84:7413-7417, 1989); liposomes (Pickering et al., Circ. 89(1):13-21, 1994;
and Wang et al., PNAS 84:7851-7855, 1987); microprojectile bombardment (Williams õ . _ et al., PNAS 88:2726-2730, 1991); and direct delivery of nucleic acids which encode the protein itself either alone (Vile and Hart, Cancer Res, 53: 3860-3864, 1993), or utilizing PEG-nucleic acid complexes.
Representative examples of molecules which may be expressed by the vectors of present invention include ribozymes and antisense molecules, each of which are discussed in more detail above.
Determination of increased bone mineral content may be determined directly through the use of X-rays (e.g., Dual Energy X-ray Absorptometry or "DEXA"), or by inference through bone turnover markers (osteoblast specific alkaline phosphatase, osteocalcin, type 1 procollagen C' propeptide (PICP), and total alkaline phosphatase; see Cornier, C., Curr. Opin. in Rheu. 7:243, 1995), or markers of bone resorption (pyridinoline, deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant acid phosphatases and galactosyl hydroxylysine; see Comier, supra). The amount of bone mass may also be calculated from body weights, or utilizing other methods (see Guinness-Hey, Mefah. Bone Dis. and Rel. Res.
5:177-181, 1984).
As will be evident to one of skill in the art, the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
Typically, the compositions may be administered by a variety of techniques, as noted above.
The following examples are offered by way of illustration, and not by way of limitation.
_ _WO 00/32773 EXAMPLES

Genetic mapping of the defect responsible for sclerosteosis in humans localized the gene responsible for this disorder to the region of human chromosome 17 that encodes a novel TGF-beta binding-protein family member. In sclerosteosis, skeletal bone displays a substantial increase in mineral density relative to that of unafflicted individuals. Bone in the head displays overgrowth as well.
Sclerosteosis patients are generally healthy although they may exhibit variable degrees of syndactyly at birth and variable degrees of cranial compression and nerve compression in the skull.
Linkage analysis of the gene defect associated with sclerosteosis was conducted by applying the homozygosity mapping method to DNA samples collected from 24 South African Afrikaaner families in which the disease occurred.
(Sheffield et al., 1994, Human Molecular Genetics 3:1331-1335. "Identification of a Bardet-Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to homozygosity mapping"). The Afrikaaner population of South Africa is genetically homogeneous; the population is descended from a small number of founders who colonized the area several centuries ago, and it has been isolated by geographic and social barriers since the founding. Sclerosteosis is rare everywhere in the world outside the Afrikaaner community, which suggests that a mutation in the gene was present in the founding population and has since increased in numbers along with the increase in the population. The use of homozygosity mapping is based on the assumption that DNA mapping markers adjacent to a recessive mutation are likely to be homozygous in affected individuals from consanguineous families and isolated populations.
A set of 371 microsatellite markers (Research Genetics, Set 6) from the autosomal chromosomes was selected to type pools of DNA from sclerosteosis patient samples. The DNA samples for this analysis came from 29 sclerosteosis patients in 24 families, 59 unaffected family members and a set of unrelated control individuals from the same population. The pools consisted of 4-6 individuals, either affected individuals, affected individuals from consanguineous families, parents and unaffected siblings, or unrelated controls. In the pools of unrelated individuals and in most of the pools with affected individuals or family members analysis of the markers showed several allele sizes for each marker. One marker, D17S1299, showed an indication of homozygosity: one band in several of the pools of affected individuals.

All 24 sclerosteosis families were typed with a total of 19 markers in the region of D17S1299 (at 17q12-q21). Affected individuals from every family were shown to be homozygous in this region, and 25 of the 29 individuals were homozygous for a core haplotype; they each had the same alleles between D17S1787 and D
I7S930.
5 The other four individuals had one chromosome which matched this haplotype and a second which did not. In sum, the data compellingly suggested that this 3 megabase region contained the sclerosteosis mutation. Sequence analysis of most of the exons in this 3 megabase region identified a nonsense mutation in the novel TGF-beta binding-protein coding sequence (C>T mutation at position 117 of Sequence ID No. I
results in 10 a stop codon). This mutation was shown to be unique to sclerosteosis patients and carriers of Afrikaaner descent. The identity of the gene was further confirmed by identifying a mutation in its intron (A>T mutation at position +3 of the intron) which results in improper mRNA processing in a single, unrelated patient with diagnosed sclerosteosis.

TISSUE-SPECIFICITY OF TGF-BETA BINDING-PROTEIN GENE EXPRESSION
A. Human Beer Gene Expression by RT-PCR:
First-strand cDNA was prepared from the following total RNA samples using a commercially available kit ("Superscript Preamplification System for First-Strand cDNA Synthesis", Life Technologies, Rockville, MD): human brain, human liver, human spleen, human thymus, human placenta, human skeletal muscle, human thyroid, human pituitary, human osteoblast (NHOst from Clonetics Corp., San Diego, CA), human osteosarcoma cell line (Saos-2, ATCC# HTB-85), human bone, human bone marrow, human cartilage, vervet monkey bone, saccharomyces cerevisiae, and human peripheral blood monocytes. All RNA samples were purchased from a commercial source (Clontech, Palo Alto, CA), except the following which were prepared in-house: human osteoblast, human osteosarcoma cell line, human bone, human cartilage and vervet monkey bone. These in-house RNA samples were prepared using a commercially available kit ("TRI Reagent", Molecular Research Center, Inc., Cincinnati, OH).
PCR was performed on these samples, and additionally on a human genomic sample as a control. The sense Beer oligonucleotide primer had the sequence 5'-CCGGAGCTGGAGAACAACAAG-3' (SEQ ID NO:19). The antisense Beer oligonucleotide primer had the sequence 5'-GCACTGGCCGGAGCACACC-3' (SEQ

ID NO:20). In addition, PCR was performed using primers for the human beta-actin gene, as a control. The sense beta-actin oligonucleotide primer had the sequence 5'-AGGCCAACCGCGAGAAGATGA CC -3' (SEQ ID NO:21). The antisense beta-actin oligonucleotide primer had the sequence 5'-GAAGT CCAGGGCGACGTAGCA-3' (SEQ ID NO:22). PCR was performed using standard conditions in 25 ul reactions, with an annealing temperature of 61 degrees Celsius. Thirty-two cycles of PCR
were performed with the Beer primers and twenty-four cycles were performed with the beta-actin primers.
Following amplification, 12 ul from each reaction were analyzed by agarose gel electrophoresis and ethidium bromide staining. See Figure 2A.
B. RNA In-situ Hybridization of Mouse Embryo Sections:
The full length mouse Beer cDNA (Sequence ID No. 11) was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) in the antisense and sense direction using the manufacturer's protocol. 'S-alpha-GTP-labeled cRNA sense and antisense transcripts were synthesized using in-vitro transcription reagents supplied by Ambion, Inc (Austin, TX). In-situ hybridization was performed according to the protocols of Lyons et al. (I. Cell Biol. ///:2427-2436, 1990).
The mouse Beer cRNA probe detected a specific message expressed in the neural tube, limb buds, blood vessels and ossifying cartilages of developing mouse embryos. Panel A in Figure 3 shows expression in the apical ectodermal ridge (aer) of the limb (I) bud, blood vessels (by) and the neural tube (nt). Panel B shows expression in the 4th ventricle of the brain (4). Panel C shows expression in the mandible (ma) cervical vertebrae (cv), occipital bone (oc), palate (pa) and a blood vessel (by). Panel D shows expression in the ribs (r) and a heart valve (va). Panel A is a transverse section of 10.5 dpc embryo. Panel B is a sagittal section of 12.5 dpc embryo and panels C and D are sagittal sections of 15.5 dpc embryos.
ba=branchial arch, h=heart, te=telencephalon (forebrain), b=brain, f=frontonasal mass, g=gut, h=heart, j=jaw, li=liver, lu=lung, ot¨otic vesicle, ao=, sc=spinal cord, skm=skeletal muscle, ns=nasal sinus, th=thymus , to=tongue, fl=forelimb, di=diaphragm EXPRESSION AND PURIFICATION OF RECOMBINANT BEER PROTEIN
A. Expression in COS-1 Cells:

The DNA sequence encoding the full length human Beer protein was amplified using the following PCR oligonucleotide primers: The 5' oligonucleotide primer had the sequence 5'-AAGCTTGGTACCATGCAGCTCCCAC-3' (SEQ ID
NO:23) and contained a HindIII restriction enzyme site (in bold) followed by start codon (ATG). The 3' oligonucleotide primer had the sequence 5'-AAGCTTCTACTTGTCATCGTCGTCCT TGTAGTCGTAGGCGTTCTCCAGCT-3' (SEQ ID NO:24) and contained a HindIII restriction enzyme site (in bold) followed by a reverse complement stop codon (CTA) followed by the reverse complement of the The PCR product was TA cloned ("Original TA Cloning Kit", Invitrogen, Carlsbad, CA) and individual clones were screened by DNA sequencing. A sequence-verified clone was then digested by HindIII and purified on a 1.5% agarose gel using a 15 commercially available reagents ("QIAquick Gel Extraction Kit", Qiagen Inc., Valencia, CA). This fragment was then ligated to HindIII digested, phosphatase-treated pcDNA3.1 (Invitrogen, Carlsbad, CA) plasmid with T4 DNA ligase. DH1OB E. coli were transformed and plated on LB, 100 1.1.g/m1 ampicillin plates. Colonies bearing the desired recombinant in the proper orientation were identified by a PCR-based screen, primer with the sequence 5'- GCACTGGCCGGAGCACACC-3' (SEQ ID NO:25) that corresponds to the reverse complement of internal BEER sequence. The sequence of the cloned fragment was confirmed by DNA sequencing.
COS-1 cells (ATCC# CRL-1650) were used for transfection. 50 pg of 25 the expression plasmid pcDNA-Beer-Flag was transfected using a commercially available kit following protocols supplied by the manufacturer ("DEAE-Dextran Transfection Kit", Sigma Chemical Co., St. Louis, MO). The final media following transfection was DMEM (Life Technologies, Rockville, MD) containing 0.1% Fetal Bovine Serum. After 4 days in culture, the media was removed. Expression of 35 monoclonal antibody.

_WO 00/32773 B. Expression in SF9 insect cells:
The human Beer gene sequence was amplified using PCR with standard conditions and the following primers:
Sense primer: 5'-GTCGTCGGATCCATGGGGTGGCAGGCGTTCAAGAATGAT-3' (SEQ ID NO:26) Antisense primer: 5'-GTCGTCAAGCTTCTACTTGTCATCGTCCTTGTAGTCGTA
GGCGTTCTCCAGCTCGGC-3' (SEQ ID NO:27) The resulting cDNA contained the coding region of Beer with two modifications. The N-terminal secretion signal was removed and a FLAG epitope tag (Sigma) was fused in frame to the C-terminal end of the insert. BamH1 and HindIII
cloning sites were added and the gene was subcloned into pMelBac vector (Invitrogen) for transfer into a baculoviral expression vector using standard methods.
Recombinant baculoviruses expressing Beer protein were made using the Bac-N-Blue transfection kit (Invitrogen) and purified according to the manufacturers instructions.
SF9 cells (Invitrogen) were maintained in TNM_FH media (Invitrogen) containing 10% fetal calf serum. For protein expression, SF9 cultures in spinner flasks were infected at an MOI of greater than 10. Samples of the media and cells were taken daily for five days, and Beer expression monitored by western blot using an anti-FLAG
M2 monoclonal antibody (Sigma) or an anti-Beer rabbit polyclonal antiserum.
After five days the baculovirus-infected SF9 cells were harvested by centrifugation and cell associated protein was extracted from the cell pellet using a high salt extraction buffer (1.5 M NaC1, 50 mM Tris pH 7.5). The extract (20 ml per ml culture) was clarified by centrifugation, dialyzed three times against four liters of Tris buffered saline (150 mM NaC1, 50 mM Tris pH 7.5), and clarified by centrifugation again. This high salt fraction was applied to Hitrap Heparin (Pharmacia;
5 ml bed volume), washed extensively with HEPES buffered saline (25 mM HEPES
7.5, 150 mM Nacl) and bound proteins were eluted with a gradient from 150 mM
NaC1 to 1200 mM NaCl. Beer elution was observed at aproximately 800 mM NaCI. Beer ¨80 degrees C.

PREPARATION AND TESTING OF POLYCLONAL ANTIBODIES TO BEER, GREMLIN, AND
DAN

_WO 00/32773 A. Preparation of antigen:
The DNA sequences of Human Beer, Human Gremlin, and Human Dan were amplified using standard PCR methods with the following oligonucleotide primers:
H. Beer Sense: 5' -GACTTGGATCCCAGGGGTGGCAGGCGTTC- 3' (SEQ ID NO:28) Antisense 5' -AGCATAAGCTTCTAGTAGGCGTTCTCCAG- 3' (SEQ ID NO:29) H. Gremlin Sense: 5' -GACTTGGATCCGAAGGGAAAAAGAAAGGG- 3' (SEQ ID NO:30) Antisense: 5' -AGCATAAGCTTTTAATCCAAATCGATGGA- 3' (SEQ ID NO:31) H. Dan Sense: 5' -ACTACGAGCTCGGCCCCACCACCCATCAACAAG- 3' (SEQ ID NO:32) Antisense: 5' -ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTCC-3' (SEQ ID
NO :33) In each case the listed primers amplified the entire coding region minus the secretion signal sequence. These include restriction sites for subcloning into the bacterial expression vector pQE-30 (Qiagen Inc., Valencia, CA) at sites BamHI/HindII1 for Beer and Gremlin, and sites SacI/HindIII for Dan. pQE30 contains a coding sequence for a 6x His tag at the 5' end of the cloning region. The completed constructs were transformed into E. coli strain M-15/pRep (Qiagen Inc) and individual clones verified by sequencing. Protein expression in M-15/pRep and purification (6xHis affinity tag binding to Ni-NTA coupled to Sepharose) were performed as described by the manufacturer (Qiagen, The QIAexpressionist).
The E. coil-derived Beer protein was recovered in significant quantity using solubilization in 6M guanidine and dialyzed to 2-4M to prevent precipitation during storage. Gremlin and Dan protein were recovered in higher quantity with solubilization in 6M guanidine and a post purification guanidine concentration of 0.5M.
B. Production and testing of polyclonal antibodies:
Polyclonal antibodies to each of the three antigens were produced in rabbit and in chicken hosts using standard protocols (R & R Antibody, Stanwood, WA;
standard protocol for rabbit immunization and antisera recovery; Short Protocols in Molecular Biology. 2nd edition. 1992. 11.37- 11.41. Contributors Helen M.
Cooper and Yvonne Paterson; chicken antisera was generated with Strategic Biosolutions, Ramona, CA).
Rabbit antisera and chicken egg Igy fraction were screened for activity via Western blot. Each of the three antigens was separated by PAGE and transferred to 0.45um nitrocellulose (Novex, San Diego, CA). The membrane was cut into strips with each strip containing approximately 75 ng of antigen. The strips were blocked in 3%
Blotting Grade Block (Bio-Rad Laboratories, Hercules, CA) and washed 3 times in IX
5 Tris buffer saline (TBS) /0.02% TWEEN buffer.
The primary antibody (preimmunization bleeds, rabbit antisera or chicken egg IgY in dilutions ranging from 1:100 to 1:10,000 in blocking buffer) was incubated with the strips for one hour with gentle rocking. A second series of three washes 1X TBS/0.02%TWEEN was followed by an one hour incubation with the secondary antibody (peroxidase conjugated donkey 10 anti-rabbit, Amersham Life Science, Piscataway, NJ; or peroxidase conjugated donkey anti-chicken, Jackson ImmunoResearch, West Grove, PA). A final cycle of 3X
washes of IX TBS/0.02%TWEEN was performed and the strips were developed with Lumi-Light Western Blotting Substrate (Roche Molecular Biochemicals, Mannheim, Germany).
C. Antibody cross-reactivity test:
Following the protocol described in the previous section, nitrocellulose strips of Beer, Gremlin or Dan were incubated with dilutions (1:5000 and 1:10,000) of their respective rabbit antisera or chicken egg IgY as well as to antisera or chicken egg Igy (dilutions 1:1000 and 1:5000) made to the remaining two antigens. The increased levels of nonmatching antibodies was performed to detect low affinity binding by those antibodies that may be seen only at increased concentration. The protocol and duration of development is the same for all three binding events using the protocol described above. There was no antigen cross-reactivity observed for any of the antigens tested.
= 25 = INTERACTION OF BEER WITH TGF-BETA SUPER-FAMILY PROTEINS
The interaction of Beer with proteins from different phylogenetic arms of the TGF-13 superfamily were studied using immunoprecipitation methods.
Purified = TGFf3-1, TGF13-2, TGFf3-3, BMP-4, BMP-5, BMP-6 and GDNF were obtained from commerical sources (R&D systems; Minneapolis, MN). A representative protocol is as follows. Partially purified Beer was dialyzed into HEPES buffered saline (25 mM
HEPES 7.5, 150 mM NaCl). Immunoprecipitations were done in 300 ul of IP buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1mM EDTA, 1.4 mM 13-mercaptoethanol, 0.5 %
triton X 100, and 10% glycerol). 30 ng recombinant human BMP-5 protein (R&D

_WO 00/32773 systems) was applied to 15 ul of FLAG affinity matrix (Sigma; St Louis MO)) in the presence and absence of 500 ng FLAG epitope-tagged Beer. The proteins were incubated for 4 hours @ 4 Cand then the affinity matrix-associated proteins were washed 5 times in IP buffer (1 ml per wash). The bound proteins were eluted from the affinity matrix in 60 microliters of IX SDS PAGE sample buffer. The proteins were resolved by SDS PAGE and Beer associated BMP-5 was detected by western blot using anti-BMP-5 antiserum (Research Diagnostics, Inc) (see Figure 5).
BEER Ligand Binding Assay:
FLAG-Beer protein (20 ng) is added to 100 ul PBS/0.2% BSA and adsorbed into each well of 96 well microtiter plate previously coated with anti-FLAG
monoclonal antibody (Sigma; St Louis MO) and blocked with 10% BSA in PBS. This is conducted at room temperature for 60 minutes. This protein solution is removed and the wells are washed to remove unbound protein. BMP-5 is added to each well in concentrations ranging from10 pM to 500 nM in PBS/0.2% BSA and incubated for 2 hours at room temperature. The binding solution is removed and the plate washed with three times with 200u1 volumes of PBS/0.2% BSA. BMP-5 levels are then detected using BMP-5 anti-serum via ELISA (F.M. Ausubel et al (1998) Current Protocols in Mol Biol. Vol 2 11.2.1-11.2.22). Specific binding is calculated by subtracting non-2() specific binding from total binding and analyzed by the LIGAND program (Munson and Podbard, Anal. Biochem., 107, p220-239, (1980).
In a variation of this method, Beer is engineered and expressed as a human Fc fusion protein. Likewise the ligand BMJI is engineered and expressed as mouse Fc fusion. These proteins are incubated together and the assay conducted as described by Mellor et al using homogeneous time resolved fluorescence detection (G.W. Mellor et al., J of Biornol Screening, 3(2) 91-99, 1998).

SCREENING ASSAY FOR INHIBITION OF TGF-BETA BINDING-PROTEIN
BINDING TO TGF -BETA FAMILY MEMBERS
The assay described above is replicated with two exceptions. First, BMP concentration is held fixed at the Kd determined previously. Second, a collection of antagonist candidates is added at a fixed concentration (20 uM in the case of the small organic molecule collections and 1 uM in antibody studies). These candidate molecules (antagonists) of TGF-beta binding-protein binding include organic compounds derived from commercial or internal collections representing diverse chemical structures. These compounds are prepared as stock solutions in DMSO
and are added to assay wells at < 1% of final volume under the standard assay conditions.
These are incubated for 2 hours at room temperature with the BMP and Beer, the solution removed and the bound BMP is quantitated as described. Agents that inhibit 40% of the BMP binding observed in the absence of compound or antibody are considered antagonists of this interaction. These are further evaluated as potential inhibitors based on titration studies to determine their inhibition constants and their influence on TGF-beta binding-protein binding affinity. Comparable specificity control assays may also be conducted to establish the selectivity profile for the identified antagonist through studies using assays dependent on the BMP ligand action (e.g. BMP/BMP receptor competition study).

INHIBITION OF TGF-BETA BINDING-PROTEIN LOCALIZATION TO BONE MATRIX
Evaluation of inhibition of localization to bone matrix (hydroxyapatite) is conducted using modifications to the method of Nicolas (Nicolas, V. (Jaleif Tissue bit 57:206, 1995). Briefly, '25I-labelled TGF-beta binding-protein is prepared as described by Nicolas (supra). Hydroxyapatite is added to each well of a 96 well microtiter plate equipped with a polypropylene filtration membrane (Polyfiltroninc, Weymouth MA). TGF-beta binding-protein is added to 0.2% albumin in PBS buffer.

The wells containing matrix are washed 3 times with this buffer. Adsorbed TGF-beta binding-protein is eluted using 0.3M NaOH and quantitated.
Inhibitor identification is conducted via incubation of TGF-beta binding-protein with test molecules and applying the mixture to the matrix as described above.
The matrix is washed 3 times with 0.2% albumin in PBS buffer. Adsorbed TGF-beta binding-protein is eluted using 0.3 M NaOH and quantitated. Agents that inhibit 40%
of the TGF-beta binding-protein binding observed in the absence of compound or antibody are considered bone localization inhibitors. These inhibitors are further characterized through dose response studies to determine their inhibition constants and their influence on TGF-beta binding-protein binding affinity.

CONSTRUCTION OF TGF-BETA BINDING-PROTEIN MUTANT
A. Mutagenesis:
A full-length TGF-beta binding-protein cDNA in pBluescript SK serves as a template for mutagenesis. Briefly, appropriate primers (see the discussion provided above) are utilized to generate the DNA fragment by polymerase chain reaction using Vent DNA polymerase (New England Biolabs, Beverly, MA). The polymerase chain reaction is run for 23 cycles in buffers provided by the manufacturer using a 57 C annealing temperature. The product is then exposed to two restriction enzymes and after isolation using agarose gel electrophoresis, ligated back into pRBP4-503 from which the matching sequence has been removed by enzymatic digestion.
Integrity of the mutant is verified by DNA sequencing, B. Mammalian Cell Expression and Isolation of Mutant TGF-beta binding-protein:
The mutant TGF-beta binding-protein cDNAs are transferred into the pcDNA3.1 mammalian expression vector described in EXAMPLE 3. After verifying the sequence, the resultant constructs are transfected into COS-1 cells, and secreted protein is purified as described in EXAMPLE 3.

ANIMAL MODELS -I
GENERATION OF TRANSGENIC MICE OVEREXPRESSING THE BEER GENE
The ¨200 kilobase (kb) BAC clone 15G5, isolated from the CITB mouse genomic DNA library (distributed by Research Genetics, Huntsville, AL) was used to determine the complete sequence of the mouse Beer gene and its 5' and 3' flanking regions. A 41 kb Sall fragment, containing the entire gene body, plus ¨17 kb of 5' flanking and ¨20 kb of 3' flanking sequence was sub-cloned into the BamHI site of the SuperCosI cosmid vector (Stratagene, La Jolla, CA) and propagated in the E.
coil strain DH10B. From this cosmid construct, a 35 kb MluI - AviII restriction fragment (Sequence No. 6), including the entire mouse Beer gene, as well as 17 kb and 14 kb of 5' and 3' flanking sequence, respectively, was then gel purified, using conventional means, and used for microinjection of mouse zygotes (DNX Transgenics; US
Patent No. 4,873,191). Founder animals in which the cloned DNA fragment was integrated randomly into the genome were obtained at a frequency of 5-30% of live-born pups.
The presence of the transgene was ascertained by performing Southern blot analysis of genomic DNA extracted from a small amount of mouse tissue, such as the tip of a tail.
DNA was extracted using the following protocol: tissue was digested overnight at 55 C in a lysis buffer containing 200 mM NaCl, 100 mM Tris pH8.5, 5 mM EDTA, 0.2%

SDS and 0.5 mg/ml Proteinase K. The following day, the DNA was extracted once with phenol/chloroform (50:50), once with chloroform/isoamylalcohol (24:1) and precipitated with ethanol. Upon resuspension in TE (10mM Tris pH7.5, 1 mM
EDTA) 8-10 ug of each DNA sample were digested with a restriction endonuclease, such as EcoRI, subjected to gel electrophoresis and transferred to a charged nylon membrane, such as HyBondN+ (Amersham, Arlington Heights, IL). The resulting filter was then hybridized with a radioactively labelled fragment of DNA deriving from the mouse Beer gene locus, and able to recognize both a fragment from the endogenous gene locus and a fragment of a different size deriving from the transgene. Founder animals were bred to normal non-transgenic mice to generate sufficient numbers of transgenic and non-transgenic progeny in which to determine the effects of Beer gene overexpression.
For these studies, animals at various ages (for example, 1 day, 3 weeks, 6 weeks, 4 months) are subjected to a number of different assays designed to ascertain gross skeletal formation, bone mineral density, bone mineral content, osteoclast and osteoblast activity, extent of endochondral ossification, cartilage formation, etc. The transcriptional activity from the transgene may be determined by extracting RNA from various tissues, and using an RT-PCR assay which takes advantage of single nucleotide polymorphisms between the mouse strain from which the transgene is derived (129Sv/J) and the strain of mice used for DNA microinjection [(C57BL5/J x SJI/J)F2].
ANIMAL MODELS ¨II
DISRUPTION OF THE MOUSE BEER GENE BY HOMOLOGOUS RECOMBINATION
Homologous recombination in embryonic stem (ES) cells can be used to inactivate the endogenous mouse Beer gene and subsequently generate animals carrying the loss-of-function mutation. A reporter gene, such as the E. coli P-galactosidase gene, was engineered into the targeting vector so that its expression is controlled by the endogenous Beer gene's promoter and translational initiation signal. In this way, the spatial and temporal patterns of Beer gene expression can be determined in animals carrying a targeted allele.
The targeting vector was constructed by first cloning the drug-selectable phosphoglycerate kinase (PGK) promoter driven neomycin-resistance gene (neo) cassette from pGT-N29 (New England Biolabs, Beverly, MA) into the cloning vector pSP72 (Promega, Madson, WI). PCR was used to flank the PGKneo cassette with bacteriophage P1 loxP sites, which are recognition sites for the P1 Cre recombinase (Hoess et al., PNAS USA, 79:3398, 1982). This allows subsequent removal of the neo-resistance marker in targeted ES cells or ES cell-derived animals (US Patent 4,959,317). The PCR primers were comprised of the 34 nucleotide (ntd) loxP
5 sequence, 15-25 ntd complementary to the 5' and 3' ends of the PGKneo cassette, as well as restriction enzyme recognition sites (BamHI in the sense primer and EcoRI in the anti-sense primer) for cloning into pSP72. The sequence of the sense primer was 5"-AATCTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCTGCAG
10 GATTCGAGGGCCCCT-3' (SEQ ID NO:34); sequence of the anti-sense primer was 5'-AATCTGAATTCCACCGGTGTTAATTAAATAACTTCGT
ATAATGTATGCTATACGAAGTTATAGATCTAGAG TCAGCTTCTGA-3' (SEQ
ID NO:35).
The next step was to clone a 3.6 kb XhoI-HindIII fragment, containing 15 the E. coli 13-ga1actosidase gene and SV40 polyadenylation signal from pSVI3 (Clontech, Palo Alto, CA) into the pSP72-PGKneo plasmid. The "short arm" of homology from the mouse Beer gene locus was generated by amplifying a 2.4 kb fragment from the BAC clone 15G5. The 3' end of the fragment coincided with the translational initiation site of the Beer gene, and the anti-sense primer used in the PCR
20 also included 30 ntd complementary to the 5' end of the 13-galaciosidase gene so that its coding region could be fused to the Beer initiation site in-frame. The approach taken for introducing the "short arm" into the pSP72-f3gal-PGKneo plasmid was to linearize the plasmid at a site upstream of the p-gal gene and then to co-transform this fragment with the "short arm" PCR product and to select for plasmids in which the PCR
product 25 was integrated by homologous recombination. The sense primer for the "short arm"
amplification included 30 ntd complementary to the pSP72 vector to allow for this recombination event. The sequence of the sense primer was 5'-ATTTAGGTGACACT
ATAGAACTCGAGCAGCTGAAGCTTAACCACATGGTGGCTCACAACCAT-3' (SEQ ID NO:36) and the sequence of the anti-sense primer was 5'-ATCATGGTCATGCTGCCAGGTGGAGGAGGGCA-3' (SEQ ID NO:37).
The "long arm" from the Beer gene locus was generated by amplifying a 6.1 kb fragment from BAC clone 15G5 with primers which also introduce the rare-cutting restriction enzyme sites SgrAI, FseI, AscI and Pad. Specifically, the sequence 35 of the sense primer was 5'-ATTACCACCGGTGACACCCGCTTCCTGACAG-3' (SEQ
ID NO:38); the sequence of the anti-sense primer was ATTACTTAATTAAACATGGCGCGCCAT
ATGGCCGGCCCCTAATTGCGGCGCATCGTTAATT-3' (SEQ ID NO:39). The resulting PCR product was cloned into the TA vector (Invitrogen, Carlsbad, CA) as an intermediate step.
The mouse Beer gene targeting construct also included a second selectable marker, the herpes simplex virus I lhymidine kinase gene (HSVTK) under the control of rous sarcoma virus long terminal repeat element (RSV LTR).
Expression of this gene renders mammalian cells sensitive (and inviable) to gancyclovir; it is therefore a convenient way to select against neomycin-resistant cells in which the construct has integrated by a non-homologous event (US Patent 5,464,764). The RSVLTR-HSVTK cassette was amplified from pPS1337 using primers that allow subsequent cloning into the FseI and AscI sites of the "long arm"-TA vector plasmid.
For this PCR, the sequence of the sense primer was 5'-ATTACGGCCGGCCGCAAAGGAATTCAAGA TCTGA-3' (SEQ ID NO:40); the sequence of the anti-sense primer was 5'-ATTACGGCGCGCCCCTC
ACAGGCCGCACCCAGCT-3' (SEQ ID NO:41).
The final step in the construction of the targeting vector involved cloning the 8.8 kb SgrAI-AscI fragment containing the "long arm" and RSVLTR-HSVTK gene into the SgrAI and AscI sites of the pSP72-"short arm"-figal-PGKneo plasmid. This targeting vector was linearized by digestion with either AscI or Pad before electroporation into ES cells.

ANTISENSE-MEDIATED BEER INACTIVATION
17-nucleotide antisense oligonucleotides are prepared in an overlapping format, in such a way that the 5' end of the first oligonucleotide overlaps the translation initiating AUG of the Beer transcript, and the 5' ends of successive oligonucleotides occur in 5 nucleotide increments moving in the 5' direction (up to 50 nucleotides away), relative to the Beer AUG. Corresponding control oligonucleotides are designed and prepared using equivalent base composition but redistributed in sequence to inhibit any significant hybridization to the coding mRNA. Reagent delivery to the test cellular system is conducted through cationic lipid delivery (PI,. Feigner, Proc. Nall.
Acad Sci. USA 84:7413, 1987). 2 ug of antisense oligonucleotide is added to 100 ul of reduced serum media (Opti-MEM I reduced serum media; Life Technologies, Gaithersburg MD) and this is mixed with Lipofectin reagent (6 ul) (Life Technologies, Gaithersburg MD) in the 100 ul of reduced serum media. These are mixed, allowed to complex for 30 minutes at room temperature and the mixture is added to previously seeded MC3T3E21 or KS483 cells. These cells are cultured and the mRNA
recovered.
Beer mRNA is monitored using RT-PCR in conjunction with Beer specific primers.
In addition, separate experimental wells are collected and protein levels characterized through western blot methods described in Example 4. The cells are harvested, resuspended in lysis buffer (50 mM Tris pH 7.5, 20 mM Naa, 1mM EDTA, 1% SDS) and the soluble protein collected. This material is applied to 10-20 %
gradient denaturing SDS PAGE. The separated proteins are transferred to nitrocellulose and the western blot conducted as above using the antibody reagents described. In parallel, the control oligonucleotides are added to identical cultures and experimental operations are repeated. Decrease in Beer mRNA or protein levels are considered significant if the treatment with the antisense oligonucleotide results in a 50% change in either instance compared to the control scrambled oligonucleotide. This methodology enables selective gene inactivation and subsequent phenotype characterization of the mineralized nodules in the tissue culture model.

SEQUENCES
Sequence ID No. 1: Human BEER cDNA (complete coding region plus 5'and 3' UTRs) AGAGCCTGT GCTACTGGAAGGTGGCGTGCCCTCCTCTGGCT GGTACCAT GCAGCTCCCACTGGCCCT GTGTC T
CGTCTGC
CT GCTGGT ACACACP.GC CT T CCGTGT AGTGGAGGGCCAC-IGC-;GT GGCAGGCGT T CAAGAAT T
GCCACGGAAA T CAT CCC
CGAGCTCGGAGAGTACCCCC-;AGCCTCCA,ccGGAGCTGGAGAACAACAAGACCATGAACCGGGCGGAGA.ACGGAGGGCGGC
CCCCACCACCCCT
TTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCAC:TTCACCCGCTACGTGACCGAT
GGGCCGTCCC.GCGCCPAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCG3CC,CGCCTGCTGCCCPACGC

C
AGCTGCTGTGT
CCCGGTGGTC;AGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACC
CGCTTCCAC.;ACCAGTCGGAGCTC:P-Ja.GC-;ACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGC.:GGCCCCG
CGCCCGGAGCGC.CAAAGCCAACCAGGCCGAGCTGGAGPACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGG
GC
GCCCCGGCCCT GAACCCGCGCCC CACATT TCTGTCCT CT GCGCGTGGT T T GAT T OT T T AT.AT T
T CAT TGTAAATGCCTGC
AACCCAGGGCAGGGGGCTGAGAC CT TCCAGGCCCT
GAGGAATCCCGGGCGCCGC;CAAGGCCCCCCTCAGCCCGOCAGCTG
AGGGGTCCCACGGG'GCAGGGGAGGGAATTGAGAGT
CACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCT
T TGCT GGTCCCACT TCAGAGGAGGCAGPAATGGAAGCAT T T T
CACCGCCCTC;GGGTTTTAAGGGAGCGGTGTGGGAGTGG
GAAAGTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTG
C
CCAGAGCACGACT GGGGGCP,ACT GT AGATGTGGTTT CTAGTCCTGGCTCTGCCACT AACTT
GCTGTGTAACCT TGAAC
TP.CACP.ATTCTCCTTCGGGACCTCPATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGRATAGGATCT
CGAGGAGACT AT
TGGakTATGATTCCAAGGACT CCAGT
GCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAAT G
CAGTTGCATTGATTCAGTGCCAAGGTCACTT CCAGAAT T CAGAGT T GT GATGCT CTCT
TCTGACAGCCAAAGATGAAAAA
CAAACAGAAAAAAAAAAGTAAAGAGT CTAT T TATGGCTGACP,TAT T T ACGGCTGACAAACTCCT
GGAAGAP,GCT ATGCTG
CT T CCCAGCCT GGCT TCCCCGGAT G T T TGGCTACCTCCACCCCTCCAT CT CAA,AGAAATAACAT
CATCCATT GGGGTAGA
AAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCP,ACTTCCCAAAGAGCAGCATCCCTCCCC
CG
ACCCATAGCCAT GT T T TAAAGTCAC CT TCCGAAGAGAAGTGAAAG GT TCAAGGACACTGGCCT
TGCAGGCCCG'AGGGAGC
AGCCATCACAPACT CACAGACCAGCACAT CCCTTT TGAGACACCGCCT TCTGCCCACCACTCACC-rGACACAT
TT CTGC'CT
AGAAA.ACAGCTT CT TACTGCT CT T ACATGT GATGGCATATCT TACACT AAAAGAA TAT T AT
TGGGGGAAAAACTACAAGT
GCTGTACATATGCTGAGAAACTGCAGP.GCATAATAGCTGCCACCCA.AAAATCTTTTTGAAAATCATTTCCAGACAAC
CTC
TTACT TT CT GT GTAGT T T T TAAT T GT TAAAAAAAAAAAGT T T
TAAACAGAAGCACATGACATATGAAAGCCTGCAGGACT
GGTCGT T T T T T T GGCAA T TCT TCCACGTGGGACT T GT CCACAAGAAT GAAAGTAGTGGTT T T
T AAAG21.GT TAAGT TACA T
AT T T AT T T T CT CACT T AAGT TAT T TATGCAAAAGT TTTT CT TGTAGAGAATGACAATGT
TAATAT TGCT T TATGAAT TAA
CAGTCTGT T CT T CCAGAGTCCAGAGACAT TGT T AATAAAGACAATGAATCA TGACCGAAAG

Sequence ID No. 2: Human BEER protein (complete sequence) MQL P LA LC LVC L LVHTA ERVVEGQGWQA PKN DATE IIPE LGEY PEPE' PE
LENNKTMNRAENGGRP PH H P FE T K DVSEYSC
RE LH FT RYVT DGPCR SAK PVT E LVC SGQCG PAR L L PNAIGRGKWWR P SG E. DFRC I P
DRY RAQRVQL, LC EGGEA. PRARKVR
LVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY
Sequence ID No. 3: Human Beer cDNA containing Sclerosteosis nonsense mutation AGAGCCT C.4T GCTACTGG.PAGGTGGCGT GCCCTCCTCTGGCT GGTACCPT GCAGCT CCCACTGGCC CT
GTGTCTCGT CTGC
CTGCTGGTACACACAGCCTT CCGTGTAGTGGAGGGCTAGGGGTGGCAGGCGTTCAAGAAT
GATGCCACGGP.P.P.T. CP.T CC
CGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAACAACAP.GACCATGAACCGGGCGGAGAACGGAGGGCG
GC
CTCCCCACCACCCCTTTGAGACCA.P.P.GACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCAC:CCGCTACGTGA
CCGAT
GGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGT
GCTCCGGCCP.GTGCGGCCCGGCGCGCCTGCTGCCCAACGC
CATCGGCCGCC_;GC.P.AGTGGTGGCGACCTAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCCCGCAGCG
CGTGC
AGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCP.P.GTGCAP.GCC;CC
TCACC
CGCTT CCACAACCAGT CGGAGCTCRAGGACTT CGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGC
GGCCCCG
CGCCCGGAGCGCCA.PAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGG
GC
GCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTG
C
AACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCT
G
AGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACC
T
TTGCTGGTCCCACTTCAGAGGAGGCAGAAATGGAAGCATTTT CACCGCCCT GGGGTTT
TAAGGGAGCGGTGTGGGAGT GG
GAAP.GTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGT
GC
CCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGT T T CT AGTCCT GGCTCT GCCACT AACT
TGCTGTGTAACCT TG.B.AC
TACACAATT CT CCTTCGGGACCT CAATT TCCACT TTGTAAAAT GAGGGTGGAGGTGGGAAT AGGA TCT
CGAGGAGACT AT
TGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAAT
G
CAGT TGCAT TGAT TCAGTGCCAAGGTCACT TCCAGAAT TCAGAGT TGT GAT GCT CTCT T
CTGACAGCCAAAGATGAAAAA
CAAACAGAAAAAAAAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCT
G
CT TCCCAGCCTGGCT T CCCCGGATGT T T GGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCAT
TGGGGTAGA
AAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCC.AACTTCCCAAAGAGCAGCATCCCTCCCC
CG
ACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAG
C
AGCCATCACAAACTCACAGACCAGCACATCCCT TT TGAGACACCGCCT TCTGCCCACCACTCACGGACACAT T
TCTGCCT
P.GAAAACAGCTT CTTACTGCT CT TACATGTGATGGCATAT CT
TACACTAAAAGAATATTATTGGGGGAAAAACTACAAGT
GCTGT ACATATGCTGAGAWT GCAGAGCATAATAGCTGCCACCCAAAAA T CT T T T TGAAAATCAT
TTCCAGACAACCT C

TTACTTTCTGTGTAGTTTTTAA TTGT TAAAAAPAAAPAGTT T TAPACAG,kAGCACATGAC:P, TAT
GAAAGCCTGCAGGAC",-GGT CGT TTTTT TGGCAATT CTTCCACGTGGGACT T GT CCACAAGAATGAAAGTAGT GGTT TT
TPAAGAGTTAAGTTACAT
ATTTATTTT CT CACTTPAGTTATTTATGCAAPAGT TTT T CT TGTAGAGAATGACP,ATGT TAATATT GCT
TTATGAATTAP.
CAGTCTGTTCTTCCAGAGTCCAGAGACATTGTTAATWGACAATGAATCATGACCGA.a.AG

Sequence ID No. 4: Truncated Human Beer protein from Sclerosteosis =
MQ LPLP LC LV C L LVHTP,FRVVEG*
Sequence II) No. 5: Human BEER cDNA. encoding protein variant (V10I) CCCACT GGCC CT GTGTCTCAT CT GC
CTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCG TT
CA.AGAP,TGATGCCACGG,AAATCATCCG
CGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAP.CPACAAGACCATGAACCGGGCGGAGP,ACGGAGGGC
GGC
CTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGA
T
GGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCT
GCTGCCCAAC(.;C:

TCCGCTGCATCCCCGP,CCGCTACCGCGCGCAGCGCGTGC
AGCT G CT G T GT CC CG GT GGT GAG GC GCCG CGCGCG CGC AAGGT G C GCCT GGT GG C
CT C GT GCAAGT GC AAGC GC CT CAC C
CGCT
TCCACAACCAGTCGGP.GCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCG
CGCCCGGAGCGCCAPAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCG.G
.GC
GCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTC-iTTTATATTTCATTGTAP,P,TGCCTGC

AACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCT
G
AGGG GT CCCACGGGGCAGGGGAGGGAATT GAGAGTCACAGACACTGAGCCACGCAGCCCCGCCT CT GGGGC
CGCCTACCT
TTGCTGGTCCCACTTCAGAGGAGGCAGAPATGGAP.GcATT.TTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAG
TGG
GAAAGTCCAGGGACTGGTTAAGAPAGTTGGATAAGATTCCCCCTTG'CACCTCGCTGCCCP.TCAG.V.AGCCTGAGGC
GTGC
CCAGAGCACARGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTIVIETTGCTGTGTAACCTTGA
P.0 CCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGP.TCTCGAGGAGACTAT
TGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAAT
GGGCAGAGGTGAGAGAGAGP,GAGAGAAAGAGAGAGAATGAATG
CAGTTGCATTGATTCAGTGCCAAGGT CACTTCCAGAATTCAGAGTTGTGATGCTCTCTT
CTGACAGCCAAAGATGAAAAA
CAAACAGAAAAAP,AAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGPAGAAGCTATGC
TG
CTTCCCAGCCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATPACATCATCCATTGGGGTAG
A

APAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCAACTTCCCAAAGAGCAGCATCCCTCCCCC
G
ACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAG
C

AGCCATCACAAACTC.;CAGP:CCAGCACATCCCITTTGAGP.CA.CCGCCTTCTGCCCACCACTCACGUACACATTTC
TGCCT
AGP.A.a.ACAGCTTOTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTAC
AAGT
GCTGTP,CATATGCTGAGA.V.CTGCAGAGCATAATP,GCTGCCACCCAWATCTTTTTGARAATCATTTCCAGACAACC
TC
TTICTTTCTGTGTAGTTTTMATTGTT.VA;AAAAMAGTTTTAPACAGAAGCACATGACATATGAAAGCCTGCAGGACT
,GC.;TCGTTTT TT TUGCAATTCT TCCACGT GGGACTTOTCCAC.A.AGAATGAAAGTAGT CGTT T
LVAGAGTTAAGTTACAT
ATT TATTTT CTCACT TP.A.GTTAT TTATGCAPAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTT
TATGA):.TTAA
CAGT CTGTTCTTCCAGAGTCCAGAGACATTGTTAATAAAGACAATC4,AATCATG.a.CCGPAn,G
Sequence ID No. 6: Human BEER protein variant (VIOI) :AQLELALCLICLLVI-ITAERVVEGQGWQAFKNDATEI I RE LGEY PE F
EEELENNKTMNRZENGGREEHHFETKDVSEySC
RELH FT RYVT DG CRSK PVT E LVC SGQCG EP.R L L GRGKWW,R F SGF DE-RC' P DRY
RAQRVQL LC P GGE A P URK.VR
INRSCKCKR LT R FI-IN Q SE L KDEGT EAARPQKGRKPRPRARSPYANQP.ELENPN
Sequence ID No. 7: Human Beer cDNA encoding protein variant (P38R) AGF,.GCCTGTC4CTIACTGGPAGGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTC,TGTCTC
GTCTGC
CTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCGTTCPGAATGATGCCACGGAAATCATCCG

CGAGCTCGGAGAGTACCCCGAUCCTCCACCGGAGCT
GGAGAACAACAAGACCATGIACCGGGCGGAGAACGGAGGGCGGC
CT CCCCACCACCCCTTTGAGACCAPAGACGT GT CCGAGTACAGCTGCCGCGAGCT
GCACTTCP.CCCGCTACC4T GACCGAT
GGGCCGTGCCGCAUCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCOCC;GCGCGCCTGCTGCCCAAC
GC
cmcGGccGc:GGcAAGTGGTGGcGAccrAGTGGGcccGAcrrccGcTGcATccccGAccGcmccGCGCGCAGCGCGTGC

AGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCOCAAGGTGCGCCTGGTGGCCTCGTGCAP.GTGCAAGCGCCTCA
CC
CGCTTCCACAACCAGTOGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGOGGCCCC
G
CGCCCGUAGCGCCAPAGCCAACCAGGCCGAGCTGGAG.V.CGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGG
GC
GCCCCGGCCCTGPACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTT CAT
TGTPAkTGCCTGC
PACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCOCTCAGCCCGCCAGCT
G
AGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACC
T
TTGCTGG T CCCACTTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCC CTGGGGTTT
TAAGGGAGCGGTGTGGGAGTGG
GAAAGTCCAGGGACTGGTTAAGAAAGTTGGAT.n.AGATTCCCCCTTGCACCTCGCT
GCCCATCAGANN,GCCTGAGGCGTGC
CCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCT
GTGTAACCTTGAAC
TACACAATTCTCCTTCGGGACCTCAATTTCCACTTTGTAP-nATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTAT
TGGCATATGATTCCPAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGRAT
G

CAGTTGCATTGATTCAGTGCCAAGGTCACTTCCAGAATTCAGA'GTTGTGATGCTCTCT T
CTGACAGCCAP.AGATGAAAAA
CAAACAGPAA.P.PAAAAAGTAPAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGPAGPAGCTATG
CTG
CTTCCCAGCCT GGCTT CCCCGGATC.2T TT GGCTACCTCCACCCCTCCAT
CTCAAAGAAPTPACATCATCCATTGGGGT.AGA
AAAGGP.GAGGGTCCGAGGGTGGTGGGAGGGATAGAPATCACATCCGCCCCAACTTCCCAPAGAGCAGCATCCCTCCCC
CG
ACCCATAG CCAT GTTT TAAAGT CACCT TCCGAAGAGAAGTGAPAGGTT CAAGGACACTGGC CT
TGCAGGCCCGAGGGAGC
AGCCAT CACAAACT CACAGACCAGCACATCCCT TTTGAGACACCGCCTT
CTGCCCACCACICACGGACACATTTCTGCOT
AGANkAcAGCTT CTTACTGCT CTTACATGTGATGGCATA T CT TACACTAAAAGAATAT TAT
TGGGGGAAAAACTACAAGT
GCTGTP.CATA.T GCT GAGAPA CT GCAGAG CATAA TAG CTG CCACCCAAP.PAT CTTT TTGAAAAT
CAT T T CCAGACAACCTC
TT ACTTTCTGTGTAGTTTTTAATT
GTTAAA.PAP_A_PAPAGTTTTPAPCAGAAGCACAT,GACATATGPAAGCCTGCAGGACT
GGTCGTTTTTTTGGCAATTCTT CCA.CGTGGGACTTGTCCACAAGAATGAAAGTAGTGGTTTTTAAAGAGTTAAGT
TACAT
ATTTATT TT CT CACT TAAGTTATT TAT GCAAAAGTTT TT CTTGTAGAGAATGACAATGTTAATATTGCT
T TAT GAAT TAA
CAGTCTGTTCTFCCAGAGTOCAGAGACATTGITAATAAAGACAATGAATCATGACCGAAAG
Sequence ID No. 8: Human Beer protein variant (P38R) MQ PLAL.0 LVC L LVHTA FRVVEGQGWQA FKN DAT EI IRELGEYEEFEEELENNKTMNRAENGGREEHH
EFETK DVSEY SC
RELHETRYVTDGFCRSAKPVTELVCSGQCGFARLLENAIGRGKWWRPSGPDERCI
PDRYRAQRVQLLCEGGEAPRARKVR
INAS CKC KR LT R FHNQSE LKDEGTE:AAR PQKGRKP RPRARSAKANQAELENAY
Sequence ID No. 9: Vervet BEER cDNA (complete coding region) ATGCAC;CTCCCACTGGCCCTGTGT CTTGT CT GCCTGCTGGTACACGCAGCCTT
CCGTGTAGTGGAGGGCCAGGGGTGGCA
GGCCTTCPAGAATGATGCCACGGAAA TCATCCCCGAGCTCGGAGAGTACCCCGAGCCT
CCACCGGAGCTGGAGAACAACA
AGACCATGP.ACCGGGCGGAGAATGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCT
GC
CGAGAGCTGCACTTCACCCGCTACGTGACCGAt GGGCCGTGCCGCAGCGCCAAGCCAGTCACCGAGTTGGTGTGCTCCGG
CCA.GTGCGGCCCGGCACGCCTGCTGCCCAACGCCATCGGCCGCGGCPAGTGGTGGCGCCCGAGTGGGCCCGACTTCCG
CT
GCATCCCCGACCGCTACCGCGCGCA.GCGTGTGCAGCTGCTGTGTCCCGGTGGTGCCGCGCCGCGCGCGCGCAAGGTGC
GC

GC
TCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGGGGGCCAPAGCCPATCAGGCCGAGCTGGAGPACGCCTAC
T
AG
Sequence ID No. 10: Vervet BEER protein (complete sequence) '73 MQL FIALCLVCL LVHAAFRVVEGQGWQAFKN DAT EIIPE LGEY F-E FPFE, LEN N KTMN RAENGGR
E-HH EFETKDVSEYSC
RE LH FT RYVT DG RSAK PVTELVCSGQCG EARL L EVAI GRGKWWR F 33 ED FRC' EDRYRAQRVQLLC PC;GAAPRARKVR
LVASCKCKRLTREHNQSELKDEGPEAAR PQKGRK PR ERARGAKANQAELENAY
Sequence ID No. 11: Mouse BEER cDNA (coding region only) GTGGAGGGCCAGGGGTGGCA
AGCCTTCAGGAATGATGCCACAGAGGTCATCCCAGGGCTTOGAGAGTACCCCGAGCCTCCTCCTGAGAACAACCAGACC
A
TG.AACCGGGCC;GAGAATGGAGGCAGACCTCCCCACCATC,CCTATGACGCCAAAGGTGTGTCCGAGTACAGCTGCCG
CGAC.;
CTGCACTACACCCGCTTCCTGACAGACGGCCCATGCCGCAGCGCCAAG C CGGT CACCGAGT TGGT GIG CT
CCGGCCAGTG
CGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGT GAAGTGGTGGCGCCCGAACGGACCGGAT
TTCCC;CTGCAT CC
CGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGGGGCGCGGCGCCGCGC'TCGCGCAAGGTGCGTCTGG
TG
GCCTCGTGCAAGT
GCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGCCGGAGACCGCGCGGCC
GCAGAAGGGTCGCAAGCCGCGGCCCGGCGCCCGGGGAGCcAAAGccAACCAGGCGGAGCTGGAGAACGCCTACTAGAG
Sequence ID No. 12: Mouse BEER protein (complete sequence) MUsLAPcLIcLLvi-IAAFazwEGQGwQAFRNDATEVI EGLGEY PE PP PENNQTMNFRAENGGR P F HH
PYDP,K DVS EYSCRE
LHYTR FLTDG PCRSAK PVT E LVCSGQCG PAR LL PNP.I GRVKWWR PNGF DFRC P DRYRAQRVQL
LCPGGAAPRSRKVRLV
ASCKCKR LT R FHNQSE LK DFG E TAR PQKGRK PR PGARG.AKANQAE LENAY
Sequence ID No. 13: Rat BEER cDNA (complete coding region plus 5' UTR) GAGGACCGAGTGCCCTTCCTCCTTCTGGCACCATGCAGCTCTCACTAGCCCCTTGCCTTGCCTGCCTGCTTGTACATGC
A
GCCTTCGT TGCTGT GGAGAGCCAGGGGTGGCAAGCCTTCAAGAATGATGCCACAGAAAT CATCCCGGGACT
CAGAGAGTA
CCCAGAGCCTCCTCAGGPACTAGAGAACAACCAGACCATGAACCGGGCCGAGAACGGAGGCAGACCCCCCCACCATCCT
T
ATGACACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCGTGACCGACGGCCCGTGCCGCAG
T
GCCAAGCCGGTCACCGAGTTGGTGTGCTCGGGCCAGT GCGG CCCCGCGCGGCTGCT
GCCCAACGCCATCGGGCGCGTGP,A
GTGGTGGCGCCCGAACGGACCCGACTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCC
G
GCGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCA
G
_ ICGGAGCTCAAGGACTTCGGACCTGAGA,ccGCGCGGccGcAGAAGGGTCGCAAGCCGCGGCCCCGCGCCCGGGGA:::
::CA.a.
AGCCA.A.CCAGGCGGAGCTGGAGAACGCCTACTAG
Sequence ID No. 14: Rat BEER protein (complete sequence) LENNQTMNRAENGGR F HH F Y DTKIYISEYS::
RE, L HYT R EVT DG PCRSAK EATTE L'ICsGQCG EAR LL PNAI GRVKWWR ENG F DERC I E
DRY RAQRVQL LC EGGAA RsRicyR
LVASCKCKRLTREHNQSELKDEGPETAR PQKGRK PR PRARGAKANQAE LENAY
Sequence ID No. 15: Bovine BEER cDNA (partial coding sequence) CGGGCGGAGAACGGAGGGAGACCTCCcCACCACCCCTTTGAGACCAAAGACGCCTCCGAGTACAGCTGCCGGGAGCTGC
A
OTT CAccCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCAC:CGP.GCT
GGTGTGCTCGGC;CCAGTGCGGCC
CGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGCCCAAGcGGGCCCGACTTCCGCTGCATCCCCGA
C
CGCTACCGCGCGCAGCGGGTGCAGCTGTTGTGTCCTGGCGGCGCGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCT
C
Sequence ID No. 16: Bovine BEER protein (partial sequence -- missing NDATEIIPELGEYPEPLPELNNKTMNRAENGGRETHHPFETKDASEYSCRELHPTRYVTDGECRSAKPVTELVCSGQCG
E, PALL PNAI GRGKINWR PSG PDPRCI PDRYRAQRVQLLC EGGAAPRARKVRLVASCKCKRLTRFHNQSE
LKDEG PEAAR QT
GRKLRPRARGTKAS RA
Sequence ID No. 17: Mlui - AviII restriction fragment used to make mouse.
Beer transgene CGCGTTTTGGTGAGCAGCAATATTGCGCTTCGATGAGCCTTGGCGTTGAGATTGATACCTCTGCTGCACAP.AAGGCAA
TC
_ GACCGAGCTGGACCAGCGCATTCGTGACACCGT CTCCTT CC.4.PACT T AT TCGCAATGGAGTGTCATT CAT
CAAGGACNGCC
T GATCGCP.PATGGTGCTATCCACGCAGCG GCAATCGAAAACCCTCAGCCG GT GACCAAT AT CTACAACAT
CAGCCT TGGT
ATCCTGCGTGATGAGCCAGCGCAGAACP,AGGTAACCGTCAGTGCCGATA.P.GTTCPAAGTTA.P.ACCTGGTGTTGA
TACCAA
CAT TGAAACGTTGATCGAAAACGCGCT GAAAAACGCTGCTGAATGTGCOGCGCTGGAT
GTCACAAAGCAAATGGCAGCAG

ACAP.G.APAGCGATGGATGAACTGGCTTCCTATGTCCGCACG'GCCATCATGATGGAATGTTTCCCCGGTGGTGTTAT
CTGG
CAGCAGTGCCGT CGAT AGT ATGCAATTGP.T.P.ATT AT TAT CAT TTGCGGGTCCTTT
CCGGCGATCCGCCTTGTT ACGGGGC
GGCGACCTCGCGGGTTTTCGCTATTTATGAAPATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTT
CGTCATAACTTP,ATGT
TTTTATTTAAPATACCCTCTGAAPAGAPAGGAPACGACAGGTGCTGAPAGCC;AGCTTTTTGGCCTCTGTCGTTTCCTT
TC
TCTGTTTTTGTCCGTGGAATGPACAATGG.4A,GTCAACAAPAAGCAGAGCTTATCGATGATAAGCGGTCAPACATGAG
AAT

GPAGCGGAGGAGCTGGACTCCGCATG
CCCAGAGACGCCCCCCAACCCCCAAAGTGCCTGACCTCP,GCCTCTACCAGCTCTGGCTTGGGCTTGGGCGGGGTCAP.
GGC

C
ACCGT GGAP,TGTCTGCCTTCGTTGCCATGGCAACGGGAT GACGTT ACAAT CTGGGTGT GGAGCTTTT CCT
GTCCGTGT CA
GGAPATCCAPATACCCTAAPATACCCTAGPAGAGGAAGTAGCTGAGCC.PAGGCTTTCCTGGCTTCTCCAGATA,PAGT
TTG
15 ACT TAGAT GaPAPAPAACAAAATGATAAAGACCCGAGCCATCT GP-AAA TT CCT CCTAA
TTGCACCACTAGGAAATGTGTA
TATTATTGAGCTCGTATGTGTTCTTATTTTAAAPAGAAPACTTTAGTCATGTTATTAATAAGAATTTCTCAGCAGTGGG
A
GAGAACCAATATTPACACCAAGATAAPAGTTGGCAT GAT CCACATTGCAGGAAG ATCCACGTTGGGT T'P
TCAT GAATGTG
PAGACCCCATTTATTAPAGTCCTAAGCTCTGTTTITGCACACTAGGAAGCGATGGCCGGGATGGCTGAGGGGCTGTAAG
G
AT CTTTCPA TGTCTTACATGTGTGT TT CCT GTCCTGCACCTAGGACCTGCT GCCT
AGCCTGCAGCAGAGCCAGAGGGGTT

TCACATGATTAGTCTCAGACACTTGGGGGCAGGTTGCATGTACTGCATCGCTTATTTCCATACGGAGCACCTACTATGT
G
TCAAACACCATATGGT GTT CACTCTTCAGAACGGTGGTGGTCATCATGGTGCATT TGCTGACGGT
TGGATTGGTGGTAGA
GAGCTGAGAT AT ATGGACGCACTCTTCAGCATTCTGTC.:AACGTGGCTGT
GCATTCTTGCTCCTGAGCAAGTGGCTAAACA
GACTCACAGGGTCAGCCTCCAGCTCAGT
CGCTGCATAGTCTTAGGGAACCTCTCCCAGTCCTCCCTACCTCAP,CTATCCA
AGAAGCCAGGGGGCT T GGCGGT CTCAGGAGCCTGCTTGCTGGGGGACAGGTTGTTGAGTTTT AT CT
GCAGTAGGTTGCCT

AGGCATAGTGTCAGGACTGATGGCTGCCTTGGAGAACACATCCTTTGCCCTCTATGCAAATCTGP.CCTTGACATGGGG
GC
GCTGCTCAGCTGGGAGGATCAACTGCATACCTAAAGCCAAGCCTAAAGCTTCTTCGTCCACCTGAAACTCCTGGACCAA
G
GGGCTTCCGGCACATCCTCTCA.GGCCAGTGAGGGAGTCTGTGTGAGCTGCACTTTCCAATCT
CAGGGCGTGAGAGGCAGA
GGGAGGTGGGGGCAGAGCCTTGCAGCTCTTTCCTCCCATCTGGACAGCGCTCTGGCTCAGCAGCCCATATGAGCACAGG
C
ACATCCCCACCCCACCCCCACCTTTCCTGTCCTGCAGAATTTAGGCTCTGTTCACGGGGGGGGGGGGGGGGGGGCAGTC
C

TATCCTCTCTTAGGTAGACAGGACTCTGCAGGAGACACTGCTTTGTAAGATACTGCAGTTTAAATTTGGATGTTGTGAG
G
GGAAAGCGAAGGGCCT CTTTGACCATTCAGTCAAGGTACCTTCTAACTCCCATCGTATT GGGGGGCTACT
CTAGTGCTAG
ACATTGCAGAGAGCCTCAGAACTGTAGTTACCAGTGTGGTA.GGATTGATCCTTCAGGGAGCCTGACATGTGACAGTTC
CA
TT CTTCACCCAGT CACCGAACATTT ATT CAGT ACCT ACCCCGT P,ACAGGCACCGTAGCAGGT ACT
GAGGGACGGACCACT
CAAAGAACTGACAGACCGAAGCCTTGGAATATAAACACCAAAGCATCAGGCTCTGCCAACAGAACACTCTTTAACACTC
A

GGCCCTTTAACACTCAGGACCCCCACCCCCACCCCAAGCAGTTGGCACTGCTATCCACATTTTACAGAGAGGPAAAACT
A
GGCACAGGACGATATAAGTGGCTTGCTTAAGCTTGTCTGCATGGTAAATGGCAGGGCTGGATTGAGACCCAGACATTCC
A
õ

CA 02352532 2001705-23 .

ACTCTAGGGT CTATT TTTCTTT TT TCTCGTT GT TCGAAT CTGGGTCTT ACTGGGTAAACT CAGG CT
AGCCTCACACTCAT
AT CCTTCTCCCATGG CT TACGAGT GCTAGGAT T CCP,GGTGTGT G CTACCATGT CTGACT CCCTGT
AGCTTGTCTAT ACC.;
TCCTCACAACAT AGGA,A TTGTGATAGCAGCACACACACCGGAAGGAG CT GGGGAAAT CCCACAGAGGG
CTCCGCAGGATG
ACAGGCGAAT
GCCTACACAGAAGGTGGGGAAGGGAP,GCAGAGGGAACAGCATGGGCGTGGGACCACAAGTCT.ATTTGGGG
AAGCTGCCGGTAACCGTATATGGCTGGGGTGAGGGGA,GAGGTCATGAGAT
GAGGCAGGAAGAGCCACAGCAGGCAGCGGC;
TACGGGCTCCTTATT GCCAAGAGGCTCGGATCTTCCTCCTCTTCCTCCTTCCGGGGCTGCCTGTTCATTT
TCCACCACTG
CcTCCCATCCAGGTCTGTGGCTCAGGACATCACCCAGCTGCAGAAACTGGC-ICATCACCCAC:GTCCTGAATGCTGCCGI-',GG
cAGGTCCIT CATGCACGTCAACACCAGTGCTAGCTTCTACGAGG IC:TGG CAT CACCT ACTTGGGCAT
CAAGGCCAAT
GATP..CGCAGGAGTTCAACCTCAGT GCTT ACTTTGAAAGGGCCACAGATTT CAT TGACCAGGCGCT
GC;CCCAT.AWAT GG
T.LAGGP:Acc; TACATT CCGGCACCCAT GGAGCGTAII,GCCCT CT GGGACCTGCTT
CCTCCAAAGAGGCCCCCACTTG.AAk.a..;
,GGT T CCAGRAAGAT CCCAAAATATGCCACCAACTAGGGATTAAGT GT OCT ACA T
GTGAGCCGATGGGGGCCACTGCA.T AT
AGTCTGTGCCATAGACATGA,CAATGGATAATAATATTT
CAGACAGAGAGCAGGAGTTAGGTAGCTGTGCTCCTTTCCCT T
TAATTGAGTGTGCCCATTTTTTTATT
CATGTATGTGTATAC.ATGTGTGTGCACACATGCCATAGGTTGATACTGAACACC
TCTTCTCGTTCCCCACCCCACCTTP.TTTTTTGAGGCAGQGTCTCTTCCCTGATCCTGGGCTCATTGGTTT.TCTAG
T
GOT GCTGGCCAGTGAGCT CT GGAGT T CTGCTTTTCTCTACCTCCCTAGCCCTGGGACT
GCAGGGGCATGTGC:TC;GGCCAG
G CTTTTAT GT CGCGTTGGGGATCTGAACTTAGGTCCCTAGGCCTGAGCAcCGTPAAGACT
CTGCCACATCCCCAGCCT GT
TTGAGCAAGTGAACCATT CCCCAG A.n.T T CCCCCAGT GGG GCT T T C CT ACCCT T T T AT T
GGCT AGGCAT T CAT GAC;T GGT C
ACCTCGCCAGAGGAA TGAGTGGCCACGACT GGCTCAGGGTCAGCAGCCT AGAGATACT GGGTT AAGT CT
TCCTGCCGCTC
GCTCCCTGCAGCCGCAGACAGAPAGTAGGACTGAATGAGAGCTGGCTAGTGGTCAGACAGGACAGAAGGCTGAGAGGGT
C
ACAGGGCAGATGTCAGCAGAGCAGACAGGTT CTCCCTCTGTGGGGGAGGGGT GGCCCACTGCAGGTGT
AATTGGCCTT CT
TT GTGCT C,CAT AGAGGCTTCCTGGGT ACACAGCAGCTTCCCTGTCCT
GGTGATTCCCIVkAGAGAACTCCCTACCACTGGA
CT TACAGAAG T T CT AT TGACTGC;TGT AACGGTTCAACAGCTTTGGCTCT T
GGTGGA,CGGTGCATACTGCT GTATCAGCTC
AAGAGCT CATTCACGAATGAACACACACACACACACACACACACACACACACACRAGCTAATTTTGAT AT
GCCTTAACTA

AACACCTTCT AA
AT CTATATT T TATCTT T GCTGCCCTGTTACCTT CT GAGAAGCCCCTAGGGCCACTT CCCT TCGCACCT
ACATT GCTGGAT
GGTTTCTCTCCTGCAGCTCTTAAATCTGATCCCTCTGCCTCTGAGCCATGGGAACAGCCCAATAACTGAGTTAGACATA
A
AAACGTCTCT AGCCAAAACTTCAGCTAAATT T AGACAATAAATCTT ACTGGTTGTGGAAT CCTTAAGATT CT
TCATGACC
TCCTTCACATGGCACGAGTATGAAGCTTTATTACAATTGTTTATTGATCAAACTAACTCATAAAAAGCCAGT
TGTCTTTC
ACCTGCT CAAGGAAGGAACAAAAT T CAT CCTT AACT GATCTGTGCACCTTGCACAAT
CCATACGAATATCTTAAGAGT AC
TAAGATTTT GGTTGTGAGAGTCACATGTTACAGAATGTACAGCTTTGACAAGGTGCAT
CCTTGGGATGCCGAAGTGACCT
GCTGTTCCAGCCCCCTACCTTCTGAGGCTGTTTTGGAAGCAATGCTCTGGAAGCAACTTTAGGAGGTAGGATGCTGGAA
C
AGCGGGTCACTT CAGCATCCCGATGACGAAT CC CGTCAAAGCTGT ACATT CTGT
AA.CAGACTGGGPA21,GCTGCAGACT TT
AAGGCCAGGGCCCTATGGTCCCTCTTAATCC CT GTCACACCCAACCCGAGCCCTTCTCCT
CCAGCCGTTCTGTGCTT CT C
ACT CTGGATAGATGGAGAACACGGCCTTGCTAGT TAAAGGAGTGAGGCTT CACCCT
TCTCACATGGCP.GTGGTTGGT CAT
CCTCATTCAGGGAACTCTGGGGCAT TCTGCCTTTACTTCCTCTTTTTGGACT
ACAGGGAATATATGCTGACTTGTTTT GA
CCTTGTGTAT GGGGAGACTGGATCTTT GGTCTGGAATGTT
TCCTGCTAGTTTTTCCCCATCCTTTGGCAAACCCTAT CTA

TATCTTP.CCACTAGGCATAGTGGCCCTCGTTCTGGAGCCTGCCTTCAGGCTGGITCTOGGGGACCATGTCC:CTGGTT
TCT
CCCCAGCA TGGTGT TCACAGTGTTCACTGCGGGTGGT
TGCTGAACAAAGCGC4GGATTGCATCCCAG.A,GCTCCGGTGCC
TTGTGGGTACACTGCTAAGATAAAATGGATACTGGCCTCTCTCTC;ACCA.CTTGCAGAGCTCTGGTGCCTTGTGGGTP
,CAC
TGCTAAGATAAAATGGATACTGGCCTCTCTCTATCCACTTGCAGGACTCTAGGGAACAGGPATCCATTACTC;AGAAAA
CC
AGGGGCTAGGAGCAGGGAGGTAGCTGGGCAGCTGAAGTGCTTGGCGACTAACCAATGAATACCAGAGTTTGGATCTCTA
G
AATACTCT TPAnATCTGGGTGGGCAGAGTGGCCTGC:CT
GTAATCCCAGAACTCGGGAC.4GCGGAGACAGGGAATCATCAC.4A
GCAAACTGGCTAACCAGAATAGCWACACTGAGCTCTGGC;CTC:TGTGAGAGATCCTGCCTTAACATATAAGAGAGAGA
_A
TAAAACATTGA-AGAAGACAGTAGAT Gr.:CA:AT
TTTAAGCCCCCACATGCACATGGACAAGTGTGCGTTTGATI.CACACATAT

CAC
ATTAGA.GTTCACAGGAAAGTGTGAGTGAGCACACCCATGCACACAGACATGTGTGCCAGGGAGTAGGPAAGGAGCCTG
GG
TTTGTGTATAAGAG.C.IGAGCCA.TCATGTGTTTCTARGGAGGGCGTGTGAAGGAGGCGTTGTGTGGGCTGGGACTGG
AGCAT
GGTTGTAACTGAGCATGCTCCCTGTGGGAAACAGGAGGGTGGCCACCCTGCAGAGGGTCCCACTGTCCAGCGGGATCAG
T
AAAAGCCC:CTGCTGAGAACTTTAGG.T.AATAGCCAGAGAGA.GAAAGGTAGGABAGTGGGGGGACTCCCATCTCTGA
TGTAG
GAGGATCTGGGCAAGTAGAGGTGC.:7TTGAGGTAGAAAGAGGGGTGCAGAGGAC;ATGCTCTTAATTCTGGGTCAGCA
GTT
TCTTTCCAAATAATGCCTGTGAGGAGC.iTGTAGGTGGTGGCCATT
CACTCACTOAGCAGAGGGATGATGATGCCCGGTGGA
TGCTGGAPATGGCCGAGCATCPACCCTGGCTCTGGAAGAACTCCATCTTICAGAAGGAGAGTGGATCTGTGTATGGCCA
G
CGGGGTCACAGGTGcTTaIGGCCCCTGGGGGACTCCTAGCACTGGGTan.TGTTTATCGAGTGCTr2TTGTGTGCCAGG
cAc TGGCCTGGGGCTTTGTTTcTGTCTCTGITTTGTTTCGTTTTTTGAGACAGACTCTTGCTATGTATCCGTGTcAATc:TT
GG
AATCTCACTGCATAGCCCAGGCTGCGGAGAGAGGGG.GGGCAAT.L,GGCCTTGTAAGCAAGCCACACTTCAGAGACTA
GAC
TCCACCCTGCGAATGATGACAGGTCAGAGCTGAGTTCCGGAAGATTTTTTTTCC,AGCTGCCAGGTGGAGTGTGGAGTG
GC
AGCTAGCGGCAAGGGTAGAGGGCGAGCTCCCTGTGCAGGAGAAATGCAAGCAAGAGATGGCAAGCCAGTGAGTTAAGCA
T
TCTGTGTGGGGAGCAGGTGGATGAAGAGAGAGGCTGGGCTTTCGCCTCTGGGGGGGGGGTGAGGGGTGGGGATGAGGTG
A
GAGGAGGGCAGCTCCCTGCAGTGTGATGAGATTTTTCCTGACAGTGACCTTTGGCCTCTCCCTCCCCCACTTCCCTTCT
T
TCCTTTCTTCCCACCATTGCTTTCCTTGTCCTTGAGAAATTCTGAGTTTCCACTTCACTGGTGATGCAGACGGAAACAG
A
AGCCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGTATGTGTGTGTGTGTGTTTGTG
T
GTP.TGTGTGTCAGTGGGAA.TGGCTCATAGTCTGCAGGAAGGTGGGCAGGP.AGGAATAAGCTGTAGGCTGAGGCAGT
GTGG
GATGCAGGGAGAGAGGAGAGGAGGGATACCAGAGAAGGAAATTAAGGGAGCTACAAGAGGGCATTGTTGGGGTGTGTGT
G
TGTGTGTGTTGTTTATATTTGTATTGGAAATACATTCTTTTAAAAAATACTTATCCATTTATTTATTTTTATGTGCACG
T
GTGTGTGCCTGCATGAGTTCATGTGTGCCACGTGTGTGCGGGAACCCTTGGAGGCCACAAGGGGGCATCTGATCCCCTG
G
P.ACTGGAGTTGGAGGAGGTTGTGAGTCCCCTGACATGTTTGCTGGGAACTGAACCCCGGTCCTATGCAAGAGCAGGAA
GT
GCAGTTATCTGCTGAGCCATCTCTCCAGTCCTGAAATCCATTCTCTTAAAATACACGTGGCAGAGACATGATGGGATTT
A
CGTATGGATTTAATGTGGCGGTCATTPAGTTCCGGCACAGGCAP.GCACCTGTAA.AGCCATCACCACAACCGCAACAG
T GA
ATGTGACCATCACCCCCATGTTCTTCATGTCCCCTGTCCCCTCCATCCTCCATTCTCAAGCACCTCTTGCTCTGCCTCT
G
TCGCTGGAGAACAGTGTGCATCTGCACACTCTTATGTCAGTGAAGTCACACAGCCTGCACCCCTTCCTGGTCTGAGTAT
T
TGGGTTCTGACTCTGCTATCACACACTACTGTACTGCATTCTCTCGCTCTCTTTTTTTAAACATATTTTTP.TTTGTTT
GT
GTGTATGCACATGTGCCACATGTGTACAGATACTATGGAGGCCAGAAGAGGCCATGGCCGTCCCTGGAGCTGGAGTTAC
A
. õ

= 78 GGCAGCGTGTGAGCTGCCTGGTGTGGGTGCTGGGAAC:C:',_==_ACTTGAAT CT AAAGCA_AGCACTTTTAACT
GCTGAGGCAEr:
T CT CAGTACCCTTCTTCATTT CT CCGCCT GGGTTCCAT T GTATGGACACATG TAGCT AGAA T.ATCTT
G'OTT AT CT AATTA
TGTACATTGTT TTGT GCT A-AGAGAGAGT AATGCTCTATAGCCTGAC_4CT
GGCCTCA?,CCTTGCCATCCTCCTGCCTCAGCC
T CCT CCTCCTGAGT GCTAGGATGACAGGCGAGTGGTAACTTACATGC.zT TT CAT GTTT TGTTCAAGACT
GAAGGATAACAT
T C AT ACAGAGAAG G T C TG GG T CACA.A_AGT GTGC AG T T CAC T GAATGGCACAAC CCGT
GAT CAAGAAACAAAACT CAGGGr..z CTGGAGAGATGGCACTGACTGCTCTTCCAGAGGTCCC4GAG:TTCAATTCCCAGCAACC.:ACATGC3TGGCTCACAGC
CATCTA
TAACGAGAT CTGACG CCCTCTT CT GGTGTGTCT GAAGACAG CTACAGTGT ACTCACATAAAA T
AAATAAAT CT TTAAAAC
ACACACACACACACAATT ACCACCCCAGAAAGCCCACTCCAT G TTCCCTCCCACGT CT CTGCCTACAGTACT
CCCAG GTT
ACCAC TGTTCAGG CTTCT,,..ACAACCTGGT TT ACTT GGGCCT CT TTT CTGCT CTGT
GGAGCCACACAT TTGT GTGCCTCAT

CCATGCATGGCACAGTGTGTGGGGATGTCAGAGTATTGTGAACAGGGGACAGT T CTT TTCT
TCAATCATGTGGGTTCCAG
AGGTTGAACTCAGGTCATCATGTGTGGCAGCAAATGCCTTTACCCACTGP.GACATC.T
CCATATTCTTTTTTTTTC:CCCTG
AGGTGGGGGCTT C;TTCCATAGCCCAAACTGGCTTTGCACTTGCAGT TCAAAGT GACT CCCTGTCT
CCACCTCTTAGAGT A
TTGC.-;AAT T ACGATGTGT ACTACCACACCTGACTGGAT CATTAAT T OTT T
GATGGGGGCGGGGAAGCGCACATGCTGCAG::;
TGAAGGG AT GACTGGACTGGACATGAGCGTGGAAGCCAGAGAACAGCTT CAGT CT AAT C;CTCTCCCAACT
GAC;CT ATTT C
GGT TT GCCAGAGAACAACTTACAGAAAGTTCTCAGTGCCATGTGGATT CGGGGT TGGAGTTCAACTCAT
CAGCTTGACAT
TGGCT CCT CTACCCACTGAGCCT TCT CACTAC:TCT CTA CCTAGAT CAT TAATTCT
TTTTTAAAAAGACT TAT TAGGG GGC
TGGAGAGATGGCTCAGCCGTTAAGAGCACCGAATGCCCTTCCAGAGGTCCTGAGTTCAATTCCCAGCATGCCATTGCTG
G
GCAGTAGGGGGCGCAGGTGTTCAACGTGAGTAGCTGTTGCCAGTTTTCCGCGGTGGAGAACCICTTGACACCCTGCTUT
C
CCTGGTCATTCT GGGTGGGTGCATGGTGATAT GCTTGTTGTATGGAAC-?.CTT T GACT
GTTACAGTGAAGTTGGGCTTCCA
CAGTTACCACGTCT CCCCTGTTT CTTGCAGGCCGGGTGCT TGTCCAT T GC CGCGAGGGCTACAGCCGCT
CCCCAACGCTA
GT T AT
CGCCTACCTCATGATGCGGCAGAAGATGGACGTCAAGTCTGCTCTGAGTACTGTGAGGCAGAATCGTGAGATCGG
CCCCAACGATGGCTTCCT
GGCCCAACTCTGCCAGCTCAATGACAGACTA,GCCAAGGAGGGCAAGGTGAAACTCTAGGGTG
CCCACAGCCTCT TTT GCAGAGGTCTGACTGGGAGGGCCCT GGCAGCCA T GT TTAGGAA-ACACAGTAT
ACCCACTCCCT G C
ACCACCAGACACGTGCCCACATCTGTCCCACT CT GGTCCTCGGGGGCCACT
CCACCCTTAGGGAGCACATGAAGAAGCT C
CCTAAGAAGTTCTGCTCCTTAGCCATCCTTTCCTGTAATTTATGTCTCTCCCTGAGGTGAGGTTCAGGTTTATGTCCCT
G
TCTGTGGCATAGATACAT
CTCAGTGACCCAGGGTGGGAGGGCTATCAGGGTGCATGGCCCGGGACACGGGCACTCTTCAT
GACCCCTCCCCCACCTGGGTTCTTCCTGT
GTGGTCCAGAACCACGAGCCTGGTAAAGGAACTATGCAAACACAGGCCCTG
ACCTCCCCATGT CT GTTCCTGGTCCTCACAGCCCGACACGCCCT GCTGAG GCAGACGAATGACATTAAGTT CT
GAAGCAG
AGTGGAGATP.GATTAGTGACTAGATTTCCAAAAAGAAGGAAAAAAAAGGCTGCATTTTAAAATTATTTCCTTAGAATT
AA
AGATACTACATAGGGGCCCTTGGGTA.AGCAAATCCATTTTTCCCAGAGGCTATCTTGATTCTTTGGAATGTTTAAAGT
GT
GCCTTGCCAGAGAGCTT ACGATCT AT ATCT GCTGCTTCAGAGCCTTCCCT GAGGAT
GGCTCTGTTCCTTTGCTTGTTAGA
AGAGCGATGCCTTGGGCAGGGTTTCCCCCTTTTCAGAATACAGGGTGTAAAGTCCAGCCTATTACAAACAAACAAACAA
A
CAAACAAACAAAGGACCTCCATTTGGAGAATTGCAAGGATT TTA T CCT GAAT TATAGT GT T GGT GAGT
T CAAGT CAT CAC
GCCAAGTGCTTGCCAT
CCTGGTTGCTATTCTAAGAATAATTAGGAGGAGGAACCTAGCCAATTGCAGCTCATGTCCGTGG
GTGTGTGCACGGGTGCATATGTTGGAAGGGGTGCCTGTCCCCTTGGGGACAGAAGGAAAATGAAAGGCCCCTCTGCTCA
C
. _ .

CCTGGCCATTT ACGGGAGGCT CT GCTGGTTCCACGGT GT CT GT GCAGGAT CCT GP-E,ACT GACT
CGCTG GACAGAAACGAG
ACTTGGCGGCACCATGAGAATGGAGAGAGAGAGAGCAn.AGAAAGAAACAGCCTTTAAAAGAACTT
TCTA.AGGGTGGTTTT
TGAACCTCGCTGGACCTTGTATGTGTGCACATTTGCCAGAGATTGAACATAATCCTCTTGGGACTTCACGTT
CTCATTAT
TTGTATGT CT CCGGGGTCACGCAGAGCCGT
CAGCCACCACCCCAGCACCCGGCACATAGGCGTCTCATAAAAGCCCATTT
TATGAGAACCAGAGCTGTTTGAGTACCCCGTGTATAGAC;AGAGTTGTT
GTCGTGGGGCACCCGGATCCCAGCAGCCTGGT
T GCCTGCCT GTAGGAT GTCTTACAGGAGT TT GCAGAGAAACCTTCCT
TGGA.GGGAAACiA.A.ATATCAGGGATTTTTGTTGA
ATATTTCAAATTCAGCTTTAAGTGTAAGACTCAGCAGTGTTCAT GGT TAAGGTAAGGAACATGCCTTTT
CCAGAGC: TG CT
GCPAGAG 'GCAGGAGAAGCAGACCTGTCT TAG GATGTCACT CCCAGGGTAAAGACCT CTGAT
CACAGC:AC;GAGCAGAGCTG
TGCAGCCTGGP.T GGT CAT TGTCCCCT11.T TCT GTGT akCCACP.GC.k.n.CCCT GGT CACAT
AGGGCT GGT CAT CCT T T TT T TT
TT TT TTTTTTTTTITTTT TGGCCCAGAAT GAAGTGACCATAGCCAAGTTGT
GTACCTCAGTCTTT.a.GTTTCCAAGCGGCT
CT CTTGCT CTACAA TGTGCP.TTTCWATAACACT GT.z,GAGT TGACAGA;CTGGTTCATGTGTTAT
GAGAGAGGAAAA
GAGAGGPAAGAACAA;ACAAAACAAzACACCACAA,ACCA,a,AAACATCTGGGCTAGCCAGGCATGATTGCAATGTCT
ACAG
GCCCAGTTCATGAGAGGCAGAGACAGGAAGACCGCCGAP..;GGTCT,AGGATA.GCATGGTCTACGTATCGA.GACTC
CAGCCP.
GGGCTACGGT CCCAAGAT CCTAGGTTTTGGATTTTGGGCT T TGGTTTTT
GAGACAGGGTTTCTCTGTGTAGCCC;TGGCTG
TCCTGGAACTCGCTCTGTAGACCAGGCTGGCCTCAAACTTAGAGAT CT GCCTGACTCT GCCTTT
GAGGGCTGGGACGAAT
GCCACCACTGCCCAACTAAGATTCCATTAAAAAAAAAAAAAGTTCAAGATAATTA.A.GAGTTGCCAGCTCGTTAAAGC
TAA
GTAGAAGCAGTCTCAGGCCTGCTGCTTGAGGCTGTTCTTGGCTTGGP.CCTGAAATCTGCCCCCAACP.,GTGT CC
P,A.C;TGCA
CATGACTTTGAGCCATCTCCAGAGAAGGAAGTGA.AAATTGTGGCTCCCCAGTCGATT
GGGA,CACAGTCTCTCTTTGTCTA
GGTAACACATGGTGACACATAGCATTGAACTCTCCACT CT
GAGGGTGGGTTTCCCTCCCCCTGCCTCTTCTGGGTTGGTC
ACCCCATAGGACAGCCACAGGACAGTCACTAGCACCTACTGGAAACCTCTTTGTGGGIA,ACATGAAGAAAGAGCCTTT
GGG
AGATTCCTGGCTTTCCATTAGGGCTGAAAGTACAACGGTTCTTGGTTGGCTTTGCCTCGTGTTTATAAAACTAGCTACT
A
TTCTTCAGGTAAAATACCGATGTTGTGGAAAAGCCAACCCCGTGGCTGCC:CGTGAGTAGGGGGTGGGGTTGGGAATCC
TG
GATAGTGTTCTAT CCATGGAAAGT GGT GGAA TAGGAATTAAGGGT GT TCCCCCCCCCCCCAACCT OTT
CCT CAC;ACCCAG
CCACTT TCTATGACT TATAAACAT CCAGGTAAAAATTACA.AACATAAAAATGGT TTCTCTT CT CAATCT
TCTAAAGT CTG
CCTGCCTTT TCCAGGGGTAGGTCTGTTTCTTTGCT GTTCT AT
TGTCTTGAGAGCACAGACTAACACTTACCAAATGAGGG
AACTCTTGGCCCATACTAAGGCTCTTCTGGGCTCCAGCACTCTTAAGTTATTTTAAGAATTCTCACTT
GGCCTTTAGCAC
ACCCGCCACCCCCAAGTGGGTGTGGATAATGCCATGGCCAGCAGGGGGCACTGTTGAGGCGGGTGCCTTTCCACCTTAA
G
TTGCTTATAGTATTTAAGATGCTAAATGTTTTAATCAAGAGAAGCACTGAT CT
TATAATACGAGGATAAGAGATTTTCTC
ACAGGARAT TGT CTTTTTCATAATT CTTTTACAGGCTTTGTCCT
GA.TCGTAGCATAGAGAGAATAGCTGGATATTTAACT
TGTATTCCP,TTTTCCTCTGCCP,GCGTTAGGTTAACTCCGTAAAAkGTGATTCAGTGGACCGAAGAGGCTCAGAGGGC
AGG
GGATGGTGGGGTGAGGCAGAGCACTGTCACCTGCCAGGCATGGGAGGTCCTGCCATCCGGGAGGAAAAGGAAAGTTTAG
C
CT CTAGTCTACCACCAGTGTTAACGCACTCTAAAGTTGTAACCAAAATAAATG
TCTTACATTACAAAGACGTCTGTTTTG
TGTTTCCTTTTGTGT GTTTGGGCTTTTTA TGTGTGCTTTATAACTGCTGTGGT GGTGCTGTTGTTAGTT T
TGAGGTAGGA
TCTCP.GGCTGGCCTTGAACTTCTGATCGCCTGCCCCTGCCCCTGCCCCTGCCCCTGTCCCTGCCTCCAAGTGCTAGGA
CT
AA.AAGCACATGCCACCACACCAGTACAGCATTTTTCTAACATTTAAAAATAATCACCTAGGGGCTGGAGAGAGGGTTC
CA
GCTAAGAGTGCACACTGCTCTTGGGTAGGACCTGAGTTTAGTTCCCAGAACCTATACTGGGTGGCTCCAGGTCCAGAGG
A

. CA 02352532 2001-05-23 TCCAGGACCTCT GGCCTCCATGGGCATCTGCT CT T AG CACAT ACC CACA
TACAGATACACACATAAAP_P.TAA TG:AAGC
CTT TAAAPACCT CCTAAAACCT .A.GCCCT TGGAGGTACGACTCTGGAAAG CT GGCATACT
GTGTPAGTCCAT CTCATGGT G
T TCTGGCTA.P.CGT AAGACT TACAGAGACAGAAPAGAACT CAGGGT GT GCT GGGGC;T
TGGGATGGAGGAAGA,GGGATGAGT
AGGGGGAGCACGGGGAACTTGGG,.2A,GTGAAAATTCTTT GCAGGACACT AGAGGAGGATAAATACCAGT CAT
TGCACCCAC

GGCCCC AGGT TGG CT GCA T TGGCT G CAT T T GCGT AAC
= AT T T TT T T PAAT TGAAAAGAAA.AAGATGTAAAT CAA.3 GTTAGATGAGTGGTTGCTGTGAGCT
GAG.P.GCTGC,,GGTGAGTG.T.,, GACATGTGC.-3ACPACTCCAT CAP.A.PAG CGACAGAAAG ---------------------- CTAC T CT
AATCTCCACCT CCGGGA G
GTGA T CA.AGGT TAGC:CCTCAGCT AGCCTGT GC_;TGCAT GAGACCCT GT T TCAAAAACT T T AAT
APAGAAATAA TGAAAAAA
GACATCAGGGCAGATCCTTGGGGCCAP,AGGCGGACAGGCGAGTCTCGTGGTAAGC;TCGTGTAGPAGCGGATC3CATG
AGCA
10 CGTGCCGCAGGCATCATGAGAGAGC2'.:CTAGGT.'-',AGTAAGGATGGATGTGAGTGTGTCGGCGTCGGCGCACTGCACGTCCT
GGC T GT GG T GC T GGACT GGC AT CT T T GGT G.A.GCT GT GGAGGGGAAATGGGTAGGGAGAT
C AP-AA T CCC T CC GAAT T P,T
TTCAAGAACTGTCT A T T ACP.A. T TATCTCAAPATA T T A-1-1'..AAPAAAGAAGAAT
TAAAAAACAAPAPACCTATCCAGGTGTG
G'TGGTGTGCA.CCT ....................................................
GCCACGGGC.:ACT T GGAAAGCTGGAGCAAGAGGAT GGCGAGT T TGAAGGTATCT GGGGCTGTACA
GCAAGA CCGT CGTCCC CAAACCA.A.P.: C.P,AACAG CAP_AC CAT T ATGT CACAC,An.GAGT GT
T T.A.T AGT GAGCG G C. CT CGCT
15 GAGAGCAT GGGC; TGGGGG T GGGGGTGGGGGACAGAAA T TCT AAACT GC.A.GTCAAT AGGGA
T CCACTGAGACCCTGGGGC
TT GACT GCAGCT T.AAC CT TGGGAAATGAT Pik GGGT T T T. GT C;T TGAGT APAP, GC:ATCGA.TT ACT GAC'T TAACC:TCAAATGA
AGPAAAP.G.A.AAAAAAGAAPACPACAAAP.GCCAPACCA.A.GGGGCTGGTC.;AGATGGCTCAGTGGGT.A.A.GA
GCACCCGACTGC
TCTTCCGAAGGTCCAGAGTTCP.P.ATCCCAGCAACCACATGGTGGCTCACAACCATCTGTAACGAGATATGATGCCCT
CTT
CTGGTGTGTCTGAAGACAGCTACAGTGTACTTACATATAATAAP,TAA.A.TCTTAPAAAAAAAAAAAPAAAAAAAAGC
CAAA

CCGAGCAAACCAGGCCCCCAAACAGAAGGCAGGCACGACGGCAGGCACCACGAGCCATCCTGTGAAAAGGCAGGGCTAC
C

C
AP,T T ACAGATAAPAT AACAPATA-AAC.,AAAAT CTAGAGCCTGGCCACTCT CTGCTCGCT T GAT T T
T TCCT GT T A,CGTCCAG
. CAGGTGGCGGA,AGT GT T CCAAGGACAGATCGCATCAT TAAGGT
GGCCAGCATAATCTCCCATCAGCAGGTGGT GCTGTGA
GAACCATTATGGTGCTCACAGAAT
CCCGGGCCCAGGAGCTGCCCTCTCCCAAGTCTGGAGCAP.TAGGAAAG,CTTTCTGGC

CCAGACAGGGTTAACAGTCCACATTCCAGAGCAGGGG.A.PAAGGAGACTGGAGGTCACAGACAAPAGGGCCAGCTTCT
AAC
P,ACTTCACAGCTCTGGTAGGAGAGATAGATCACCCCCA_ACA,ATGGCCACAGCTGGTTTTGTCTGCCCCGAAGGP,A
ACTGA
CT T AGGAAGCAGGTATCAGAGTCCCCT T CCT GAGGGGACT T CTGTCTG CCT TGT AAAGCTGT
CAGAGCAGCTGCAT TGAT
GTGTGGGTGACAGAAGATGAAAAGGAGGACCCAGGCAGATCGCCP.CAGATGGACCGGCCACTTACAAGTCGAGGCAGG
TG
GCAGAGCCTTGCAGAAGCTCTGCAGGTGGACGACACTG.ATTCATTACCCAGTTAGCATACCACAGCGGGCTAGGCGGA
CC

GT C T CAGT GCAG CT T CCGCCAGCCCCTCC
TCCTTTTGCACCTCAGGTGTGAACCCTCCCTCCTCTCCTTCTCCCTGTGGCATGGCCCTCCTGCTACTGCAGGCTGAGC
A
T TGGAT T TCT T T GT GCT TAGATAGACCTGAGATGGCTT TCTGAT T TATATATATATATCCAT CCCT
TGGATCTTACATCT
AGGACCCAGAGCTGT T TGTGAT ACCP.TAAGAGGCTGGGGAGATGAT A TGGTAAGAGTGCT
TGCTGTACAAGCATGAAGAC
ATGAGT TCGARTCCCCAGCAACCAT GTGGAAAAATAACCT
TCTAACCTCAGAGTTGAGGGGAAP,GGCAGGTGGAT TCT GG
35 GGGCTTA.CTGGCCAGCTAGCCAGCCTAACCTAAATGTCTCAGTCAGAGAT
CCTGTCTCAGGGAATAACTTGGGAGAATGA
CTGAGAPAGACACCTCCTCAGGTCTCCCATGCACCCACACAGACACA.CGGGGGGGGGGTAATGTAATP.AGCTAAGAA
ATA

. WO

ATGAGGGAAATGATTTTTTGcTAAGApATGAAATTc7:3TGTTGGccGcA.A.GAAGccTGGcc.;GGGAAGGAAcTGc crrTc;
GCACACCAGCCTATA..AGTCACCATGAGTTCCC:TGGCT.kAGAAT
CACATGTAATGGAGCCCAGGTCCCTCTTGCCTGGTGG
TTGCCTCTCCCACTGGTTTTGAAGAGAAATTCAAGAGAGATCTCCTTGGTCAGAATTGT.AGGTGCTGAGCAATGTGGA
GC
TGGGG TCAATG GGA TTCCTT TAAAGGCATCCTTCCCAC.4GGCTGGGTCATACTT CAA T AGT AGG'GTG
CTTGCACAGCAAGC
GT GAGACCC T AGGT TAGAGTCCCCAGAATCTGCCCCCA.ACCCCCCAAAAAC;GCATCCTTCT
GCCTCTGGGTGGGT GGGGG -GAGCAAACACCTTTAACTAAGACCATTAGCTGGCAGGGGTAACAAATGACCTTGGCTAGAGGAATTTGGTCA.a.GCTG
GAT
TCCGCCTTCTGTAGAAGCCCCACTTGTTTCCT T TGTT AAG CT GC.-;CCCACAGTTTGTTT TGAGAAT
GCCTGAGGGGCCCAG
GGAGCCAGAC.kAT T AAAAGCCAAGCTCAT T TT GAT AT CiakAAACCACAGC:CT GACTC;CCCT
CCCGTGC;GAGGT ACTGG
GAGAG CTG3 CT GTGTCCCTGCCTCACCAACC;CCCCCCCCCCC.AACACACACTCCTCGGGTCA OCTGGGAGGT
GCCAGCAG
CAATTTGGPAGTTTACTGAGCTTGAGAAGT CTT GGGAGGGCTGACGCT AAGCACACCCCT TC T
CCACCCCCCCCCACCCC
ACCCCCGT GAGGAGGAGGGTGAGGAAACAT GGGACCAGCCCT GCTCCAGCCCGTCCT T AT
TGGCTGGCATGAGGCAGAGG
GGGCTTTAAAAAGGCAACCGT AT CT AGG CT GGACACT GGAGCCT GT GCT ACCGAGT GCCC T
CCTCCACCT GGCAGCATGC
AGCCCTCACTAGCCCCGTGCCTCATCTGCCTACTTGTGCACGCTGCCTT
CTGTGCTGTC;GAGGGCCAC;GGGTGGCAAGCC
T TCAGG.AA TG:ATGCCACAGAGGT CAT
CCCAGGGCTTGGAGAGTACCCCGAGCCTCCTCCTGAGAACAACCAGACCA TGA.A.
CCGGGCGGAGRA T GGAGGCAGACCT CCCCACCATCCCTA T GACGCCAAAGGTACGGC-LATGAAGAAGCACATTAGTGGGGG
GGGGGGTCCTGGGAGGTGACTGGGGT GGTTTTAGCATCT T CTTCAGAGGTTTGTGT GGGTGGCTAGCCTCTGCT
ACATCA
GG:2CAGGGACACATTTGCCTGGAAGAATACTAGCACAGCP.TTAGPACCTGGAGC;GCAGCATT
GGGGGGCTGGTAGP.GAGC
ACCCAAGGCAGGGTGGAGGCTGAGGTCAGCCGAAGCTGGCATTAAC.ACGGGCATGGGCTTGTATGATGGTCCAGAGAA
TC
TCCTCCTAAGGATGAGGACACAGGTCAGATCTAGCTGCT
GACCAGTGGGGAAGTGATATGGTGAGGCTGGATGCCAGAT
CCATCCATGGCTGTACTATATCCCACATGACCACCACATGAGGTAA.AGAAGGCCCCAGCT
TGAAGATGGA.GAAACCGAGA
GGCTCCTGAGATAAAGTCACCTGGGAGTAAGAAGAGCTGAGACTGGAAGCTGGTTTGATCCAGATGCAAGGCAACCCTA
G
ATTGGGTTTGGGTGGGPACCTGAAGCCAGGAGGAATCCCTTTAGTTCCCCCTTGCCCAGGGTCTGCTCAATGAGCCCAG
P.
GGGTTAGCATTAAAAGAACAGGGTTTGT AGGTGGCATGTGACATGAGGGGCAGCT GAGT GAAATGTCCCCT
GTATGAGCA
CAGGTGGCACCACTTGCCCTGAGCTTGCACCCTGACCCCAGCTTTGCCTCATTCCTGAGGACAGCAGAAACTGTGGAGG
C
AGAGCCAGCACAGAGAGATGCCTGGGGTGGGGGTGGGGGTATCACGCACGGAACTAGCAGCAATGAAT
GGGGTGGGGTGG
CAGCTGGAGGGACACTCCP,GAGW,TGACCTTGCTGGTCACCATTTGTGTGGGAGGAGAGCTCATTTTCCAGCTTGCCA
C
CACATGCTGT CCCT CCTGTCT CCT AGCCAGTAAGGGAT GTGGAGGAAAGGGCCACCCCAA.AGGAGCAT
GCAATGCAGTCA
CGTTTTTGCAGAGGAAGTGCTTGACCTAAGGGCACTATTCTTGGAAAGCCCCAAAACTAC;TCCTTCCCTGGGCAAACA
GG
CCTCCCCCACATACCACCTCTGCAGGGGTGAGTAAATTAAGCCAGCCACAGAAGGGTGGCAAGGCCTACACCTCCCCCC
T
GTTGTGCCCCCCCCCCCCCCGTGAAGGTGCATCCTGGCCTCTGCCCCTCTGGCTTTGGTACTGGGATTTT
TTTTTTCCTT
TT ATGT CATATTGATCCTGACAC CATGGAACTTTTGGAGGTAGACAGGACCCACACATGGA TTAGTT
AAA,AGCCTCCCAT
CCATCTAAGCTCATGGTAGGAGATAGAGCATGTCCAAGAGAGGAGGGCAGGCATCAGACCTAGAAGP.TATGGCTGGGC
AT
CCAAC CCAAT CT CCTTCCCCGGAGAACAGACTCTAAGT CAGATCCAGCCACCCTTGAGT AACCAGCT
CAAGGTACACAGA
ACAAGAGAGTCTGGTATACAGCAGGTGCTWCAAATGCTTGTGGTAGCAAAAGCTATAGGTTTTGGGTCAGAACTCCGA
CC CAAGT C GCGAGT GAAGAGCGAARGGC CCT CT ACT C G CCACCG CCC CGC CCCCACCT GGGGT
C CT ATAACAGAT CACT T
TCACCCTTGCGGGAGCCAGAGAGCCCTGGCATCCTAGGTAGCCCCCCCCGCCCCCCCCCCGCAAGCAGCCCAGCCCTGC
C
. .

TT TGGGGC:PAGTTCT TT TCT CAG C:CTGGACCT GT GATAATGAGGGGGT
TGGACGCGCCGCCTTTGGTOGCT T TCAAGTCT
AAT GPATT CTT AT CCCT ACCACCT GCCCTTCTACCCCGCTCCTCOACA.C;CAGCT
GTCCTGATTTATTACCTTCPAT TAAC:
C.:TCCACTCCT
TTCTCCATCTCCTGGGATACCGCCCCTGTCCCAGTGGCTGGTAPAGGAGCTTAGGAAGGACCAGAGCCAG
GT GTGGCT AGAGGCT ACCAGG CAGGGCTGGC;GAT GAGGAGCTAAACTGGAAGAGTGT TTGGT
TAGTAGGCACAAAGCCTT
G G GT GGGPN CC C TAGT C C.; GC GCT G T C PAGAC C CCATC CT T P.A.CT AC
AC P.GC: C T
TTGAGGCCAGCCTGGGCT AC.ATAAAAACCCA,AT CTCPAAAGCTGC OPAT TCT GATT
(:TGTGCCACGTAGTGCCCGATGTA
GTGGATGAAGTCGTTGPATCCTGOGGCAACCTATTTTP.CAGATGTGGGGAAPAGC:AACTTTAAGTACCCTGCCCACA

GATOACPAP. GAPAGT AAGT GAOAGP.GOT CC P.GT GT TTCAT CCC:T G G GTT CCAAG GAC: AG
GGAC;AGA.C;AAGCCAG GGT GGG .
!1:1" C.,TCACT GCTCC(_:CGGTGCCTCCTTCCTATAATCCATACAGATT
CGAAAGCGCAGGGCAGGTTTGGAAAPAC;AGAGAAG
GGTGGPAGGAGOAGACCAGTCTGGCCTAGGCTGCAGC:OCCTCACGCATCCCTCTCTCCGCAGATGTGTCCGAGTACAG
OT
GCCGCGAGCTGCACTACACCCGCTT C.:CT GACAGACG GCCCAT GCCGCAGCGCC AAGCCGGTCACCGAGT
TGGT GTGCTCO
GGCCAGTGCGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGTGPAGTGGTGGCGCCCGAACGGACCGGATTTCC
G
CT GOATCCCGGATCGCTACCGCGCGCAGCGGGTGCP.GC:TGOTGTGCCCCGGGGGCGCGGCGCCGCGCT
CGCGCAAGGTGC
GT CT GGT GGCCT CGT GC.A.P.GT GCAAGC:GCCTCACCCGCT TCCA.C.AP,CCAGT CGGAGCT
CPAGGACTTCGGGCCGGAGACC
GCGCGGCCGCAGAAGGGTCGCAAGCCGCGGCCCGGC:OCCCGGGGAGCCAAAGCCAACCAGGCGGAGCT
GGAGFACGCCT A
CT AGAGCGAGCCCGCGCCTATGC.A.GCCCCCGCGCGATCCGATTCGTTT T

GCCAPACTTTCCAGACCGTGT GGAGTTCCCAGCCCAGT AGAGACCGCAGGT CCTT CT GCCCGCTGCGGGGGAT
GGGGP.GG
GGGTGGGGTTCCCGCGGGCCAGGAGAGGAAC;CTTGAGTC:CCAGACTCTGCCTAGCCCCGGGTGGGATGGGGGTCTTT
CTA, CCCTCGCCGGACCTATACAGGACAAGGCAGTGTTTCCACCTTAAAGGGPA'GGGAGTGTGGPACGAPAGACCTGGGACT
GG
TTAT GGA.CGTA.CAGT A.A.GATCT ACT CCTTCCACCCA.P.P.T GT
A.A.A.GCCTGCGTGC;GrCTAGAT AGGGT TTCT GACCCTGACC
TGGCCACTGAGTGTGATGTTGGGCTACGTGGTTCTCTTTTGGTACGGTCTTCTTTGTAAAATAGGGACC:GGAACTCTG
CT
GAGA.TT CCAAGGATTGGGGTACCCCGTGTAGACTGGT
GAGAGAGAGGAGAACAGGGGAGGGGTTAGGGGAGAGATTGTGG
TGGGCAACCGCCTAGAAGPAGCT
GTTTGTTGGCTCCCP.GCCTCGCCGCCTCAGAGGTTTGGCTTCCCCCACTCCTTCCTC
TCP-AATCTGCCTICAAATCCATATCTGGGATAGGGAAGGCCAGGGTCCGAGAGATGGTGGAAGGGCCAGAPATCACACTC
CT GG CCCCCCGAAGAGCAGTGTCCCGCCCCCAACTGCCTTGTCATATT 'GT APAGGGATTTT CT
ACACAACAGTT TAAGGT
CGTTGGAGGAAACTGGGCTTGCCAGTCACCTCCCATCCTTGTCCCTTGCCAGGACACCACCTCCTGCCTGCCACCCACG
G
ACACATTTCTGT
CTAGAPACAGAGCGTCGTCGTGCTGTCCTCTGAGACAGCATATCTTACATTAPAPAGAATAATACGGG
GGGGGGGGGCGGAGGGCGCAAGTGTTATACATATGCTGAGAAGCTGTCAGGCGCCACAGCACCACCCACAATCTTTTTG
T
A.kATCATT T CCAGACACCTCTTACTTTCT GTGT AGATTTTAATTGT TPAAAGGGGAGGAGAGAGAGCGT
TTGT AP.CAGAA
GCACATGGAGGGGGGGGTAGGGGGGTTGGGGCTGGTGAGTTTGGCGAACTTTCCATGTGA,GACTCATCCACAAAGACT
GA
AAGCCGCGTT TTTTTTTTTAAGAGTTCAGTGACATATTTATTTTCTCATTTAAGTTATTTATGCCAACATT
TTTTTCTTG
TAGAGAAAGGCAGTGTTAATATCGCTTTGTGAAGCACPAGTGTGTGTGGTTTTTTGTTTTTTGTTTTTTCCCCGACCAG
A
GGCATTGTTAATAPAGACAATGAATCTCGAGCAGGAGGCTGTGGTCTTGTTTTGTCAACCACACACAATGTCTCGCCAC
T
GT CAT CTCACTCCCTT CCCTTGGT
CACAAGACCCAPACCTTGACAACACCTCCGACTGCTCTCTGGTAGCCCTTGTGGCA
ATACGTGTTTCCTTTGAAAAGTCACATTCATCCTTTCCTTTGCAP,ACCTGGCTCTCATTCOCCAGCTGGGTCATCGTC
AT
ACCCTCACCCCAGCCTCCCTTTAGCTGACCACTCTCCACACTGTCTTCCAPAAGTGCACGTT TCACCGAGCCAGTT
CCCT

GGTCCAGGTCATCCCATTGCTCCT CCT TGCT CCAGACCCT T CTCCCACAPAGATGT TCAT CT CCCACT
CCAT CAAGCCCC
AGTGGCCCT GCGGCT ATCCCTGTCTCTTCAGTTAGCT G.P.ATCTAC_:T TGCTGACACCACAT GAAT T
CCT TCCCCTGT CT T
AGGT T CATG GAAC TCTT G cc T GCCC CT GAACCT T C CAGGACT G T CC CAG CG T CT GA
T GT GT C CT CT CT C T TG TAAAGC CC
CACCCCACT AT T TGATTCCCAATTCTAGAT CT TCCCTTGT TCAT TCCT TCACGGGATAGT GTCTCAT
CTGGCCAAGTCCT
GCTTGATAT TGGGAT AAAT GCAP.AGC CAAGT ACAAT T GAG GACCAG TT CAT CAT T GGG CCAAG
CT T TT TC.A.P.A.ATGTGAP.
TT T T ACACCT ATAGPAGTGT PAAAGCCT TCCAAAGCAGAGGCAATGCCTGGCT CT T CCT
TCP,ACATCAC;GGCT CCTGCT T.
TATGGGTGTGGTGGGGTAGTACATTCATAAACCCAACACTAGGGGTGTGAAAGCPAGATG.ATTGGGAGTTCGAGGCCA
AT
CT T GGCT ATGAGGCCCTGTCTCAACCTCTCCTCCCTCCCTCCAGGGT T T T GT T T TGT T T TGT T
T T T T TGAT T TGAAACTG
CAACACTT TAPATCCAGT CP-AGT GCAT CT TT GCGTGAGGGGAACTCTATCCCTAATATAAGCT T
CCATCT TGAT T TGT GT
ATGTGCACACTGGGGGTTGAACCT GG G CC T T T GT ACCT G CC GGG CPAG CT CT CT ACT GCT
CT AP-AC CCA GCC CT CACT GG
CT T T CTGT T T CAACT CCCAATGAAT TCCCCTAAATGAAT T AT
CAATATCATGTCTTTGAAAAATACCATTGAGTGCTGCT
GGTGTCCCTGTGGTT CCAGAT TCCAGGAAGGACTT T TCAGGGAAT CCAGGCAT CCTGAAGRA TGT CT T
AGAGCAGGAGGC
CATGGAGACCT TGGCCAGCCCCACAAGGCAGTGTGGTGCAGAGGGTGAGGATGGAGGCAGGCT T GCAAT
TGAAGCT GAGA
CAGGGT ACTCAGGAT TAAPAAGCT TCCCCCAP:AACAAT T CCAAGATCAG T TCCTGGT ACT T GCACCT
GT T CAGCTATGCA
GAGCCCAGT GGGCATAGGTGAAGACACCGGT T GTACTGT CAT GT ACTP.ACTGT GCT
TCAGAGCCGGCAGAGACAP.P.TAA T
GT TATGGTGACCCCAGGGGACAGTGAT TCCAGAAGGPACACAGAAGAGAG T GCTGCT
AGAGGCTGCCTGAAGGAGAAGGG
GTCCCAGACTCTCTAAGCAAAGACTCCACTCACATAAP.GACACAGGCTGAGCAGAGOTGGCCGTGGP.TGCAGC;GAG
CCCP.
TCCACCATCCTTTAGCATGCCCTTGTATTCCCATCACATGCCAGGGATGA.GGGGCATCAGAGAGTCCAAGTGATGCCC
AP.
ACCCAAACACACCTAGGACTT GCT T TCTGGGACAGACAGA TGCAGGAGAGACT AGGT TGGGCT
GTGATCCCAT TACCACA
AA GAGGGAPAPAACAAPAAACAPACAAA CAAA CAAAAAAAAACAAAACAAAAC AAAAAAAAAC CCAAGG T C
CAAAT T GT A
GGTCAGGT TAGAGT T TATT TATGGAAAGT TATAT TCT P.CCTCCAT GGGGTCT ACAAGGCT
GGCGCCCATCAGAAAGAACA
NkcACAGGCTGATCTGGGAGGGGTGGTACTCTATGGCAGGGAGCACGTGTGCTTGGGGTACAGCCAGJCACGGGGCTTG

TAT TAATCACAGGGCT TGTAT TAP.
TAGGCTGAGAGTCAAGCAGACAGAGAGACAGAAGGAAACACACACACACACACACA
CACACACACACACACACACACATGCACACACCACT CACT T CTCACTCGPAGAGCCCCT ACT TACAT T
CTAAGAACA.APCC
AT TCCTCCT CAT AAAGGAGACAAAGTTGCAGAAACCCAAAAGAGCCACAGGGTCCCCACTCTCTT TGAAATGAC
TTGGAC
TTGT TGCAGGGAAGACAGAGGGGTCTGCAGAGGCT TCCTGGGTGACCCAGAGCCACAGACACTGAPATCTGGTGCT
GAGA
CCTGTATAAACCCTCTTCCACAGGTTCCCTGAAAGGAGCCCACATTCCCCAACCCTGTCTCCTGACCACTGAGGATGAG
A
GCACTTGGGCCTTCCCCATTCTTGGAGTGCACCCTGGTTTCCCCATCTGAGGGCACATGAGGTCTCAGGTCTTGGGAAA
G
TTCCACAAGTATTGAAAGTGTTCTTGTTTTGTTTGTGATTTAATTTAGGTGTATGAGTGCTTTTGCTTGAPTATATGCC
T
GTGTAGcATTTAcAAGccrGurGccTGAGGAGATcAGA,AGATGGcATcAGATAcccTGGAAcTGGAcTTGcAGAcAGT
TA
TGAGCCACTGTGTGGGTGCTAGGPACAGAACCTGGATCCTCCGGAAGAGCAGACAGCCAGCGCTCTTAGCCACTAAGCC
A
TCACTGAGGTTCTTTCTGTGGCTAAAGP.GACAGGAGACAAAGGAGAGTTTCTTTTAGTCAATAGGACCATGAATGTTC
CT
CGTAACGT GAGACTAGGGCAGGGTGAT CCCCCAGTGACP,CCGATGGCC CTGT GTAGT TAT
TAGCAGCTCTAGTCT T ATT C
CT TAAT AAGTCCCAGT T T GGGGCAGGAGATAT GTAT TCCCTGCT T TGAAGTGGCTGAGGTCCAGT
TATCTACT TCCAAGT
ACT TGT T T CTCT T TCTGGAGT T GGGGAAGCTCCCTGCCTGCCTGTAPATGT GTCCAT T CT T
CAACCT T AGACAAGATCAC
T TT CCC T GAG CAGT CAGGC CAGT C CAAAGCCCT T CAAT T TAGCT TT CA TAAGG AACAC CC
CT TT T GT T G GG T GGAGGT AG

- WO

C ACT TGCCT TGAATCCCAGCAT TAAGAAGGCAGAGACAGTCGGATCT CTGT GAGT
TCACAGCCAGCCTi7,3GT CT ACGGAGT
.GAGTTCCPAGACP.GCCAGGCCTA.CACAGAGAAACCCTGTCTCGAAAAAP.ACAAAAACAAAGAAATAAAGA.aj,-AAGAAAA
CAAAAACGAACAAACAGAAAAAC.V.GCCAGAGTGTTTGTCCCCGTP.TTTT.a.TTAATCATATT T T TGTCCCT
T. TGCCAT T T
TAGACTAAAAGACT CGGGAAAGCAGGTCTCT CTCT GT T TCT CATCCGGACACACCCAGAACCAGA.TGT
ATGGAAGAT GGC
TAT GT GC T GC AGT T G C ACP.T C T GGGGCTGGGTGGATT GGTTAGATGGCAT C.;GG CT CI
GGT GT GG T TACC.TGACTGCAGG
AGCA.AGGAGT A T GTGGT GCATAGCAAACGAGGAAGT TTGCACAGAACAACACTGTGTGT ACT
GATGTGCAGG T A TGGGCA
CATGCAAGCAGAAGCCAAGGGACAGCCT T AGGGTAGTGT T TCCACAGACCCCTC.:CCCCC:T T T T
AACATGG GCAT CT CTCA
T TGGCCTGGAGCT T GCCAACTGGGCTGGGCTGGCTAGCT TGTAGGTCCCAGGGAT CT GOAT ATCT
CTGCCT CCCTAGTGC
TGGGATTACAGTCATATAT GAGCACACCTGGCTTT T T TATGT GGGTT CT C.4GGCT T T
GAAC:CCAGATCT GAGT GCT TGCAA
GOCAATCGGTT GARTGACTGCT TCP,TCT CCCCAGACCCT GGGAT TCT ACT T TCTAT TAAAGTAT T T
CTAT T AAATCAATG
AGCCCCTGCCCCT GCACTCAGCAGT T CT TP.GGCCTGCTGAGAGTCAAGT
GGGGAGTGAGAGCAAGCCTCGAGACCCCATC
AGCGAAGCAGAGGACAAAGAAA TGAAAACTTGGGATTCGAGGCTCGGGAT AT GGAGAT ACAGAAAG
GGTCAGGGAAGGAA
AT GAACCAGAT GA2kTAGAGGCAGGAAGGGT AGGGCCCTGCATACATGGAACCTGGT GTACAT GT T ATCT
GCA T GGGGT T T
GCATTGCAATGGCTCTTCAGCAGGTTCACCACACTGGGAIACAGAAGCCAzAAAGAAGAGTAGGTGGTGTTG
GAGTCAGA
TACTGTCAGTCATGCCTGAJ,,,GAAATGGAAGCAATTAACGATGCGCCGCAATTAGGATATTAGCTCCCTGAAGAAAG
GCAA
GAAGCT GGGCT GT GG GCACT GAAG GGAG CT T T GPAT GAT GT CACAT T C TC T GTA T G
CC TAGCAGG G C AGT AT T GGAGA CT
GAGACTTGACTTGTGTGTCCATATGATTCCTCCTTTTCCTACAGTCATCTGGGGCTCCTGAGCTTCGTCCTTGTCCAAG
A
ACCTGGAGCTGGCAGTGGGCAGCTGCAGTGATAGATGTCTGCAAGAAAGAT CT GAPAAGAGGGAGGAAGAT
GAAGGACCC
AGAGGACCACCGACCTCTGCT GCCTGACAAAGCTGCAGGACCAGTCT CT
CCTACAGATGGGAGACAGAGGCGAGAGATGA
AT GGTCAGGGGAGGAGT CAGAGAAAGGAGAGGGTGAGGCAGAGACCAAAGGAGGGAAACACT TGT
GCTCTACAGCT ACTG
ACTGAGTACCAGCTGCGTGGCAGACA.GCCAATGCCAAGGCTCGGCTGATCATGGCACCTCGTGGGACTCCTAGCCCAG
TG
CTGGCP,GAGGGGAGTGCTGAATGGTGCAT GGT T TGGATAT GATCTGAAT GTGGTCCAGCCCT AGT T
TCCT TCCAGT TGCT
GGGATAPAGCACCCTGACCAAAGCT ACT T T TT TGT TT GT T TGT T T TGGT T TGGT T T TGT T
TGGT TTTTCGAGGCAGGGTT
T CT CT GTATCACCCTAGCTGTCCTGGAACT CACTCTGTAGACCAGGCTGG CCT CGAACT
CAGAAATCCCCCTGCCTCTGC
CTCCTAAGTGCTGGAP,T TAAAGGCCTGCGCCACCACT GCCGGCCCPAAGCTACT TT AAGAGAGAGAGAGGAA
TGT AT AAG
TAT TATAAT TCCAGGTTATAGT T CAT T GCTGTAGAAT TGGAGT CT TCP.TAT TCCAGGT.AAT
CTCCCACAGACATGCCACA
AAACAACCTGT T CT ACGAAATCT CT CATGGACTCCCT TCCCCAGTAAT
TCTAAACTGTGTCAAATCTACAAGAAATAGTG
ACAGTCACAGTCTCTAACGTTTTGGGCATGAGTCTGAAGTCTCATTGCTAAGTACTGGGAAGATGAAAACTTTACCTAG
T
GT CAGCAT T TGGAGCAGAGCCTT T GGGAT T TGAGP,TGGT CT T T TGCAGAGCTCCT AATGGCT
ACATGGAGAGAGGGGGCC
TGC,GAGAGACCCATACACCT TT TGCT GCCT TAT GTCACCTGACCTGCT
CCTTGGGAAGCTCTAGCAAGAAGGCCTTCCCT
GGAT CACCCACCACCT TGCACCTCCAGAACT CAGAGCCAAAT TAAACTT T CT TGT
TACTGTCGTCAAAGCACAGTCGGTC
T GGGT TGTAT CACTGTCAAT GGGAAACAGACT TGCCTGGAT GGATAACT T GTACAT T GCAT
AATGTCTAGAAATGAAAAG
TCCTATAGAGAAAAAGAAAATTAGCTGGCACACAGATAGAGGCCCTGGAGGAGGCTGGCTTTGTCCTCCCCGAGGAGGT
G
GCGAGTAAGGTGTAPATGT T CATGGATGTAAATGGGCCCAT AT AT GAGGGTCTGGGGT
AACAAGAAGGCCTGTGAATAT A
AAGCACTGAAGGTATGTCTAGTCTGGAGAAGGTCACTACAGAGAGTTCTCCAACTCAGTGCCCATACACACACACACAC
A
CACACACACACACACACACACACACACACACCACAAAGAAAAAAAGGAAGAAAAATCTGAGAGCAAGTACAGTACTTAA
A
. .

TGIGTC4.,,..T T
3TGTGTGTGACTCTGATGTCACATGCTCATCTTGCCCTATG.A.GTTGPAPACCAAATC;GCCOCTC4P,GAGG
CAT.a.P.C.L.P.COACACTGTTGGCTGTGTGCTCACGTTTTTCTLWGCGTCTGTCTGGTTTGCTGCTAGCATCAC.;
GCAGACT
T GCAGCAGACT.L.CAT ATGCTCAGCOCTGPAGTCCT TCTAGGGTGCATGTCTCT T CAGAAT T T
CAGRAL.GT CAT CT C.4TGGC:
TCCAGGACC.:CTGCACTCTCCCTOTGCCGCGAGGCTGCAG.A.CTCTAGGCTGGGGTGGPAGCAACGCTTP.C:CTCT
GGGAC
5 AAGTAT.A.A.C.ATGTTGGCTTTTCTTTCCCT CTGT
GGCTCCP,ACCTGGACATAAAATAGATGCAAGCTGTGT FAT P.P.AT,rk.T T
TCCTCCCGTCCP,CTTAGTTCTCAACPATAACTACTCTGAGAGCACTTATTAATAGGTGGCTTAGACATAP.GC:TTTG
GCTC
AT T C.CCCC.L.r,7 AGCTCT TACT T CT T TAACT CT TTCAAACCAT T CTGTGT CT
TCCACATGGT T.A.GTT ACCTCTCCT CCAT
CCTGGTTCCTTCTTCCTTCGAGTCGCCCTCAGTGTCTCTAGGTcTGCTTGTPCATATTCTTTCTAcGCTGAC4C4 TGGTGGCACTCTGGGAGTT CAAAGCCAG C CT GAT CTACACAGCAAGCT C CAG G ATAT CCAGG GCAAT
GT TG C.4GAA.z...AC
10 T T CT C.n...nAC.LAAAAGAGGGGT TCAGT TGTCAGGAGGAGA,CCCATGGGT T AAGYLAGT
CT AGACGAGCCATGGTGATGCAT A
CCTT TCP.TCaLAGCACT TAGGAGGC :.'-',.a.GP,1,..n..GUTGA.AACT CT T T GACTIT
GAGGCCAGCTP,GGTTACATAGTGATACCC
TGCT TP.GT T TGT GTGTGTGTGTGTGTGTGTGTUTGT GTGTGTGTAAT T T AAAAGTCTAAAAATGCAT T
CT T
TATGTATAAGT.L.T T TGCCTGCACATATGTATGTATGTATGT ATACCA TGTGTGT GTCT GGT
GCTGINAGGACTAGGCT AG
A.C.T
CCCTAG.a.ACTAGAGTC.P.TAG.A.C..213TTGTGACACTCCCCAACCCCCCACCATGTGGGTGCTTGAAGCT.a.
A.ACTOCTGT
15 CCT TT GTP.A.a.GCAGCAGGT GTCTATGAACCCTGAACCAT CTCTCCAGT CTCCAGATGT GCAT
T CT C.n.AAGAGGP.GTOCT T
CP.T T TCC(717,..P.ACTGFACATCCT T CAGTGAGCATCCTCGAGTCACCAAAGCTACT GCAAACCCTCT
TAGGGAACAT
TCACT A T T CP.CT TCT ACT TGGCT CAT G.V.ACT
TAAGTACACACACACNAACACACACACACACACAGAGTCATGCACTCA.
CAAA.P.GCAT GCATGTACACCAT TCT T AT TAGACTA TGCT TTGCTAAPAGACT T TCCTAGATACTT
T.11,13At.CAT CACT TCT
GCCTTTTGGTGGGCAGGTTCCAAGATTGGTACTGGCGTACTGGPAr..CTGAACY:AGGTAGA.GATCTAGAPATCACA
GCAGG

TCAGPAGGGCCAGCCTGTACAAGAGAGAGTTCCACACCTTCCAGGIAACACTGAGCAGGGGGCTGGGACCTTGCCTCTC
AG
CC CAAGNAACT AGTGCGT TTCCTGTAT GCATGCCT CT CAGAGAT T CCATARGATCTGCCT T
CTGCCATAAGATCTCCTGC
ATCCAGACAP.GCCT AGGGGPAGT T GAGAGGCTUCCT GAGTCTCTCCCP,CAGGCCCCT T CT
TGCCTGGCAGT AT T TT T T TA
TCTGGAGGP.GAGGAATCAGGGTGGGAATGATCAAATACAATTATCAAGGAA.MAGTAAAAAACATATATATATATATA
TT
AACT GAT C T AG G p,.GC T C.3GCT CAGCAGT TAAGAGT T CT GGCTGC C CT T GC TT
CAGAT C T T GC T T T GAT T.CCCAG CACCCA

CATGATGGCTTTCAACTGTATCTCTGCTTCCAGGGGATCCAACAGCCTCTTCTGACCTCCATAGACAAGACCTAGTCCT
C:
TGCAAGAGCACCAAAT GCTCT TATCTGT TGATCCATCT CT CTAG CCTCATGCCAGATC:AT T
TAAAACTACTGGACACTGT
CCCAT T T T AC GAAGATGTCACTGCCCAGTCATT T GCCATGAGTGGATATT TCGAT TCTT TCTATGT
TCT CACCCT T GCAA
TTTATAAGPAAG.n.TATCTGCATTTGTCTCCTGAGAGAACAAAGGGTGGAGGGCTACTGAGATGGCTCTAGGGGTAPA
GGT
GCTTGCCACATCTGACAACTTAAGTTTGGTCTTGGAATCCACATGGTGGAGAGAGAGAAGAGATTCCCGTA.n.GTTGT

CCTCAPACTTCCCACACATGTGCTGTGGCTTATGTGTAACCCCAATAAGTAPAGATAGTTTTAAACACTACATAAGGTA
G
GGTTTCTTCATGACCCCAAGGAATGATGCOCCTGATAGAGCTTATGCTGA.A.P.CCCCATCTCCATTGTGCCATCTGG
APAG
AGACAAT TGCATCCCGGAAACAGAA T CT TCATGAATGGATTAATGAGCT AT T PAGAAAGTGGCT TGGT T
AT TGCACATGC
TG'GCGGCUTPAT GACCTCCACCATG.kTGTTATCCAGCATGAAGGTCCTCP,CCAGAAGTCAT ACPAATCT.TCT
T AGGCT TC
CAGAGTCGTGAGCAPAMAAGCACACCTCTAAATAAATTAACTAGCCTCAGGIAGTTAACCACCGAAAATGAACCAAGGC

AGTTCTAP.TACACCACTTCCCTTCCCTGTTCAAACCACAGTGCCCTATTATCTAAAAGATAAACTTCAAGCCAAGCT

TT TAGGT TGCCAGTAT TTATGTAACAACAAGGCCCGT T
GACACACATCTGTP.ACTCCTAGTACTGGGCCTCAGGGGCAGA
. _ . .

GACAGGTGGAGCCCTGGAGTTTGAATT CCAGGT TC.7.2,T GAGAAACICT
GAAAAGACAATANGGTGAGTGACCCGGC, AGGATATCTGATATTGACTTCTGGCCAACACACAGCCATCTCT GCACAT GTAGTTG CAAGCCTT-TTGCACTAAGT TT G
GCCAGP.GTCAGAGTTTGCAAGTGTTTGTGGACTG.A.a.TGCACGTGTTGCTGGTGATCTACAAAGTCACCCTCCTTC
TC.
cTAGCAGCACTGGCTTCGGCCAGCTGCTCATTCAAGCCTCTTTOCAGAGTCATCACGGGGATGGGGGAGCAGGGCCCOT
C
CCTAGA,ACACCAAGCCTGTGGTTGTTTATTCAGGACATTATTGAGGGCCA.a.GATGACAGATAACTCTATCACTTGG
CCAA
CAGT CGGGT GT TGCGGTGTTAGGTTATTT CTGTGT
CTGCAGAA_kACAGTGCAACCTGGACAAAAGAAATA.AATGATATCA

GGGGA.A.CGAGGCCGGGACAGCGCGGCTCCTGAGTCC.;
CTAT T T CCGTCTGTCAACTT CTCT?_;T CT CTTGAT T T C'CT CCCT CTGTOTGTTT CCTTCCT

GTCTGTGTACTCACAGGGAGGAGGGTGGCAAAGCCC.7TC;GTCCTCTACGGGCTGG'GGGAA.GGGGGGAAGCTGTCG
GCCCAG
TGACTTTTTCOCCTTTCTCTTTTTCTTAGAAACCAGT
CTCAATTTAAG:Trf_a_ATGAGTCTCCTCATTCACGTOTGCTCACT
ATTCATAGGGACTTATCCACCCCCGCCCTGT CAATCTGGCTAAGTAAGACAAGTCAAkTTTAAAAGGGA-ACGTTTTTCTA.
.r=AAATGTGGCTGGACCGTGTGCCUG:CACGAAACCASCI4 3.11,TGGCGGT CT.k.a.GTTACAT GCT CT
CTGCCIGCCCCGGTGCCT
T TT CCT TTCGGAAAGGAGACCCGG.a.GGTPAAACGAAGT T GCCAACT TTT GAT GATGGTGT
GCGCCGGGT GACTCTTTAAA
ATGTCATCCATACCTGGGATAGGGAAGGCT CT TCAGGGAGT CATCTAGCCCTCCCTTCAGGAAAAGA TT
CCACTTCCGGT
TTAGT TAGCTTCCACCTGGTCCCT TAT CCGCTGT CT C.T GCCCACTAGTCCT CATCCATCCGGTTT
CCGCCCTCATCCACC
TTGCCCTTTTAGTTCCTAGAAAGCAGCACCGTAGT CTTGGCAGGTGGGCCAT T
GGTCACTCCGCTACCACTGTTACCATC;
GCCACCAAGGTGTCATTTAAATATGAGCTCACTGAGTCCTGCGGGATGGCTTGGTTGOTAATATGCTTGCTGC:AAAAT
CG
TGAGAACTGGAGTTCAATTCCCAGCACATGGATGTATTTCCAGCACCTGGAAGGCAGGGAGCAGAGATCTTA.A.AGCT
CCT
GGCCAGACAGCCCAGCCTAATTAGTAATCAGTGAGAGACCCTGTCTCAAGAAACAAGATGGA-ACATCRAAGGT
CAACCT
TTGTCTCCACACAC.ACA-AATACACACATGCACATACATCCACACACAGGCAACACATGCACACACCTGA.A,CACCCTCCA
CAAATACATACATAAAAAAATAAATACATACACACATACATACATACACCA.ACATTCCCTCTCCTTAGTCTCCTGGCT
AC
GCTCTTGTCACCCCCACTAAGGCTTCAACTT CT TCTAT TT CTTCAT CTTGACTCCT CTGTACTT
TGCRTGCCT TTTCCAG
CAAAGGCTTTTCTTTAAATCTCCGTCATTCATAAACTCCCTCTAAATTTCTTCCCCTGCCCTTT
TCTTTCTCTCTAGGGA
GATAAAGACACACACTACAAAGTCACCGTGGGACCAGT TTATTCACCCACC CACCCCTGCTTCTGTT CAT
CCGGCCAGCT
AAGTAGTCCAACCTCTCTGGTGCTGTACCCTGGACCCTGGCTTCACCACAGCTCCTCCP.TGCTACCCAGCCCTGCAAA
CC
TT CAGCCTAGCCTCT GGTTCTCCAACCAGCACAGGCCCAG TCTGGCTTCTAT GT CCTAGAAATCTCCT
TCATTCT CTCCA
TTT CCCTCCTGAAT CTACCACCTTCTT TCTCCCTT CTCCTGACCTCTAAT GT CT
TGGTCAAACGATTACAAGGAAGCCAA
TGAAATTAGCAGTTTGGGGTACCT CP.GAGT CAGCAGGGGAG CT GGGATGAAT T CACATTT
CCAGGCCTTTGCTTTGCTCC
CCGGATTCTGACAGGCAGTTCCGAAGCTGAGT CCAGGAAGCTGAATTTA-AAAT
CACACTCCAGCTGGGTTCTGAGGCAGC
CCTACCACATCAGCTGGCCCTGACTGAGCTGTGTCTGGGTGGCAGTGGTGCTGGTGGTGCTGGTGGTGCTGGTGGTGGT
G
GTGGTGGTGGTGGTGGTGGTGGTGGTGGTGTGTGTGTGTGTTTTCTGCTTTTACAAAACTTT
TCTAATTCTTATACAAAG
GACAAATCTGCCTCATATAGGCAGAAkGATGACTTATGCCTATATAAGATATAAAGATGACTTTATGCCACTTATTAGC
A
ATAGTTACTGTCAAAAGTAATTCT ATTTATACACCCTTATACATGGTATTGCTTTT GTTGGAGACTCTAAAAT
CCAGATT
ATGTATTTAAAAAAAAATTCCCCAGTCCTTAAAAGGTGAAGAATGGACCCAGATAGAAGGTCACGGCACAAGTATGGAG
T
CGGAGT GT GGAGT CCTGCCAATGGTCTGGACAGAAGCATCCAGAGAGGGT CCAP,GACAAATGCCT
CGCCTCCTAAGGAAC
ACTGGCAGCCCT GAT
GAGGTP,CCAGAGATTGCTAAGTGGAGGAATACAGGATCAGACCCATGGP.GGGGCTTAAAGCGT GA
¨ ¨ ¨ _ G
GAGTCACCTCGGGTACCATTGTTTAGGGAGGTGGGGATTTGTGGTGTGGAGICAGGCA,GCCTCAA,GGATGCTTITCA
ACA
AT GGTT G,A T GP.GTTGGAACTAAAACAGGGGCCATCA.CACTGGCT CCCATAGCT CTGGGCT TG
CCAGCT TCCACATCTGCC
C.CCCACCCCCTGTCTGGCACCAGCTCAAGCT CTGTGATTCTACACAT CCAANkaa.GGAAGAGTAGCCT
ACTGGGCATG CC
ACCT CTT CT GGACCATCAGGTGAGAGTGT GGCAAGCCCT AGGCT CCTGT
CCAGC4ATGCAGGGCTGCCAGATA,GGATGCTC
AGCT ATCT OCT GAGCTGGAACTATTT T AGGAATAAGGATTAT
GCCCGCCCGGGGTTGGCCAGCACCCCAGCAGCCTGTGC
TT GCGT AAAAGCAAGTGCTGTTGAT TTATCTAWACAGAGCCGTGGACCCACCCACAGGACAAGT ATGTAT
GCATCTGT
TTCATGTATCTGARAAGCGACACPACCATTTTTCACATcATGGCATCTTCCTAACCCCCATIcTTTTTTGTTTTGTTTT
T
TTGAGACAGGGTTTCTCTGTGTAGT CCT GGCTGTCCTGGP.A.CT CP.CTT T GT P.GAC CAGGCT GGCCT
CGAACTCAGPAATC, CTGGGATTAAAGGTGTGTGCCACCACGCCCGGCCCTAACCCCCP.TTCTTAATGGTGATCCAGTGGTTGAAATTTCGGG
CC
ACACACATGTCCATTAGGGATTAGCTGCTGTCTT
CTGAGCTACCTGGTACAATCTTTATCCCCTGGGGCCTGGGCTCCTG
AT CCCTGACTCGGGCCCGAT CAAGTCCAGTTCCTGG'GCCCGATCAAGTCCAGTT CCTGGGCCCGAACAA.G T
CCAGTCCCT
A.GCT CGATT AGCT CATCCT GGCTCCCT GGCCTGTTCTT ACTTP.CACT CT TCCCCTT GCTCT
GGACTTGTT GOTTT CT TTA

CAAGCACGGAPAGT CCTG
AGTCACCACACCCTCTGGAGGTGTGTGGACACATGTTCATGCGTGTGGTTGCGCTTACGTACGT GTGC
Sequence ID No. 18: Human Beer Genomic Sequence (This gene has two exons, at positions 161-427 abd 3186-5219).
tagaggagaa gtctttgggg agggtttgct ctgagcacac ccctttccct ccctccgggg 60 ctgagggaaa catgggacca gccctgcccc agcctgtcct cattggctgg catgaagcag 120 agaggggctt taaaaaggcg accgtgtctc ggctggagac cagagcctgt gctactggaa 180 ggtggcgtgc cctcctctgg ctggtaccat gcagctccca ctggccctgt gtctcgtctg 240 cctgctggta cacacagcct tccgtgtagt ggagggccag gggtggcagg cgttcaagaa 300 tgatgccacg gaaatcatcc ccgagctcgg agagtacccc gagcctccac cggagctgga 360 gaacaacaag accatgaacc gggcggagaa cggagggcgg cctccccacc acccctttga 420 gaccaaaggt atggggtgga ggagagaatt cttagtaaaa gatcctgggg aggttttaga 480 _ _ 09s1 BoolpoopBE poo5poo56p 5.651pablae vo55Buevep qa6q556Bze qa2.65.6qq6q 5E
00sT DOVPDP0a00 .600D010035 eo33np5.63p 3-43.66ep3o5 qoeoo6-e-ef. p6e3.66I6Bv 017VT 0Dleq1P005 6qD3D04640 301105eBev ;a2Beoq;op p6q.e.sebqp qba665.6.6qp OE
08E' Do6be63ooq ev55vo66T1 pvqeqp1356 qolvo51.64.6 vot,51.66epo obepftEpop ozET a6qoo56voq 561=116.65 qopopv66eE. ve5fiep5p6.6 BeE.E.6.6eDoq TeoqDoElqqo onT ovepooqbeo 300PD35QD5 eol.pol&goq pae.6.65vq5v 56ep6qE1D4 polvo5po5p gz on Be6565e6.6q pona,epolB pE.D5PD54.6 651eo6100 035P11TI5P 0e0B50qq1 opt' 66E5 pp6.33pD56 lpEre565eDo a6q33.661.eo Do6qqva6q.6 q5eD6q3pp3 OZ
ogoi pp.6.6p6vbp a65pp6Ece66 6.6q5PoBeep oepooTe5v. DeMBE5q.61 5E5.656.6q51 ozoT 6366qop3bv ze&epo6qop voolE.B1115 5403PP&35q 6.6a6qo65P P5v515P16.6 096 llpeelfto; opaMvooll 6poppe6ve 6566pe5oqp 5Po3popee6 -1.66PBBeoDD SI
006 D1PPE.EqP31 PDPDOOP6P0 POPOOPODOD 4623.6.6q.6.64 66qa63.2-loq 3Poe.663ov3 or 555.6..q6qooP oBvoep6Poe DoTeoopqop 5e55Ecepq55 QeoeebBErep BE,513popel Of 08L 56qoBlo6pD oqBovoobp6 buqeqeeBE6 lpoloolole ebeppoqT3E. 5eeuv5ppee on 6vala666.6e Dlo66povp5 eolva5.66PD 1v36.61Pq55 6P5155q55.6 610602.66P5 099 E5PD566pDo 36e5p6z651 0EDP6PpeP0 55P5qP055v 6v051.56PD qv331pa6 g = 009 p3515.61o53 qa6.5qpe5ep E56.6555pE. 5loobqqp3.6 tove65e5oq .66qopp5pop ops 53.qop56lo Eqqe5pv5q6 epoop5eq.6.6 55 j555 qlo55.e.666-1 1-43.4oqqoey 066LZ/66S1UIDd ELLVDDO Clitt -EZ-g0-TOOZ ZEgZgEZO VO

tcttcaaaat ggaggacaag ggcgcctccc cccacagctc cccttctagg caaggtcagc 1620 tgggctccag cgactgcctg aagggctgta aggaacccaa acacaaaatg tccaccttgc 1680 tggactccca cgagaggcca cagcccctga ggaagccaca tgctcaaaac aaagtcatga 1740 tctgcagagg aagtgcctgg cctaggggcg ctattctcga aaagccgcaa aatgccccct 1800 tccctgggca aatgcccccc tgaccacaca cacattccag ccctgcagag gtgaggatgc 1860 aaaccagccc acagaccaga aagcagcccc agacgatggc agtggccaca tctcccctgc 1920 tgtgcttgct cttcagagtg ggggtggggg gtggccttct ctgtcccctc tctggtttgg 1980 tcttaagact atttttcatt ctttcttgtc acattggaac tatccccatg aaacctttgg 2040 gggtggactg gtactcacac gacgaccagc tatttaaaaa gctcccaccc atctaagtcc 2100 accataggag acatggtcaa ggtgtgtgca ggggatcagg ccaggcctcg gagcccaatc 2160 tctgcctgcc cagggagtat caccatgagg cgcccattca gataacacag aacaagaaat 2220 gtgcccagca gagagccagg tcaatgtttg tggcagctga acctgtaggt tttgggtcag 2280 agctcagggc ccctatggta ggaaagtaac gacagtaaaa agcagccctc agctccatcc 2340 cccagcccag cctcccatgg atgctcgaac gcagagcctc cactcttgcc ggagccaaaa 2400 ggtgctggga ccccagggaa gtggagtccg gagatgcagc ccagcctttt gggcaagttc 2460 ttttctctgg ctgggcctca gtattctcat tgataatgag ggggttggac acactgcctt 2520 tgattccttt caagtctaat gaattcctgt cctgatcacc tccccttcag tccctcgcct 2580 ccacagcagc tgccctgatt tattaccttc aattaacctc tactcctttc tccatcccct 2640 _WO 00/32773 PCT/US99/27990 gtccacccct cccaagtggc tggaaaagga atttgggaga agccagagcc aggcagaagg 2700 tgtgctgagt acttaccctg cccaggccag ggaccctgcg gcacaagtgt ggcttaaatc 2760 5 ataagaagac cccagaagag aaatgataat aataatacat aacagccgac gctttcagct 2820 atatgtgcca aatggtattt tctgcattgc gtgtgtaatg gattaactcg caatgcttgg 2880 ggcggcccat tttgcagaca ggaagaagag agaggttaag gaacttgccc aagatgacac 2940 ctgcagtgag cgatggagcc ctggtgtttg aaccccagca gtcatttggc tccgagggga 3000 cagggtgcgc aggagagctt tccaccagct ctagagcatc tgggaccttc ctgcaataga 3060 tgttcagggg caaaagcctc tggagacagg cttggcaaaa gcagggctgg ggtggagaga 3120 gacgggccgg tccagggcag gggtggccag gcgggcggcc accctcacgc gcgcctctct 3180 ccacagacgt gtccgagtac agctgccgcg agctgcactt cacccgctac gtgaccgatg 3240 ggccgtgccg cagcgccaag ccggtcaccg agctggtgtg ctccggccag tgcggcccgg 3300 cgcgcctgct gcccaacgcc atcggccgcg gcaagtggtg gcgacctagt gggcccgact 3360 tccgctgcat ccccgaccgc taccgcgcgc agcgcgtgca gctgctgtgt cccggtggtg 3420 aggcgccgcg cgcgcgcaag gtgcgcctgg tggcctcgtg caagtgcaag cgcctcaccc 3480 gcttccacaa ccagtcggag ctcaaggact tcgggaccga ggccgctcgg ccgcagaagg 3540 gccggaagcc gcggccccgc gcccggagcg ccaaagccaa ccaggccgag ctggagaacg 3600 cctactagag cccgcccgcg cccctcccca ccggcgggcg ccccggccct gaacccgcgc 3660 cccacatttc tgtcctctgc gcgtggtttg attgtttata tttcattgta aatgcctgca 3720 acccagggca gggggctgag accttccagg ccctgaggaa tcccgggcgc cggcaaggcc 3780 cccctcagcc cgccagctga ggggtcccac ggggcagggg agggaattga gagtcacaga 3840 cactgagcca cgcagccccg cctctggggc cgcctacctt tgctggtccc acttcagagg 3900 aggcagaaat ggaagcattt tcaccgccct ggggttttaa gggagcggtg tgggagtggg 3960 aaagtccagg gactggttaa gaaagttgga taagattccc ccttgcacct cgctgcccat 4020 cagaaagcct gaggcgtgcc cagagcacaa gactgggggc aactgtagat gtggtttcta 4080 gtcctggctc tgccactaac ttgctgtgta accttgaact acacaattct ccttcgggac 4140 ctcaatttcc actttgtaaa atgagggtgg aggtgggaat aggatctcga ggagactatt 4200 ggcatatgat tccaaggact ccagtgcctt ttgaatgggc agaggtgaga gagagagaga 4260 gaaagagaga gaatgaatgc agttgcattg attcagtgcc aaggtcactt ccagaattca 4320 gagttgtgat gctctcttct gacagccaaa gatgaaaaac aaacagaaaa aaaaaagtaa 4380 agagtctatt tatggctgac atatttacgg ctgacaaact cctggaagaa gctatgctgc 4440 ttcccagcct ggcttccccg gatgtttggc tacctccacc cctccatctc aaagaaataa 4500 catcatccat tggggtagaa aaggagaggg tccgagggtg gtgggaggga tagaaatcac 4560 atccgcccca acttcccaaa gagcagcatc cctcccccga cccatagcca tgttttaaag 4620 tcaccttccg aagagaagtg aaaggttcaa ggacactggc cttgcaggcc cgagggagca 4680 gccatcacaa actcacagac cagcacatcc cttttgagac accgccttct gcccaccact 4740 cacggacaca tttctgccta gaaaacagct tcttactgct cttacatgtg atggcatatc 4800 -=ttacactaaa agaatattat tgggggaaaa actacaagtg ctgtacatat gctgagaaac 4860 tgcagagcat aatagctgcc acccaaaaat ctttttgaaa atcatttcca gacaacctct 4920 tactttctgt gtagttttta attgttaaaa aaaaaaagtt ttaaacagaa gcacatgaca 4980 tatgaaagcc tgcaggactg gtcgtttttt tggcaattct tccacgtggg acttgtccac 5040 aagaatgaaa gtagtggttt ttaaagagtt aagttacata tttattttct cacttaagtt 5100 atttatgcaa aagtttttct tgtagagaat gacaatgtta atattgcttt atgaattaac 5160 agtctgttct tccagagtcc agagacattg ttaataaaga caatgaatca tgaccgaaag 5220 gatgtggtct cattttgtca accacacatg acgtcatttc tgtcaaagtt gacacccttc 5280 tcttggtcac tagagctcca accttggaca cacctttgac tgctctctgg tggcccttgt 5340 ggcaattatg tcttcctttg aaaagtcatg tttatccctt cctttccaaa cccagaccgc 5400 atttcttcac ccagggcatg gtaataacct cagccttgta tccttttagc agcctcccct 5460 ccatgctggc ttccaaaatg ctgttctcat tgtatcactc ccctgctcaa aagccttcca 5520 tagctccccc ttgcccagga tcaagtgcag tttccctatc tgacatggga ggccttctct 5580 gcttgactcc cacctcccac tccaccaagc ttcctactga ctccaaatgg tcatgcagat 5640 ccctgcttcc ttagtttgcc atccacactt agcaccccca ataactaatc ctctttcttt 5700 aggattcaca ttacttgtca tctcttcccc taaccttcca gagatgttcc aatctcccat 5760 gatccctctc tcctctgagg ttccagcccc ttttgtctac accactactt tggttcctaa 5820 ttctgttttc catttgacag tcattcatgg aggaccagcc tggccaagtc ctgcttagta 5880 Q5,02352532 2001-05-23 ctggcataga caacacaaag ccaagtacaa ttcaggacca gctcacagga aacttcatct 5940 tcttcgaagt gtggatttga tgcctcctgg gtagaaatgt aggatcttca aaagtgggcc 6000 agcctcctgc acttctctca aagtctcgcc tccccaaggt gtcttaatag tgctggatgc 6060 tagctgagtt agcatcttca gatgaagagt aaccctaaag ttactcttca gttgccctaa 6120 ggtgggatgg tcaactggaa agctttaaat taagtccagc ctaccttggg ggaacccacc 6180 cccacaaaga aagctgaggt ccctcctgat gacttgtcag tttaactacc aataacccac 6240 ttgaattaat catcatcatc aagtctttga taggtgtgag tgggtatcag tggccggtcc 6300 cttcctgggg ctccagcccc cgaggaggcc tcagtgagcc cctgcagaaa atccatgcat 6360 catgagtgtc tcagggccca gaatatgaga gcaggtagga aacagagaca tcttccatcc 6420 ctgagaggca gtgcggtcca gtgggtgggg acacgggctc tgggtcaggt ttgtgttgtt 6480 tgtttgtttg ttttgagaca gagtctcgct ctattgccca ggctggagtg cagtgtcaca 6540 atctcggctt actgcaactt ctgccttccc ggattcaagt gattctcctg cctcagcctc 6600 cagagtagct gggattacag gtgcgtgcca ccacgcctgg ctaatttttg tatttttgat 6660 agagacgggg tttcaccatg ttggccaggc tagtctcgaa ctcttgacct caagtgatct 6720 gcctgcctcg gcctcccaaa gtgctgggat tacaggcgtg agccaccaca cccagcccca 6780 ggttggtgtt tgaatctgag gagactgaag caccaagggg ttaaatgttt tgcccacagc 6840 catacttggg ctcagttcct tgccctaccc ctcacttgag ctgcttagaa cctggtgggc 6900 acatgggcaa taaccaggtc acactgtttt gtaccaagtg ttatgggaat ccaagatagg 6960 . _ agtaatttgc tctgtggagg ggatgaggga tagtggttag ggaaagcttc acaaagtggg 7020 tgttgcttag agattttcca ggtggagaag ggggcttcta ggcagaaggc atagcccaag 7080 caaagactgc aagtgcatgg ctgctcatgg gtagaagaga atccaccatt cctcaacatg 7140 taccgagtcc ttgccatgtg caaggcaaca tgggggtacc aggaattcca agcaatgtcc 7200 aaacctaggg tctgctttct gggacctgaa gatacaggat ggatcagccc aggctgcaat 7260 cccattacca cgagggggaa aaaaacctga aggctaaatt gtaggtcggg ttagaggtta 7320 tttatggaaa gttatattct acctacatgg ggtctataag cctggcgcca atcagaaaag 7380 gaacaaacaa cagacctagc tgggaggggc agcattttgt tgtagggggc ggggcacatg 7440 ttctgggggt acagccagac tcagggcttg tattaatagt ctgagagtaa gacagacaga 7500 gggatagaag gaaataggtc cctttctctc tctctctctc tctctctctc actctctctc 7560 tctctcacac acacacacag acacacacac acgctctgta ggggtctact tatgctccaa 7620 gtacaaatca ggccacattt acacaaggag gtaaaggaaa agaacgttgg aggagccaca 7680 ggaccccaaa attccctgtt ttccttgaat caggcaggac ttacgcagct gggagggtgg 7740 agagcctgca gaagccacct gcgagtaagc caagttcaga gtcacagaca ccaaaagctg 7800 gtgccatgtc ccacacccgc ccacctccca cctgctcctt gacacagccc tgtgctccac 7860 aacccggctc ccagatcatt gattatagct ctggggcctg caccgtcctt cctgccacat 7920 ccccacccca ttcttggaac ctgccctctg tcttctccct tgtccaaggg caggcaaggg 7980 ctcagctatt gggcagcttt gaccaacagc tgaggctcct tttgtggctg gagatgcagg 8040 , .

aggcagggga atattcctct tagtcaatgc gaccatgtgc ctggtttgcc cagggtggtc 8100 tcgtttacac ctgtaggcca agcgtaatta ttaacagctc ccacttctac tctaaaaaat 8160 5 gacccaatct gggcagtaaa ttatatggtg cccatgctat taagagctgc aacttgctgg 8220 gcgtggtggc tcacacctgt aatcccagta ctttgggacg tcaaggcggg tggatcacct 8280 gaggtcacga gttagagact ggcctggcca gcatggcaaa accccatctt tactaaaaat 8340 acaaaaatta gcaaggcatg gtggcatgca cctgtaatcc caggtactcg ggaggctgag 8400 acaggagaat ggcttgaacc caggaggcag aggttgcagt gagccaagat tgtgccactg 8460 ccctccagcc ctggcaacag agcaagactt catctcaaaa gaaaaaggat actgtcaatc 8520 actgcaggaa gaacccaggt aatgaatgag gagaagagag gggctgagtc accatagtgg 8580 cagcaccgac tcctgcagga aaggcgagac actgggtcat gggtactgaa gggtgccctg 8640 aatgacgttc tgctttagag accgaacctg agccctgaaa gtgcatgcct gttcatgggt 8700 gagagactaa attcatcatt ccttggcagg tactgaatcc tttcttacgg ctgccctcca 8760 atgcccaatt tccctacaat tgtctggggt gcctaagctt ctgcccacca agagggccag 8820 agctggcagc gagcagctgc aggtaggaga gataggtacc cataagggag gtgggaaaga 8880 gagatggaag gagaggggtg cagagcacac acctcccctg cctgacaact tcctgagggc 8940 tggtcatgcc agcagattta aggcggaggc aggggagatg gggcgggaga ggaagtgaaa 9000 aaggagaggg tggggatgga gaggaagaga gggtgatcat tcattcattc cattgctact 9060 gactggatgc cagctgtgag ccaggcacca ccctagctct gggcatgtgg ttgtaatctt 9120 ggagcctcat ggagctcaca gggagtgctg gcaaggagat ggataatgga cggataacaa 9180 ataaacattt agtacaatgt ccgggaatgg aaagttctcg aaagaaaaat aaagctggtg 9240 agcatataga cagccctgaa ggcggccagg ccaggcattt ctgaggaggt ggcatttgag 9300 From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING
<110> Brunkow, Mary E.
Galas, David J.
Kovacevich, Brian Mulligan, John T.
Paeper, Bryan W.
Van Ness, Jeffrey Winkler, David G.
<120> COMPOSITIONS AND METHODS FOR INCREASING
BONE MINERALIZATION
<130> 240083.508 <140> US
<141> 1999-11-24 <160> 41 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 2301 <212> DNA
<213> Homo sapien <400> 1 agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac tggccctgtg tctcgtctgc ctgctggtac acacagcctt ccgtgtagtg gagggccagg ggtggcaggc gttcaagaat gatgccacgg aaatcatccc cgagctcgga gagtaccccg agcctccacc ggagctggag aacaacaaga ccatgaaccg ggcggagaac ggagggcggc ctccccacca cccctttgag accaaagacg tgtccgagta cagctgccgc gagctgcact 300 tcacccgcta cgtgaccgat gggccgtgcc gcagcgccaa gccggtcacc gagctggtgt ETZ <TTZ>
Z <OTZ>
SE

5vvp5nae5Te aqevbluppe Ereee3Pv116 Tappv5pBeo 315e5epoqq p346qp1.6po pellpe51v1 11o51E-apv OZZZ
qq.63evov5q .e.e5G.Bvq51q oqTaT4Bvee ep5Telqq.eq 4Bvv.33ovol pq3q2q1.2 lvDpq&evq 4EceEpepqqq qq5b15E.5.e. Peblpeaepo ppol54op5 5.6q5aeop41 OE
OOTZ
3qqP*23.6544 Talqq53q55 lopbElpDblo obvPe.61.2. peEcTeaeo6P -e6vopev.aqq ODOZ
15PPPPPPPE' ePP116TIPE) q11;5P'151 6101140T1 oqoae,epeBp 0311T231Pe pp6.111q1o1 VPPPPOOOPO DEclobeqe-el po5p6vp5o pep6pblo5q plpop.1535 OZ6T 5DD 28555552Telp.e6vv peqopopqqo 16Teovlqpq 351pell3qq Dbeopevebe qo35.131qqp oppv653voq DpooppopEca plqoD6oppo SZ

p6e5qqqqoo o;e3vo5voo eBegovoqoep paeoqpoo6p D5e5.65v5po o55p35-11Do 0T7L1 563opae55e 15E.P5p5epE. poq3ppoq.6 epplqq15p po5plpo3pe Boopooloop qvaEreobebv e-epooqqopp oppoBooTeo poqE.p.e6pq 665e&65165 q.655p5op.6 56.25p55epv v5eq65.65-4q poolEolvae Pl.eppEipevo qpqpaoqoop aepoqDoeqo 55.11q51e55 opoolgo66.4 DoSepooqlo 5qa5qela6e p6p.e.6.6q331 OZ

3PPP06105 Eoe'MP1P0 PEq35E.Teal 1p3315p5ee eq6npvpvpe 2PP5POPETO

epevv61p5p peop5poe,51 oqlooqoE. v.65T3.6.e..6v plqpe5Popq aDvoq5Beep 08E1 DE.5-eolqp5 qE.361q6.e.o 5Tee5p.25e BeErebePpBe 5e525e5p5e 6p61B6e52D
OZET 556Tee5.4 q33615e3pq 355 DD
vE.eleo5.6q leqDP5P56P E01012bEel ve655166p5 54.66P5qpP pwq5.1.Dvo olqqpppqoo pBEIBoqqopq Dqlevoepvl gi Dppfiloope Erq61D.6.1qo eplopoo63 1366.apoqBe goqqE,Eq.B. p6e1.6;ovep Of/TT
.66656.1ov5p eoep6p5e3o D5.435.6.2.61 po6ppp6Poq PDDO5D503 00'2051130D

oaTTeBeeqp 65T162-epEce p1366qov65 SpooqBev.G.E. 55q5p.655.45 165o5p555E.
OZOT
elqq.15.556q DppEopepqq q1235pp.661 peafto5.6p6 5a623T4aeo pol.55.1D51q lnoe;Do6D3 5656q3q3o5 poopEva6D.2 oD6e5-loppv 5pD2oq6v5e 5.41-e25552.6 OI

55.6e0E6650 popo-1.6555-e. Bqa&epoboo oftpappoop 366ppo55oo 5a6653o3qp pUeBqoppEr Be3Dl3pe6 eElo.66565p a6.65pooppp 05qopEqp22 frI1P0q11P

4PqT1511P6 T3.55.16o.63 Eqoqooq6D 11xeoppoop baeopopyBq 0005500305 OZL
3555366302 3333qo3o36 353335333.6 .e.6.e133D5 3PPE255q36 e5oa66e33p P3352P2006 35e5.633353 5333355353 3522553355 5ee5po5oo6 53qp53355t, 5 533P6553qq De55ee3qD6 .2,563.4.6.2=p vovo3qq353 33.2p336a6 22D536evo5 0f75 q53q3355-15 5l335a6155 Peo6a6p5o5 o5oo6D56e5 45.6q5.6Dp3l 51.6q35q35e.
08t, DE,BoBoEceo 536DE33eD Bpoeboopol po&apbooqq 325303E654 622=25355 16.6152236.6 3.53366o4eD obov000Bq 3543353E35 5333653536 2=66o0W6 066a/66Sn/1341 CLLMOOOM
EZ-go-TOOZ ZEgZgEZO VD

ak 02352532 2001-05-23 "WO 00/32773 <212> PRT
<213> Homo sapien <400> 2 Met Gin Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr 1 5 ' 10 15 Ala Phe Arg Val Val Glu Gly Gin Gly Trp Gin Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Lou Val Cys Ser Gly Gin Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gin Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gin Ala Glu Lou Glu Asn Ala Tyr <210> 3 <211> 2301 <212> DNA

eeBlqqqaq PPPPPODOPO 05235eTepq poBeBeoEqo eav5e6q361 eqepel6q36 0z61 qBeeoeqoev eve566B6q1 P1TP1PPE,PQ ee.apeoello leqeo66qP6 3.61Poel4D1 gE

DEcqoeqq3q.1 oBepeeeebe qoo&lol.ale peov.66oeol peopeopo61 ol3Dobopeo e6e6qqqqaD oTepeoBeop eBeoeDlpee PDP0'4P005V obe556e5Do ob5eo6mloo 5.54oepe.66e t.qq.E.6eve6 15ee5e6ee5 ooqgooeoq5 PPPqT3151P DOBele000P

6Do3ooq3p3 -leobeoBeBe eepooTIDee opoofippleo PD4PPPE.PTe B55e666.466 465.6e5DDqb 6ne6p.65vee e5e.56.651q epoqeoqeop eaeve5eve3 qoqpoolDoo OE

DE30qD0P10 6blqq6qe6.6 opooll3561 oobeopoqqo 6.1o5eqD5e e5ee5.6poq 3eep3e5qa6 5oe'4qq.eqeD e5qn.6.6;e11 'leqolbebee p1BPPPPVPV PPQBPOPPPD

eveeeBgebe eepobeoeBq Dlloqoqp61 ebq6qq.6e6e olTeefieopq weDq6Bee3 p615eolqeB lqeD611Ba3 Eqee6qee5e BeZebeeeEe 5e6e6ebeEce 6e5q56eBeo OZET
.656Tee6q3 DoE.q5eopq oe65evoolq eblelea6Bq lewe5e5.6e Boqolebbel gZ

ve6E056.6e6 555.6e6lee eplbqqloeo oqqlepoloo a6BSolqool oqq-evovoaq oon pee6qao3ee 15161p.6110 ealopoo5q3 qa6.6q=1.6e wqqq.66'15q aBe151DeQD
OVTT
66555qoe5e epeo6e6epo p6q6o55a61 DoBeeeBeoq upooBloboq opeofralopo ooqqpbeeTe 65qq6eve5e eq15E.I.oeB5 5eopl5eee.6 55 P5555 T65o5e5.6.6e OZOT
P1'33-455651 Do050Deolq lleobee55q pea5ea6.6e6 BeZeoqqpeo oo55q3511 OZ

looagooBoo 5.666qoloo6 opopbeoboe oa6861peoe 5eopoq6e6e 6.1Tee.656e5 .655eD656.63 POD 555w 5loBeDo6Do pEceoz000po DE,Beep56o3 506.65opoe e66e6lo336 5epoqoae5 e61o55555e o555.eopoee o6qop5qeee q61qeoqlqa leqq.45qe.6 qq-166q8p50 5loqopE.13 lqqvaap000 BoBoopee5; 33356=36 OZL
36B6a65poe D000qoppo6 p600p6o336 e5eloeqoa6 o55535 e60055e00P SI

epoBeeepo5 o5ab5ooD6o 50=35.6053 oBeeMoo&E, 6ee5eo5=6 Bo.qoboobBe 533e.66531q oeBBevolob e65olBeooe eoeopqloBo DDeoqo3536 eeo.6q5ev3.6 Ot.S
6D.qop66.15 Bwo53.655 aeo6363535 o5oo6o5.6e5 .155q663p3q 515q3.6135e 08f7 oBgEloboBeo 6o535Doeqo 6Doe533p3l eoBlabooql oe5000.6651 5e3oe63.65 On, q56q5pe35.6 o6o35.6pqeo oBoappoo51 o6qop5o6o5 E3p35536q5 aDDE.60DDE. OI

1.6,66qa5e5 opeoqbBoo6 peop5o6eD5 op615po666 le5ooe5160 elo500Deol l3eD5-4o5e5 abooEclobeo eq6e5ool5q BoeBevepoe Befiqql0000 epoepopoqo OVZ
0.650565e56 3ee6a66D56 63ove5lepo aBeepeepee 5e561p6e66 DovooloD5e Eop3peq5e5 e55oqp6p53 pooleoqeee 55oepo5le5 qeeEceeoqq5 o55eo5Eca55 OZT
.66e36.65e5 5.6eq54533 ql335eoepe oe1561o5lo 061D15o133 E151D00551 3P3OD4D6PD 5qapoe155q 06.6q3woq3 =5150E616 6ee651oeq3 6q5l335e5e E <00T7>
uaTdes owoH <UTZ>
06612/66SWIDd EZ-go-TOOZ ZEgZgEZO VO

Q5,02352532 2001-05-23 aatcatttcc agacaacctc ttactttctg tgtagttttt aattgttaaa aaaaaaaagt tttaaacaga agcacatgac atatgaaagc ctgcaggact ggtcgttttt ttggcaattc ttccacgtgg gacttgtcca caagaatgaa agtagtggtt tttaaagagt taagttacat atttattttc tcacttaagt tatttatgca aaagtttttc ttgtagagaa tgacaatgtt 5 aatattgctt tatgaattaa cagtctgttc ttccagagtc cagagacatt gttaataaag acaatgaatc atgaccgaaa g <210> 4 <211> 23 <212> PRT
<213> Homo sapien <400> 4 Met Gin Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr Ala Phe Arg Val Val Glu Gly <210> 5 20 <211> 2301 <212> DNA
<213> Homo sapien <400> 5 agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac 60 tggccctgtg tctcatctgc ctgctggtac acacagcctt ccgtgtagtg gagggccagg ggtggcaggc gttcaagaat gatgccacgg aaatcatccg cgagctcgga gagtaccccg agcctccacc ggagctggag aacaacaaga ccatgaaccg ggcggagaac ggagggcggc ctccccacca cccctttgag accaaagacg tgtccgagta cagctgccgc gagctgcact tcacccgcta cgtgaccgat gggccgtgcc gcagcgccaa gccggtcacc gagctggtgt 360 gctccggcca gtgcggcccg gcgcgcctgc tgcccaacgc catcggccgc ggcaagtggt ggcgacctag tgggcccgac ttccgctgca tccccgaccg ctaccgcgcg cagcgcgtgc agctgctgtg tcccggtggt gaggcgccgc gcgcgcgcaa ggtgcgcctg gtggcctcgt gcaagtgcaa gcgcctcacc cgcttccaca accagtcgga gctcaaggac ttcgggaccg aggccgctcg gccgcagaag ggccggaagc cgcggccccg cgcccggagc gccaaagcca 660 accaggccga gctggagaac gcctactaga gcccgcccgc gcccctcccc accggcgggc cp, 02352532 2001-05-23 gccccggccc tgaacccgcg ccccacattt ctgtcctctg cgcgtggttt gattgtttat atttcattgt aaatgcctgc aacccagggc agggggctga gaccttccag gccctgagga atcccgggcg ccggcaaggc ccccctcagc ccgccagctg aggggtccca cggggcaggg gagggaattg agagtcacag acactgagcc acgcagcccc gcctctgggg ccgcctacct ttgctggtcc cacttcagag gaggcagaaa tggaagcatt ttcaccgccc tggggtttta 1020 agggagcggt gtgggagtgg gaaagtccag ggactggtta agaaagttgg ataagattcc cccttgcacc tcgctgccca tcagaaagcc tgaggcgtgc ccagagcaca agactggggg caactgtaga tgtggtttct agtcctggct ctgccactaa cttgctgtgt aaccttgaac tacacaattc tccttcggga cctcaatttc cactttgtaa aatgagggtg gaggtgggaa taggatctcg aggagactat tggcatatga ttccaaggac tccagtgcct tttgaatggg 1320 cagaggtgag agagagagag agaaagagag agaatgaatg cagttgcatt gattcagtgc caaggtcact tccagaattc agagttgtga tgctctcttc tgacagccaa agatgaaaaa caaacagaaa aaaaaaagta aagagtctat ttatggctga catatttacg gctgacaaac tcctggaaga agctatgctg cttcccagcc tggcttcccc ggatgtttgg ctacctccac ccctccatct caaagaaata acatcatcca ttggggtaga aaaggagagg gtccgagggt 1620 ggtgggaggg atagaaatca catccgcccc aacttcccaa agagcagcat ccctcccccg acccatagcc atgttttaaa gtCaCCttCC gaagagaagt gaaaggttca aggacactgg ccttgcaggc ccgagggagc agccatcaca aactcacaga ccagcacatc ccttttgaga caccgccttc tgcccaccac tcacggacac atttctgcct agaaaacagc ttcttactgc tcttacatgt gatggcatat cttacactaa aagaatatta ttgggggaaa aactacaagt 1920 gctgtacata tgctgagaaa ctgcagagca taatagctgc cacccaaaaa tctttttgaa aatcatttcc agacaacctc ttactttctg tgtagttttt aattgttaaa aaaaaaaagt tttaaacaga agcacatgac atatgaaagc ctgcaggact ggtcgttttt ttggcaattc ttccacgtgg gacttgtcca caagaatgaa agtagtggtt tttaaagagt taagttacat atttattttc tcacttaagt tatttatgca aaagtttttc ttgtagagaa tgacaatgtt 2220 aatattgctt tatgaattaa cagtctgttc ttccagagtc cagagacatt gttaataaag acaatgaatc atgaccgaaa g <210> 6 <211> 213 <212> PRT
<213> Homo sapien <400> 6 Met Gin Leu Pro Leu Ala Leu Cys Leu Ile Cys Leu Leu Val His Thr Q5,02352532 2001-05-23 Ala Phe Arg Val Val Glu Gly Gin Gly Trp Gin Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Arg Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Lela Val Cys Ser Gly Gin Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gin Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gin Ala Glu Leu Glu Asn Ala Tyr <210> 7 <211> 2301 <212> DNA
<213> Homo sapien <400> 7 agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac tggccctgtg tctcgtctgc ctgctggtac acacagcctt ccgtgtagtg gagggccagg 120 ggtggcaggc gttcaagaat gatgccacgg aaatcatccg cgagctcgga gagtaccccg Pee5poe6q. olvp5Tepop onz 6epplve335 qqpop5e5vo oq6p5epoqq oll5q315e3 evqqvp5Tel 410511qP2 g OZZZ
115W23P6q e'e6Q6Pq5qq 0'1'1114.6-epe po6qeql.Tel 15pplqoPol olqqpqqqp 091z qvp.eqq5-epl qBeBuepqqq ..qq.55q5-25v ppEcqev5n,e3 pooq5qqoe5 5535ovooTa 3qqppo6611 11q11.6pq.55 .43e55p3523 a6ppe5TeTe opEcl.pova5p e.6.2pev1.1 Of7OZ
BPPPePPPP evq.4.61qee qq3115s35q Bloql1Dell oqoaepae5p 33-13Teolep ep5T4-3qq33 PPE,V23DOPD D5qo6eqt.q po5e5vo6lo Pes6vBa61 eleDP151D6 01 pveb56.65qq Q14qeP5PP ePqDVaellD le1PD651V6 1.6qepPqq01 oBlopq-qoql 35eoveepbe 13D5'43q1ze oppe65ov3; appoppobq Dq3D3153peo P62611-1100 plvDP35epo p5epeD;oev 2De3qop5E. p5v566e600 355no6lqo3 Ot,LT
55qo2aeB6e p3lq56eep5 15v.e5e5pv5 DoqqopE.oqb ueseqqq3.61E, poBelvooDe BOODOD;DDO 4po6po5v5p peopoqqa2P 00D05001PD P34PPv5Ple 6666E656 gZ

1.556p53015 .65e6e55vee e6eq.55564q vooTeoTeoe 2leev6veep .1Dq2331333 DvoolDoelo 66666 opoo.11o551 33.6poopl;o 51o6qpqo6e p5ve561Dol opeppe5q35 53eTITeleD p51o55qt,q1 qeqoqbe52p PlEcePPPVVP PPPBPDPVPD

ppeppBlebe ptopEceoe51 3.1pqn6q eb.4.6qq5e5p olTee5v001 lorolEEP2D
, 08E1 D5.15vol;p5 TTeo5T45e3 5Tep.6qep5e .625v5.2-2e5v Bv5p5pBe5e 6e5255e5po OZ
ozET
6561ep5qqq qp3635popq 3v55ve33qq p5.1-eqe355 lelop5e65p .63o3p5E.e.1 p256.655e5 5.555v632E. eeq6qq.qopo oqqlveoqop e555pqqp0a D'34PPDP0-21 opeEclqopev .4.65q3511D eelopoo5qo qp5blopq5e q3;q15515.1 e6pq5.4oevo 556553v5e e3e36v5eD3 D61535.6p5q 3o6vve5p3l Po3351353.q 33e353q3po 0801 3pqT25E-e3e 55345ppp62 eqq55qDp5E. 5e3pl5ppe5 55-15p555q6 q66o6v555e gi .elqqq5555.1 o3oboovDT4 11e06PP661 PPP6e066P6 5P5e3lqDp3 3pE.61.3531 loppl33533 .655.6qpoo6 opp36eD53v DD5p6lopoe 5povoq5e5p 636556 555eD6555o p33pq55562 5135voo6oo obeoloDopo DB5evo5Boo 535563331e 0T78 p56e54o335 5poolqoop5 e5ao55555P D555popopp DE.Doblvev TelT451e5 T1.5545353 5;plopa6lo 1.eoppoop 5D5oDoee51 =5533=5 01 OZL
D5553653ov o333lpo3o5 3533353335,e5el3pl3o6 3ee5e551D5 E.50365-203P

c335ppp335 35e5533353 5333355363 DE2P55op55 5ppEceo5oo5 5656E.

600p55531 a265ep3q35 p66o1.6epoe va2=11053 opeplop5D5 P.235.15peD5 Of7S
q5333o55q5 63363565 eP3535o5o5 35036o55p5 155z6.6333 545131535e 35153535P3 53.63533el3 6Doe5ooDol p3543.633q; 3vEo33E.564 5eq33e6355 On, 155q5ve355 a53o.55pqeo o6aevopo51 o5loofia5o5 53op553515 2o35533.a35 15155qp5u5 p3e3356336 ppoDE.D5v3E. 335z6Do555 qPboae615o pqp5opovol q3e351D5e6 p5DDE.I.D5po pq5u5ooq5.1 Boe5peepoe 5p5;qqopoo P3DP3330q3 3553555e56 3vp5e65355 533pv.61vop v5evovpovp 6p55D5E.65 3opoolDo5e 066LZ/66SIVI3d ELLVDVO CM&
-EZ-g0-TOOZ ZEgZgEZO VO

_W0 00/32773 <210> 8 <211> 213 <212> PRT
<213> Homo sapien <400> 8 Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr Ala Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Arg Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg =

Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 20 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr .WO 00/32773 <210> 9 <211> 642 <212> DNA
<213> Cercopithecus pygerythrus <400> 9 atgcagctcc cactggccct gtgtcttgtc tgcctgctgg tacacgcagc cttccgtgta gtggagggcc aggggtggca ggccttcaag aatgatgcca cggaaatcat ccccgagctc ggagagtacc ccgagcctcc accggagctg gagaacaaca agaccatgaa ccgggcggag 10 aatggagggc ggcctcccca ccaccccttt gagaccaaag acgtgtccga gtacagctgc cgagagctgc acttcacccg ctacgtgacc gatgggccgt gccgcagcgc caagccagtc accgagttgg tgtgctccgg ccagtgcggc ccggcacgcc tgctgcccaa cgccatcggc cgcggcaagt ggtggcgccc gagtgggccc gacttccgct gcatccccga ccgctaccgc gcgcagcgtg tgcagctgct gtgtcccggt ggtgccgcgc cgcgcgcgcg caaggtgcgc ctggtggcct cgtgcaagtg caagcgcctc acccgcttcc acaaccagtc ggagctcaag 540 gacttcggtc ccgaggccgc tcggccgcag aagggccgga agccgcggcc ccgcgcccgg ggggccaaag ccaatcaggc cgagctggag aacgcctact ag <210> 10 <211> 213 <212> PRT
<213> Cercopithecus pygerythrus <400> 10 Met Gin Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Ala Ala Phe Arg Val Val Glu Gly Gin Gly Trp Gin Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser . VR) 010277.3 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gin Cys Gly Pro Ala Arg Leu lieu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Pro Glu Ala Ala Arg Pro Gin Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Gin Ala Glu Leu Glu Asn Ala Tyr <210> 11 <211> 638 <212> DNA
<213> Mus musculus <400> 11 atgcagccct cactagcccc gtgcctcatc tgcctacttg tgcacgctgc cttctgtgct gtggagggcc aggggtggca agccttcagg aatgatgcca cagaggtcat cccagggctt 120 ggagagtacc ccgagcctcc tcctgagaac aaccagacca tgaaccgggc ggagaatgga ggcagacctc cccaccatcc ctatgacgcc aaaggtgtgt ccgagtacag ctgccgcgag ctgcactaca cccgcttcct gacagacggc ccatgccgca gcgccaagcc ggtcaccgag ttggtgtgct ccggccagtg cggccccgcg cggctgctgc ccaacgccat cgggcgcgtg aagtggtggc gcccgaacgg accggatttc cgctgcatcc cggatcgcta ccgcgcgcag 420 cgggtgcagc tgctgtgccc cgggggcgcg gcgccgcgct cgcgcaaggt gcgtctggtg gcctcgtgca agtgcaagcg cctcacccgc ttccacaacc agtcggagct caaggacttc gggccggaga ccgcgcggcc gcagaagggt cgcaagccgc ggcccggcgc ccggggagcc aaagccaacc aggcggagct ggagaacgcc tactagag <210> 12 <211> 211 <212> PRT
<213> Mus musculus <400> 12 Met Gin Pro Ser Leu Ala Pro Cys Leu Ile Cys Leu Leu Val His Ala Ala Phe Cys Ala Val Glu Gly Gin Gly Trp Gin Ala Phe Arg Asn Asp Ala Thr Glu Val Ile Pro Gly Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Asn Asn Gin Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Tyr Asp Ala Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Tyr Thr Arg Phe Leu Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gin Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gin Lys Gly Arg Lys Pro Arg Pro Gly Ala Arg Gly Ala Lys Ala Asn Gin Ala Glu Leu Glu Asn Ala Tyr <210> 13 <211> 674 ak 02352532 2001-05-23 <212> DNA
<213> Rattus norvegicus <400> 13 gaggaccgag tgcccttcct ccttctggca ccatgcagct ctcactagcc ccttgccttg 60 cctgcctgct tgtacatgca gccttcgttg ctgtggagag ccaggggtgg caagccttca agaatgatgc cacagaaatc atcccgggac tcagagagta cccagagcct cctcaggaac tagagaacaa ccagaccatg aaccgggccg agaacggagg cagacccccc caccatcctt atgacaccaa agacgtgtcc gagtacagct gccgcgagct gcactacacc cgcttcgtga ccgacggccc gtgccgcagt gccaagccgg tcaccgagtt ggtgtgctcg ggccagtgcg 360 gccccgcgcg gctgctgccc aacgccatcg ggcgcgtgaa gtggtggcgc ccgaacggac ccgacttccg ctgcatcccg gatcgctacc gcgcgcagcg ggtgcagctg ctgtgccccg gcggcgcggc gccgcgctcg cgcaaggtgc gtctggtggc ctcgtgcaag tgcaagcgcc tcacccgctt ccacaaccag tcggagctca aggacttcgg acctgagacc gcgcggccgc agaagggtcg caagccgcgg ccccgcgccc ggggagccaa agccaaccag gcggagctgg 660 agaacgccta ctag <210> 14 <211> 213 <212> PRT
<213> Rattus norvegicus <400> 14 Met Gln Leu Ser Leu Ala Pro Cys Leu Ala Cys Leu Leu Val His Ala Ala Phe Val Ala Val Glu Ser Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Gly Leu Arg Glu Tyr Pro Glu Pro Pro Gin 30 Glu Leu Glu Asn Asn Gln Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Tyr Asp Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Tyr Thr Arg Phe Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala ¨

ak 02352532 2001-05-23 Arg Leu Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gin Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Gin Ala Glu Leu Glu Asn Ala Tyr <210> 15 <211> 532 <212> DNA
<213> Bos torus <400> 15 agaatgatgc cacagaaatc atccccgagc tgggcgagta ccccgagcct ctgccagagc tgaacaacaa gaccatgaac cgggcggaga acggagggag acctccccac cacccctttg agaccaaaga cgcctccgag tacagctgcc gggagctgca cttcacccgc tacgtgaccg 180 atgggccgtg ccgcagcgcc aagccggtca ccgagctggt gtgctcgggc cagtgcggcc cggcgcgcct gctgcccaac gccatcggcc gcggcaagtg gtggcgccca agcgggcccg acttccgctg catccccgac cgctaccgcg cgcagcgggt gcagctgttg tgtcctggcg gcgcggcgcc gcgcgcgcgc aaggtgcgcc tggtggcctc gtgcaagtgc aagcgcctca ctcgcttcca caaccagtcc gagctcaagg acttcgggcc cgaggccgcg cggccgcaaa 480 cgggccggaa gctgcggccc cgcgcccggg gcaccaaagc cagccgggcc ga <210> 16 <211> 176 <212> PRT
<213> Bos torus <400> 16 Asn Asp Ala Thr Giu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro 5 Leu Pro Glu Leu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Ala Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gin Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro 15 Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gin Arg Val Gin Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gin Ser Glu Leu Lys Asp Phe Gly Pro Glu Ala Ala Arg Pro Gin Thr Gly Arg Lys Leu Arg Pro Arg Ala Arg Gly Thr Lys Ala Ser Arg Ala <210> 17 <211> 35828 <212> DNA
<213> Mus musculus <220>
<221> misc_feature <222> (1)...(35828) <223> n = A,T,C or G
<400> 17 09TZ Bpp3op65;o 3loveR613o poolboqol l36evel3o6 P2DOE.PP20 ovq.po5o2E.
ocaz olp56e5551 ofippqa6a6 0555,6532pp 64q33pb;o1 ppeo6Teqpq opoBlqqopq gE
OtOZ popope5p55 qq3351365.3 e5Inv55eDq BqbvleoBBv qp361155e3 6P0.5qD1e11 0861 115P511541 5.5eov66.655 bD6D3 Ece55Paqob Bo6fia.656 65epoSpvbp 0z6T ep3l2l3evo looplopoqo 3eoDD.431 Doev.65.6e1 al&eqeo5lo 5p15-epo5.2 0981 001005P315 55e0epqpv6 Povepla6.61 62up.6.251D3 lo5;qaq.po 616.4a65.153 0081 veolEqoqqr, oft.ol-aDqDe D5D2651-1-e. PE,e5q35,P5 p5v1.5.6551 OVLI 65235 15Bleolvol 651551.5.6De p5voqqoloe oqq555q-eq 2DOPOPPDq 0891 51BwapPlo DE.35e5Eopl pooqlleqqo Bolvo5lael 5qvp6qq6EP a6.666Eqqa2 0Z91 De5po.1315e lqp5TeDpoq 15555p5po o6p6p35vD5 qpoBeq335; oblpopEbvq 0991 33eoblooq5 lopq11516q BTepelloq5 1evD11101v 55Qu'161D56 66e5q065qv 0091 556036.61v5 o5p.e.6.6vov 356 qoaa5ploo qbevplqpql ze3333p6vv 5z OttT 5qBqee51P0 1111555115 DPDaqP5.2.26 6vD6q1voe3 D1.261v3.66q 1Beepv1v5E.
08E1 epopopvqlp lep3oPe5v5 P555.16vD6, Dollqpv5e P1PP11P115 1e01521113 OZET e22VBPPPPP Tqlqvqq3qq 5q5.avq.5olo Bp5.4qpqvq eq5.161vep6 5pqopopea6 09Z1 ql-evlDoqop T1p2ve5q3q Poo6p5oppv 5PeeTe5qep eeopve.evev 65.wE.Pqqop 0OZT 6q3q.62ppt, Eepol.ollob 5qopqq1D55 pv=5.25qp5 uq.62.255a6E, e&eloopeqe oz OtTT eeeqopovze veDoqppe56 voq6qEopT6 qopqqqqp5t) 5646q6.6.64o qpPoP1153e 0801 6125,55ovpo 66.12Do5q15 qpebEraBoDe 5p56p 5qoqq5eqpo OZOT 64a6eDE,516 qvp45D53.63 3.6641Dqp-a6 qp56.61o55q 56eaev13pq oll.53v00P1 096 355pe31655 63655q13.66 5qq36.6qp3 Elvopploloo BeoloopBqo p515veep3o 006 DoPPoponoo 6De6e5vo3D Bqeo600qos5 55qp5p.65e6 5o5Pe5qeD5 o5opoolvq Ot8 33Eop6D1e5 55v4eloppq pe63-e3eelp p5=563631 lpe5e6qppE. 2pol6Bobep 08G le51.e5opq 106P6PDEPQ PPPOPPD45P 256qvepe26 Te,e553.6opq 66431 OZL olq3oqq.15 oq61313355 1Taqqp5v53 6v.e.e.6q35-36 E..eae5pepv6 5PPP52PPPE, 099 1D2o33vTee ppqqwq4q4 -Th6Te-elqpee 1E0153110q qp11500111 656E
009 53DPPp p6qeqqeqo 5oqq15.650 EoqoppEo55 3665 p5 qq335D31P5 01 OtS D553331q33 q6.65pEcTqTe oqpqq.Ppv qa6TqveoBq elEvlpEoq5 DoE,q5po6eD
08t .65q3qp.51 6.6155D333 163ep.661p 6Polpoo56 3vo53oz6le qopT4DE.5qp OZt ece5.1e5.6125 z5PPP5PP3P 6p36p36.6E. eepBeppopo q51266136D 5.605-461-2-09E 1051050vv-2 Pu5106o53e PPQ5ale5.1q Boevv533Po epopeleEcal 535.6qopv.ep 00E 115PeP0115 evqe633616 eo5o3e.-2.5 5epope5o5 35epo5p512 .65051301P
OtZ q65qqop5e3 qvaevopqpq PTeppovE5 Booftoqpoo v'e.PEolepo 5536p353t3 081 oqe1354554 eeep5oqe6 p351102.66ve oTealqvol5 1.6p55lee35 3Tqv.313ep5 OZT DqloololEo 3p 553 vo.635epoP5 5q35P5opP5 34-evoMeve ppeo510510 09 qD3e4e541v Ece.644.6D561 4D35pElpEo qap5o61q2q pe35p35-261 5511115050 066CD66SWIDd ELLVDDO OM
EZ-go-TOOZ ZEgZgEZO VD

OE t 31555E.E.le oqlvD65plo 66-1.4.eq;.4qo poplopqqqo 665636vo33 opqlvp52D3 09Zt o3Tgropp.e5 q5epo6p5qq 6qpoE,E.Doo Dqpoppa6qa qpeEceePq63 peo5e5q3D6 cc 00Zt 5vqoo3q65e qove6lnqv 65.65q1b363 q5q4q1o5 .6vD3BE.Bqp6 -15qe3.665be OtTt pEcgop.656-43 op5pqa3pqo peoqoqlqq DEcq3qq.E.v55 loo5pElBp 33551.36qa6 080t 5pqolellq5 64pol366B blonqv5q= oqlo2Dq656 vo5.6p5qqqq 'ITeqqopeDa OZOt 3pp3opoq1E. 3qPpolloq6 ODE3VP510P leblz6.6eqv poBqpopoeo 6.46153.610 096E pTe,16.6qe1 6T2333p3qq lqqvpoofiq 536p61qpel 11=31qqop 1361.5305eq OE
006E 66pqq5P65E, a6poebeoPb poqqqvzeel e-elv5Bweo eblvor,fivr. op.61.6q36v Ot8E qt.e361Dpo DE6556qpbo o6v5q5qpop qopl5qBpq qe.656poPe pae03.61P1Q
08LE PPRDOO'IPBV Pe6eDoqq6E, pvppvblae ooppobbeBv ePooloolqo EqoppE65qo OZLE loopE,Pvq6o 6P5 3D obbooTavo q6DeP6Bepl 551peeppqp opo55qp635 099E Bepaebqlpo TavEceoPoo 666pvp61qq pelpEcq5up loovoTIEre 55poEo2qp.6 cz 009E qpppo65.ev3 qoppolvD55 lolleBbvfo .e33lo5eqo .6z6eopeope OtSE 316Dea6Te3 1-433155eo6 56p5op5q35 lpeEq3D163 vopopolvD5 .6.6.3epa5ro 08tE B3D5vopaeo lvop.65poqo 66q5loq65p oploopqop 6OPODVD3q -qqva415qo OE 05qa656533 qopapoqqo looqoolqoq e663q355-2.6 eepoblqvlq Doqp.65.6ovq 09CE 66635-23E5p o5eotoo5PE ve55P365p5 qa&e.BlvDqE, 5eEre6.656e5 .465.65'3055q oz 00EE elez6opp23 66o35136.2p 55651q-app qft.paeope5 66q5o5.6.6.Te D5pope56.6v OtZE
5po5et.656e PE.5554.66pp 623.ekoeopE. qvu6a6BeDe Blva,poBoo qoBBEeftop 08TE poozepv655 .6.35p55ep.6 5DOPOPOPDP 3bv3buqp61 BTWQ662We OPPOPOIDDq OIEpoovle.l3q6 qqa6eq6qop 3qoeb-lo3.61 pooploB-15q 6.156PooTaP .66v10.615P5 090E 3eq3661po op-qopqe Teoloeopol o36Pq355.23 lovve46551 pell05561 ci P3q.6.652q33e epolqeop6p oppeBe&ale 0t6Z 6.61D656e06 61PPP156P a6qp16q3.6 evqqp5qq36 5q5PP-qPqR6 ap56.eppo56 088Z elpeppeuBB .26p6eovq-qq qeppoolelo Blovo5.6q;.6 POBVPOODOP 000=20030 OZ82 DPB5P0q3P0 PPql=o5.6 voqDpop1.1. loqopopebe oppoo6qolo .65eolva6pp 09LZ eDOPDPPPqP lev,651o3.6 ppboovBeop Ecloee6evo qoPoof,502 5665 OI
OOLZ 5eo6vq5Doe 35.6eoepq.63 333p33pq6 poqleqlqya ep6opeoqfip poppo'lloTI
Ot9Z ep3qq5P3p6 -6qp3e6l33 6e666p3qq3 ple61qp55e q66q646e33 Plq6P613P
08SZ e5e33.6.25 e6pD6.11pop 5.2q351.6po qae1355.655 5qqpq531Po DDE.D3q.
OZSZ
33p1.66.ep34 6e3ap33P6 1q13133655 ePboBepe5E. 55e5Q6161 P651qqPeeq 09tZ q1E.P3.613-2 PS P63 5l3e3p5p56 ea6l3l3p55 p5p66pi C
00tZ poqBeo5565 5555556655 65.6.6.6DPDT1 6q3qp6.6Pqq TevE.pobloo q.6.qpoqloo OtEZ POODODPDOO 3P0000q23P 356p3p35P5 le3p3op5p.o 5poqD55131 3.636v3e.664 33y3333331 1-13136e3.6q 33.6e6p3.6.6 56.515.6p556 P6up56PEce5 15D656e3q3 OZZZ 1E,v3031 36236.2515 513.4.6p666e 6q.6e336Ere3 qolpolPovo 6EID3113555 LI
0156M166SIIA2d ELLVE/00 CV&
EZ-go-TOOZ ZEgZgEZO VO

089 leqeDeoPoP e61qq53516 EPPDP.6.6qP DPDEqPDQDD 3oD6E.eqqT3 pvDDE.Tebe OZV9 BPDPEIPP5VP 53qeDpvpvq pe5p5v5e5e eqvqpoe.ell 005;001PBP 5eE1.61D4DE
cc 09E9 .65DloBebq 3POPePVD5P zevaepopP; D55opepo5 e6poleowv 66Beaebu66 00E9 36.6e.665oqD eebvpoogpe 16qDoEqoa6 6q6ebvD666 q65.61oTeeP e1q010PqeP
0VZ9 .6e;pq3W65 1qq6p5poop qpp5Tepope eqoa63.66qq 355 P0555106P3 0819 66p5B5vo5p 5bp.436555-E. DopepP6v6 ael;PoDqPv 56e3ee555v la'ao5BPD5 0Z19 qqovooplo lolol336Bq peqe551pet. eTe5e'ela6; peDv1555q6 330361553D of 0909 105P520611 Depos6pqo qqopE.Blop 3e68'3eppel 26peqo5ov 3plE.B6.61q.
0009 po6q5600qo Ece6p33pleo 5qqp66.6535 .e.pDp.e6.435 4I5BEBE.o6 loP3-43.6q5v OV6S ovoqq565q PTGOBPODDO oqq;bElloo al6q2o3.255 6.6D-4D3q.66q oBbeoqqopb DD5P5Bl3q ;Boqoao564 BvqvobBeqD pooplqaTel PqD1PqD0OP PE0.65111op OZ8S geoppoqqq 1Buoblool 116'4-2P6510 1B51110'avE, 6655 leq6q6qqop gz 09LS 2.6q31q5q10 ep.665upe-40 eB61143101 0031DP1D 35qD1-2356 OOLS 66qoq3ve65 BepTIE,oqop 1PD166166 .152a65'wou. 010T4DODVD qq055PB5P
0V9S 65vvPqq5pq oBaqop5Elop ovv5e6.64P6 pqv-6.6qoqop ogolqp5q6q 3qq.6oDBpop 08SS qoplo.qqopo 5p5o3otToo Depeo'16q3p o.32eqlo43o 3;6Eq2q0DD 556PDDE6vu OZSS lqq3p6po6q o5epv56Elo p6vDevq513 11PoP-4B136 eveol5opol ev6ovE.vbo OZ
09VS Dole05e03 ppo4E6E35p ova66135qe 55 55855P qqqopQ36pr, 6.6,34DETee 00VS a5eP.6.6q11 Eq35E,p6qpq loopqoopoo 5ep3lq5q3E. loop546285 336Te.66.6q1.
OVES opqe35q55e po263.1135e opEcTeeBeo eTq.6Te3poq Be6e51613.6 6q11qeEvel 08ZS opqBeftpll oqPTeeBoel e33Tep3e35 qlopeo6151 Dqv53pee41 DoTeoTTeve OZZS eovu56ea65 etOqD51DDP 0411016q5 PODEI2PPPel POlOPPqa2V PO1P5111q gI

.6,4qpe3pq Plqqobvebq e5e53e3.65 qPp.eDlqopq Do2.6.23.4qp 1Te5peloo COTS ;Pea6153'4.6 E.3P0Tee eqeP3e6t,1 qpep.1.35po laepveopEt. qD13;53vve CVOS veTeopEQ13 6p.61.o.epTee oDo63e-e55 5leop5v5q3 133.613l333 le610TePP1 086V 10105P3630 01013'111155 qp56qa6q1e ovlopeo53 qpoolqaepo .6.65Pq0300.6 .2.6v6q3.4qo 3elq5q3=5 36-qqp.qp-1 Tqqvqvoqe p.e.33l33v3 pvqq365130 01 098V qq.6q651D13 opTaleoppv 3be.q15Pe61 33D.4p3ee5q DqqqP055.61 DP615P0405 008V e3ve;33E. PP6JP PlOBPPOPae DPOPDPDPOP pEoepeovoe DPOPDPOPDP
OVLV p6qve53eD; qpaqobeBep 3q36v33e16 36q3e3eD6 q5.63e55.155 loqoBBTql 089V 35t3e.23116 Bove-a6q.654 op61.4ploqg BevE.Poelqo e651oeo3P4 333q3pe5p5 0Z9V eep333qe6 q6Blool5q3 33lq36v35e 3p3pq.66513 olqp6.6P5P copq05;5qq 099V 1314305631 PP45q.66PD5 peop366q6 6.6.6p55656q 6qolopolol 16BpoeBpa6 00SV ebeDbeoqbq ebP3666p3e 3I6E5p6e51 365pp5v3e5 5P3e6v3455 q5-21055q06 OVVV p5e5lve633 p662q5epp5 eoebe36336 P36qoppq36 336335333 1q31.6epqq5 08EV 5.633pTe6p5 pqopaeofto q55.6pDqo55 qou53.23356 q5p5Te.E.56p BeDDEolDDp 066LZ/66Sf1/13d ILLINAO OW
EZ-go-TOOZ ZEgZgEZO VD

O'9 8 151qq5qqlp q.111.2.4eoe eel;;10 1005010'10 lqpp5qap16 1Deqopovnv 0858 ple;DE.qoqp e6qoqq6E61 11.pqbp6pq Bblooqapo ovo6qpobvp vDeDqbe-e5q gE
OZS8 6pD1.6qo qopoeD6oq po5q.64Ecepe p5a66-4D5o1 513qpobloq 35-3;0q00PD
09T78 5eepo;q23 poolepolo popabqopop 164v3qq3lq 5Teop3ppeo leopp5q6qe 008 pEqbeaepo5 DOQ:e0POOP3 ;PopEreep3E, 3oopp6E.DE Bv3e3.6633; 4.6Qvqqeoq6 017E8 Ba65q6.1euq qp.651pl6o elqze6.6.61e BTeDeBeSeD 561B3pDpqv 08Z8 PoolvPP510 DqEcepolDlo lp33Ep5q05 1.3.3eqq&e.o6 gEpe66vo6e b-e361P103 oE
qbbooppe2B qopv5551o6 Tql6;vou.6.3 poopqBvE,E, qq56p5E,p55 .3q6e5.6.13vp 0918 b5lopop;p5 qaqPD66655 PpopoofiE.P.6 5q100DPE.5 53615161E0 P336q515qP
00T8 33q6.2.5.1p3.6 opE,15q.6:16 16oP3636Te qTqle1.11P qq1u0o1P
aelepppepq 0T08 11q0113pq epp.661qe.16 111pe4q.45 4q61616q6q 616q6.4bq6.6 66qq61qeD6 086G 56P6ePoul3 Be.6552P;Te pp55.2p6p6v 3aelebE5p6 566656 p656v36qa6 gz 0Z6L 6516q5v365 v633562151 o6ev2eeaft p55eD5651E. 5Pe55e36qo qBelvoqo68 098G que56516eo qbq6q5qp3E. q.65q1q.61.6 46T636q516 qe-1.5q51.6q3 q6.T6.16.6 008G 1615-4515q6 q635q.615q5 qB16q.615q5 q545;Boa5p p5e3vvp653 2E2364.2E1.B
0i7LL EqpPoqqaeo D11-16P63l. eve5p5.110 3151q00111 3.600P00 D130111001 089L q1D-qq33oql ovoopooqpo 333qoa66qq qopp6152op Ere.615q5;.6. oZ
0Z9L po6;33oq36 eo555p66e5 e5165e5T2.6 56.6q6555p5 q556555555 Boloo6013 09SL -13556365p 6e5e6pe53p .65356e36.2.5 6561635apq leafivqq6P 61Beap6eeo 00SG .661e6p5evo 6ppoEcqeeQ6 e55o616;o p3qp5e6o56 6p5eq5.65eP 36,535pl36p 017VG 355 5p656 -452E6q65eD DE.o5po331 1T1113P5vp 56Doqq5e51 o5v5voq.6.6.2 08EG ae6qp5qee6 3.6q3p000l op6E-IDeEpb pDqpeoeoo ftepEcepBq 133682qvp3 çj OZEL 5.65e6655e6 e5e615363o5 Ecepoo6eqeD 64aeolo;vp .66qqa;Pe3q 5q533lvq51 09ZL pqD611043e 6e3P5v&I-44 111.601Tq5 1;EcIDD1E. q011a.511q0 5656q00.661 00ZL aeD6BeDD5.1 61.51qop6q 6s633.2Tqq.6 p55553p vlopqoPb E566q0D006 0f71L 156511D5155 Qoppa.6.6.6.6o 6233661e1.6 z6q.Dqv55q5 p6E.52pEep ;13Te33q3 080L pe5Pp6Eq3q D6.633oppo leofipboo5.6 ee.e5.6q3E1 e56q6533DE. le.6qP6Te55 oT
OZOG 526e35P043 epqpeo;Teo 35616546.6p q6-156e.6.5e6 .E.;DDE.2eq Pevpoqqpq 0969 115P35PD-15 6.60T4eP13 3D51e5P66 25e055656 pEceep6p1.66 v611q5061E.
0069 Ee6pq5vpD6 55qDqe66e5 BelEcIP6.13 p;epo3lpeE. 565551Eppe 6.62q65pve6 0t,89 pbeBeEvoob ppeq6.6eql ;3PP5p5q35 q333p6epev 16p3qp6563 6e33q6q3e3 08(.9 3355.68523 613332=5.6 .1.6.55p56eop pp6.66.46q33 3;D64p35e6 qDppq6Tqa6 g 0ZL9 lvobvbElqae 65636555 q6qq636BeE. 5ve6154536 6.6pfibev.apq TIE,q5T23-3t, 0999 006e66.6P5P P1P'365q1q 565133.6e66 pev56e35e6 66v33.615.6 q23e6P3e3p 0099 3&4P333P3P 36P6q5e61.6 q6vPp66eoP 311.6e5eq1e 3P3616e6e5 5pbe5e6q6p 0T7S9 5e5.262.2B3 qTevappepv Dp3qp-q435.6 BoqpeopoBq 23.6633p25 a60133.E.

066LZ/66S11/I2d Z-go-TOOZ ZEgnZ0 730 Q5,02352532 2001-05-23 gtgtatgcac atgtgccaca tgtgtacaga tactatggag gccagaagag gccatggccg 8700 tccctggagc tggagttaca ggcagcgtgt gagctgcctg gtgtgggtgc tgggaaccaa 8760 acttgaatct aaagcaagca cttttaactg ctgaggcagc tctcagtacc cttcttcatt 8820 tctccgcctg ggttccattg tatggacaca tgtagctaga atatcttgct tatctaatta 8880 5 tgtacattgt tttgtgctaa gagagagtaa tgctctatag cctgagctgg cctcaacctt 8940 gccatcctcc tgcctcagcc tcctcctcct gagtgctagg atgacaggcg agtggtaact 9000 tacatggttt catgttttgt tcaagactga aggataacat tcatacagag aaggtctggg 9060 tcacaaagtg tgcagttcac tgaatggcac aacccgtgat caagaaacaa aactcagggg 9120 ctggagagat ggcactgact gctcttccag aggtccggag ttcaattccc agcaaccaca 9180 10 tggtggctca cagccatcta taacgagatc tgacgccctc ttctggtgtg tctgaagaca 9240 gctacagtgt actcacataa aataaataaa tctttaaaac acacacacac acacaattac 9300 caccccagaa agcccactcc atgttccctc ccacgtctct gcctacagta ctcccaggtt 9360 accactgttc aggcttctaa caacctggtt tacttgggcc tcttttctgc tctgtggagc 9420 cacacatttg tgtgcctcat acacgttctt tctagtaagt tgcatattac tctgcgtttt 9480 15 tacatgtatt tatttattgt agttgtgtgt gcgtgtgggc ccatgcatgg cacagtgtgt 9540 ggggatgtca gagtattgtg aacaggggac agttcttttc ttcaatcatg tgggttccag 9600 aggttgaact caggtcatca tgtgtggcag caaatgcctt tacccactga gacatctcca 9660 tattcttttt ttttcccctg aggtgggggc ttgttccata gcccaaactg gctttgcact 9720 tgcagttcaa agtgactccc tgtctccacc tcttagagta ttggaattac gatgtgtact 9780 20 accacacctg actggatcat taattctttg atgggggcgg ggaagcgcac atgctgcagg 9840 tgaagggatg actggactgg acatgagcgt ggaagccaga gaacagcttc agtctaatgc 9900 tctcccaact gagctatttc ggtttgccag agaacaactt acagaaagtt ctcagtgcca 9960 tgtggattcg gggttggagt tcaactcatc agcttgacat tggctcctct acccactgag 10020 ccttctcact actctctacc tagatcatta attctttttt aaaaagactt attagggggc 10080 tggagagatg gctcagccgt taagagcacc gaatgccctt ccagaggtcc tgagttcaat 10140 tcccagcatg ccattgctgg gcagtagggg gcgcaggtgt tcaacgtgag tagctgttgc 10200 cagttttccg cggtggagaa cctcttgaca ccctgctgtc cctggtcatt ctgggtgggt 10260 gcatggtgat atgcttgttg tatggaagac tttgactgtt acagtgaagt tgggcttcca 10320 cagttaccac gtctcccctg tttcttgcag gccgggtgct tgtccattgc cgcgagggct 10380 acagccgctc cccaacgcta gttatcgcct acctcatgat gcggcagaag atggacgtca 10440 agtctgctct gagtactgtg aggcagaatc gtgagatcgg ccccaacgat ggcttcctgg 10500 cccaactctg ccagctcaat gacagactag ccaaggaggg caaggtgaaa ctctagggtg 10560 cccacagcct cttttgcaga ggtctgactg ggagggccct ggcagccatg tttaggaaac 10620 acagtatacc cactccctgc accaccagac acgtgcccac atctgtccca ctctggtcct 10680 cgggggccac tccaccctta gggagcacat gaagaagctc cctaagaagt tctgctcctt 10740 agccatcctt tcctgtaatt tatgtctctc cctgaggtga ggttcaggtt tatgtccctg 096t vo5qbve3oq Blapoeeopo 336q3weeb lo3p56qq35 6qqoTq5qp.6 5e6qq361.a6 006T 133.66eoqpq 5eo6pv5pq5 vpqa6evvqq 5313.6epobq 4.6p5t.PPe le6PP03-6P
SE
0178zi eevveevvpv peqqeopq4Q bpPqoePoop 5q3vope3o6 qQp6oe.56.5q. D6EE.E.q11 08Lzi ,o6o.4DP5qD oBqpqe.E.p6p qqoeveoqpo E.BobEfeoDv Bel5qp1D53 laee65qoD4 onzi Ecapb&qopob pq.615qp.q3q qq.656pDpEe 5q114q551q .1a6E.E.q11qp 66.1.qqq66eq 099n poqefivP000 16.53eqD666 ep36p33qop EmboTeq5a2 1.345.6.1vobv le.65peaaa6 009zi pevEoo6ope Ecep5BepeBe 5p356pBefq epqqEpoop6 5vopqpq.61p ea&a..6Teo opszT 66eoo.6qD5 .65qoleaeep 2POOPPPOPO opopPvepev peoRpveoep Bevv55e6.2.6 08f,z-E pePP56p5v5 p5qvq1Bq5q volqa6-4DPP 5e0P5q16e6 elBlaeoPPq ePPe3111P0 ozpzT 616wepe3u. epq351.qoqo qobElp&evoo 3116vq1qpq Epoqooeq5q 6.1.15vvoo6v 09EZT TeopP61.6QP Blve6P0006 EcIlqlqq11 11q1q44qq. '1111q3DD 4P03.65q355 00En 5le,qv3eq5E, '1300VP36PD epov.65151 OTTeq30301 BqlvolE.Ble 6613DEQ36q gz of7uT 513.6p6Po5t. .6.6po6vppoq efiqolDoeBp pel&65epoo qopoq.62.6.6 vq.13-45qopv ogin 6uoBee.566 pofi6p5ep36 qp5405v6eo oqlqqopE.p aevE.E,Pv;a6 Pvq1B61P01 oz-Ezi 1616.2D5epq 3n,6pvq.66v elqlo5polq epvolTle4P e5q1E,12qq v6.66vo1e1E
090T Pe5PeP6662 56q1Dplqoo evebu6p361 qq5e6bepeq qoq51p5aeq 6q00513061 000ZT 1651006e06 QD03qe5EDD DP0.6555153 q6TqBqq5e6 p6vBeqvq.6.1 SopoopqBeB
OZ
0V6TT qqq6105P5e DoPP5e61E-1 31qopoSpe Peqpoloqbo E6eqeovo6.6 33peo8poo3 08811 OPODPODEPD lEopoe6e36 ot,pq.65.6603 q31.6qQ.q51q Teqlepqa44 60QD1q0P66 0Z8TT 5q10qo31PP eope641e5 ebeo3Eclqq-2 pe3.651.6.qp q5.1433pE.6q oBoloDeR61 09L11 q;15.6q.65E, epqo-14Dep 6eepeq;qop Bepeee5evp 5Peep6e6e6 e5pbv6e.661 00Lit Pe6e6Te33y 3.6.6o5.643E. 5p5oevR6po v55qo6pqpv .6qpsvE.qpo ge65poBI6.1 gi 0D.9-ET 3.151.65peop 14.65106qo OBBe56.63e 1qPDDE,Bloo pepqa6qolo Doo.65eee.61 ossiT PUPP66PP6P ag5.65.6q30 paT6qoa6q6 666Pe66a4.6 3eqP0B1665 peD515-4615 OZSIT 551530qEcle 3q0BPD5Q1P PD05Pqpoee .65e6.6.eB6e leeqppbepz Dqqeqp.61q6 0917TT 6qoDqp0361 q364.6poo6 DepTeDq&ev pq.EreEz6Bq q.615eqvqle P5q0Dqeqqq.
clovET ge6Ecep3613 ep6p.66qqqe opope5.6.ep PDPPPOPPPO epeD2evove epeevo.eqqp 01 VETT '4005-200'16P eeq61565po elpe6v3qq1 qpoono6 553566D DbleB36P6e 08ZTT e6P116q0b lqq0011643 qa65E.B5eb gooplqooBe 5eoqlo61.DE qpleqP0qP
OEM BDP10.6e5P 5P0D.60.6 1.6q5pepqgq 6Tee6Eqqo qqe6q4oqpq Da6e&e0001 0911T 1114eDoe-e ea&e.eq5.651 q00066.6.6e1 voeqp-e4E.Be pe342p5elq pollqvqqpp 00111 ep3111236q p5Ereppee2e P65.2e6.2pee epolqqe5v pe5q6eqqa6 pqR6R66q6e opoit EpoBvp6q31 q6evleop5 Tep6oeBeob 6p.6136qopo BoPoPEopo5 vDto100465 08601 l3ollbq01.6 W0000100E' 513D356Q3P 3PPPO6T210 PP6BPPPq6E. q005VE3PD3 0z601 uP5Poo1561 6q51031q0q q56Blopeop opoq33o3p5 woqqaqopo 666peova6.6 09801 p3o6B,23E. .665Poe'qD6 65266645155 Poope.616po qplvoeq26p qeoBB.a.6qoq 1z 066a/66S(1113d ELLZE/00 -go-TOOZ ZEgZgEZO VD

ozTsT vve.epe6qpv TePPBPV'eqP p311Devepe 3qqq63p3pe bpElpablEB q5qop5qa6 090sT Q030DD6Pq 65 D55 625563o-43o eopqowew qoppw6pop .6q6B.q6qp.65 gE
oposT Bove&eepae avEobeeeev oqeop3pepo E.B6q61.2a25 e.E.EQ.6q6.6.6 51p5s6p6ao Ot6tT 6v5q51353q 56E.2 qqE,BpPaWe vq61e6PPPe e5PPPPB1P eeqqqq4qqe 088t1 3vPq.63.6q1 PaEl.o5541v p61355.32pq 6Eceopoo55e v5p5pvvp6.1.
E.55.4o642lq 0Z8tT PP5a6v0D1D Pv0u6613e3 0P000Qo5lq epq&epovle evle6Bp5Ece BvappoeBBe 09a7T D.631o3Tee ev646eD655 q332p.65.65o 'ea6a66.565e .45e5Te66.6e 6PeBBP66Te 0E
ooLfq 6.6611.6.6665 qp5q5456.6E. oqovvE,E,Ppp Ecepe6p6voe qlopftcelBo evla6B1343 0t9tT 616.6qPogoq eop1.6pplBq 53E.Teo.66q a6pppBE.qoa ou5oeqE.E.e5 Bqwoo6plo 08sT7T opeuplooqp opvepeqqo 36ve6qppee qppevpqeov oepeqeEpoP poppoovle ozsf7T aeoBw;qpqo 6-qoqpo55.6.1 PoDloa6Eqo t33 630 p66vEepoq5 Eep3qo5fiq5 09W1 56333 PP6P000445 pq115e5103 P55P455.613 ow6lpeopo .6-35vEipeq35 cZ
00T7T7T PD3655P5 5P.663a656 Beqoppoqee TP48 op31 PD5pae35s3 ODEtT pepepot306 qpopoBeeve qae.65v13.6.1 6pe33q336q. 3333510003 613035100 08zwc 33510333.6q opEos6qoq qovp6.4q33.6 5qa6b.epqol v66e155p61 qqz6P3T6q1 OZZtT 6305155'4E6 q5q0.610vPq Pq11051.6q5 4Pqqq11065 63q5E,161 qqqopq1.6q 09TpT 56346 aebepeop.lq Paelqpq6qe vPqnvevooe v151qBeeeq oqpeoBoesq oz 00TtT '35q5eopp3o eqpI6eqpqo obPqq15.evp 55eppp56e6 MoolpooBq 33165e5563 OtOtT P365epa61D peoq.6qopo6 R6P35.6p6q6 5E6q55Te66 55po556e5.e. o3366e5ETE.
086E1 33p65q5.20 3p5.15eepee q5pawepaq 6.6p1.16a6eo o5wqop114 1vooTlez6q 0Z6ET 10eeqqaple 56a6PTee5 p5u6eqeobe 3.60TP51001 61q066PDP q11131PP1 098E1 epTqlqqpq6 qeep65.eop oloqq11p5v EQvTe55.2.6o eqpeqeqq3-1 e51DP0BPP6 gi 008ET e5PepTereq '1.64ePP106 P.E.PeqT4pq 62.1e13.6qq 5pelqoov33 qqqpa6q566 of,LET 0.66a6q5qo vo5E65.6e35 eop56Teop5 qpeqe55.464 66516eP0D0 DDP005D3DP
089ET 0P06PT4100 65330e0q0q qPpE,Pvlqq e'lq6PPq104 pe35e33q36 55335 oz9ET 5PplaeqpDo a6.61qopep 665'251PPPO DE'lqDPOPP1 oP6ppea6pE
09SET 10q1643511 q31q151015 5e165.56P33 111100610D 510qBePeqo 3101t'V3l3q OI
oosET q3qoqq6.6q PPPPP1POPP Pael3PPPPP 165Pooqeoe vt>zeqqop5q eqoqqlovoo OttET 6P333p6p3 003103Ge 0303000000 30q6656p eqle.25.6E.1-2 pEE.q&EqBee 08EET p56Te33.lpq 3ql6q5elp6 633gpe56.6 q6.66.6.1565 5521E26150 33505553 ozEET oppeepoBee pe5.6q5qq.6q 12633eqeePp q56e3ll3qq pqovloBeo epeeTeqq15 09zET 1.631=6q11 365qq5Eqq3 115.6oppopl 5ePu51D555 eqqpooqlqo 6.6qop13p5e g oozET 6661qq335p BeppbevEQp aepEE.51611 loqopeeebb qDeqpooBe goupbuDE.E.
ot,TET BeDVODBeOP 66eqe3p33p pl56q16.651 ol;ploa5qo opooqoopqq 3.655.3656e6 080E1 qploppoqoq oppElTeoBe qpDrop6q66 qp3e3peq56 p431541q31 3q016e0P3e ozoET .65.6qTe5pq.6 v033336.6q 6qweee.616 ep65ee6e6e Doqogpoofiu 6qll3e64e ZZ
066a/660/.10d CLaCTO (PA
-go-TOOZ ZEgZgEZO VO

08u1 e6zee6v6.66 qqoPvleP66 Sepqpq6433 qeb-ebvp.q.6Q pao.461Ppeq poPP1DD5up ozzLi p6e13.60o5 6ovqqp5.5.6 6.6.1pq;e55.3 .65eD66eee5 6.6.6efiqz6e6 Poloae.P30q gE
091L1 qooeeTeeev eBEq6qepae, PD6PD000q PE3lq6e5Te op6E,26.4e3.6 eE.aefiloBq 00-ELT lob3E.Q.6.2pq .6.64-eTebqefi p66.6.6q385v 5PvqeDDQ1P fica611'1.605 P5eDDDP6BP
VOLT 13TeoPq03 26E113001P opTeqe1P1P leleqq1P5q Dllqp5Eqefi Q5qD0P5P1P
08691 .6v1q3.6q5q3 qoTqqef.51q. vobeBqo.6.6e n6loPlo6qo p;poo85Teo 155qb.aooDD
0E691 qq.poqoqoD; 3p3qo3Dee6 q5-3.5.6voqoa pa644.11Doq polopoofto pEopTIDEep 0E
09891 6q6po3q.E..1 ol2DpuPpoq poqbeEBB.5 DEBbpooqop qp46eopol qD3l336v3e 00891 pov653.65P; o65.6o6ppeo Depa5pqq..6 pDoopqlepq TebqopopEo v6.6q5Beabq 0T7L91 3q3Be.26p36 lqoo6P6p35 5q5.5e366v6 oqBeepeqqo eop5.6onE,55 qt.EpoppoBD
08991 le6eDE5epo op65p65e.ep pEgv6pebeo efq..665qB35 1.2.6q1e3513 5ea6vEv01.6 obvepq.frlq 00.601540q qov.6565p5q poqqo3ool5 P6eoleq.66e DE.E.v6.6vq0 sz 0991 P.610Pee66P e6003061D1 541q4.65q36 eppoo5.6.ape OPPDODOOPD
ebPqE.E,E.E.E.
00591 66pq66.4pqo 6e0P0120Pe ORPqplqD5V po.6.65veevo 3Z&6 26P65 0f71,91 PePEI6E6e06 PBPDpq1PDP 001Ecepeeql. 56.6eaeB2oo 36643qqlofi epe5.6eqpeo 08E91 Be6.6qpq.E.pe pooqpqooD6 qpBuBbvpoo .666opplepE. ppEoq3.6q.66 qele00PP6 OZE9T P61.6q35465 -1B6e3.6vo4P 00D3Dleele DB200E6156 e214E01P05 0P5P0e.6.6P
OZ
09z91 e3D11.6q6E.P 6E066466PD 5P001.5oPT1 503311q1P 6-31D6o4a6q ololopoDE.5 00Z91 1005PBP'131 evp.eDvp-ele evoeeqpeve qvBeopq4eP Dq0U1OPOP 51161q0q14 Ot.191 '11op3ele6 p;pq-446-23 'qp.E.Ere1.66o 65eqebv5p6 ppo355.6v66 pEo35.65qeo 08091 opplo.66.6eD 56pppe62.61 o3qp335e.63 vopeo56p3.6 6oe63p356v DE.BppEeppe 0091 P30D3066PD Depeo5P6o3 ppe3p6vve'e veeeet,Pepe PPPETVPTI oqpPelvpuq.
gi 09651 eP1PqPnP11 pe3.616P3el 3E.De6es..6.q 015.161E6.43 qq3a.03D612 00651 e.q6apep3p p3polo6.5.6 Bqppepovvo 5poDowevo q.BeEce.D045 EIPPB0D1q0q 0t,8s1 oBorboopv oBeE.pe.666 .4.6vD1oBBle 6p5q5.6qDB.6 5BevooPPep DEcevPPOPPO
08L51 ppev6vppe peBeppvpBe e6weepq00 Pe11DPB1DP 11P.601e0Be et,P1Ere6qq6 OZLST 15q.11565e eqP6Teev55 Eqq.00eela.o BppEclovEl DEB651.poo2 6e5qaeop3P

09951 55.6elpeoq5 PD&QOPPPO lpaeevEvoe 6.6556'46.656 BqE666.6.6.6 BEcTeoBefivE.
00951 '10-60q00.6.60 Ece5q5eleT1 161BeEpPoe peoq.6.1p4q.E. D3oveuD6vo eeepaeveop opssT pepoopolBa q.Boopbevob povq52DE65 EcqoPq.6.6Pe 51115P5055 E.E,6P6PP0.6 081751 v651pEppe.6 5qqoep56.6o eop6p1P1Do yo646q6.615 .6q615E.voo.1 elpoeveeep npsT oppevveTqv ebee5eppee eppeqwlee PPO'qDqP1T2 p3elqu-qpq.5 lopebveolq g 09E51 TegltreE,Dol Doolpepe3p DI.P6p56.6eq 555 655 526546'405P 61.6511101v QUEST 3615qou.5.63 666656 -lopq5aeo6q aeobo56o;5 o.65o1.6q.6.16 e6161.25.6.1e ODZST 66eP46eP16 6e333Ece.Ece 6e.5-ae3Teo6 Evo5oo.61.6o pobv.6qp36.1 p.66o.6.e.e&eq 08151 6.6a15.6ppl .66163.q.D4p 6o5Eceop55o 56eeepo566 51.4Doqefiep 6E.E.P0eDP6 EZ
066i2/66S1*a2d ELLZOWOM
-go-TOOZ ZEgZgEZO VD

0f7v61 oep3.61.3.6e 3pqqq1po1.o 5.26p56PE.65 1.6qEqqqepo pp465q3.6.qq pot6Tepsft 08E61 Beopl3e3e6 66 6D5 6615.666q66 66 62o Beo5Pop6 6Dpo53voqp gE
0zE6T
1E66661656 .6616.666qop 5qp5p6a6po p35v3a6p5v o6e.6.64.6q3 ppebeoBeoe 08z61 5.6e6qopqqp o4o35aqq05 DopoP6qD0 0Q061106v6 q000.6qqopo ovobbq6i5vo 00z61 vo5EMe151 oDooq6qe2e 5.62B3o5e3 .66.6EceBlvae 61.6qeo.66-q6 6pq6q1166.6 0t,161 eaevBept,p1 1Po6eq46.66 EBPDpa6vbq vvoqa63o45 BE.Popo51.43 opooqq6eqq.

qoDolvp.6.6p 5.6pDa5pe5q nov265B.1.6.6 51qq.656.4qp 6eqpooPpo6 5ue351pEep 01e5qq16Bq 06ea6513eb PE.qa6p6pe6 peqbe6.651D opol.ftevl 6a6Qp3qp6.6 vBeEopuea6 u6.6qp6pp.63 q36n33o366 ee5pupq6Be 61POP03P33 P6TeDPO30q 00681 elel3Pq610 6.6qP00qP0D B1-26eop6qt, 666 6qpq.e&a6G.E. 6666 633 0f7881 5qobl3Belo qebPol-65e3 vop56P61p.6 Bvvqp01D01 DTeP6P5P00 1551p5Te;5 113556;p36 65opaepqqP 3663o6ev63 3bv3155e6q 36666566 paMpvpoos,cz 0u8T
35p6e5eq65 '1355655511 po6po666.e6 E.qpnee6Pq. Po6P3P05P1 DP1PP6P266 133.613qe3p 3p666e05.66 poqpaewbq oloo5v.13.65 1B66q61.6q1 1-65P.6311D

1101v0.64 qq.65q6.666q o26.55E.6.66 qopq5BE665 66.6.6616eq voE,35e.e6E.E, 0i7s8T Eqp&E.5aeq6 ftpeopbopE, 'Pl0003.230 poopoqopp5 36 66 6.660655op peb3pooeft Doue3ee6s6 qoplooqoa6 EtEo33ovq5e, Ece6.6qqp66.6 P3001P01.65 OZ
0n,81 eBeoPooBle .63ppb6poql paftvo6E.q5 665.eop556e Eibq5135q61 011305106D

e35.3.6-3e3 na6qoqv3l3 3663333Ece, govpqopaft 36Teo5p366 loovoolopQ

333646E633 eq361.6q338 v66q3poP66 la66v3aTel 5ooee36E.pe evr.11305.65 66e5P36.6P6 le0661356q 1e1103a5po 36eo3w6l3 33633e66.6 1PPee.6.E.P.6 .6.6.6.e.6.6e6.6 e6.463p3pot, ODOOPOOODO poope33l3q lo333e3e36 vuqD6o.2610 gi 2.6P6qqo 5e5qovqqq5 pp5.64qqpvp 5poBpo351.5 5P5553or.o 1.65.6D1333 POPOPOPPOD 300030035 DPVDDPOOD 53D 56 D564D6p6p6 226.6e6 6516=36'1D 336qo26l33 5ROPD3EPPP .60Te4P5qq 11P0106vu3 06PePel4Qv op5ean5e55 6e303.655.6e BooEizep.E.p 556 eovpooff,qo E.Pe151-110 3111B1;Deo 3336E.p6p1.6 qolqopEpoq Te.55.136Ppo 1561q'IPP5.6 Of 0z8LT
p5v13.66113 ovEqeeeoPp 1656.6e365q 3.6-eqqe3os6 PPlaeP110 DeopepoBEB
09LLI 65656566 .6.6.613q33.6 ollooqeo65 PP 3333 ouPopopoBq 3lvp.623333 OOLLT
5eE.E..1156E. q333e6P6-16 ofteDbeoep 51q0.61555p 1.6.Q1PP3q3 2le3q_66613 5563333 oqvpbBevPq qlooqp.655 ze.P3q6.666q ofivE.BqBwe 35e6436466 eq511ve5e0 TE.Eq.33lo.1 .e.6p6pEvu3q Tepe.6.25eP6 qq;15613e3 33l3q33611 g 0z9L1 5555.4336q 13.4=3156p 3336p5.64ep 151p3eolPp 5vPla6.6qop 3q46e61P00 p3l6pvTel3 35P3DP3P36 54T400610P .2.66pe.656v3 0.6.64336Pv6 ep3633.66.41 00t.L1 Bqoqoqqpep 5qeeeEpvq3 5qqqqq1p.6 2PP566e5e vqP.evbveqo 6Ppqep4EcTe DELT
P366665666 65peoppp5p 3p3p333v36 3233333365 epqoploopo pEpp.E.p.633 066CE/6601a3d ELLZ1/00 OM
-go-TOOZ ZEgZgEZO VD

, B5p6.6.6.6qt..6 6.6.6.6o5Qa63 poElqoqopq .65.en6DoeBe fivq6eop3.Elt.
po33bvE.5q .6q6opp6p33 qqqaeppoo5 .16666-epo6.6 voDoBPDEqo Daevvq51.6e plqq3.63qE. cc 08vEz 5303v536D6 poopobpo.6.4 pqooBobooD 6e5o6P6el3 uqoo53eeBe .6.6qp5.26.63.6 oz1,-Ez Beopepoobv ppooBeEBBB 3oD5D.56Doo 5635oo5ppo Bo.5.6.6-ep&e. DEoD.6.5o6o.5 09Eiz 3pv6v6booE. 66 66 Polo5e6bal 6Po3ePDP00 TI3E0Daepq 30506ee36q 00ETz 6eP3BlEolo 3.66q.E.E.q.36 36 56D 63qo53.63D6 D.65oB36666 600ppEq6qo otzTz Eq.3.5p36q6.5 5o6p360.636 povqoboqvb BooaquoSqo 633qqqp6.6o opaEopsEop oc 0FITTz 3536E665 ve6q636366 634e3oBaev opp6qoBqp6 56 EDBqBeDDEB
oz-nz 3olo6q.6155 qq5pBoovoq 6655 oBvp5po6lv 000660Q6P0 e6130140.60 P3P13P05 qp&e.5363o5 qp6pop-1.6e6 Do161.6qe6e obpalo1040 00P050PDq 0001z popoftoBqo 66pqo36BqD qBE,Doa6o.6 .26.6Pp66q66 6pp6v6efte pep6.6qqq.66 otGoz E.3.665e363.6 peeboqqp.ft oeqeopwe Pqool.opqo D5.1.6.6op3ol 36qovaqpqe çZ
0880z 656 6653 Svp6p6v5p5 .6.6e3v65epo 3q5E..6q030 1-e0q45q6P 0010.6e6P3e ozgoz 5q5ev5eve 6pepovoqp5 POP3006100 De.a5p-eqq1D wan6ePEP65 5553P
09L0z qqqqegooev o66.65133.e u61q6oq6ep 6qv6.615e1P elbqe53005 q5e50P006 q5q0qqe5qp qqpeop6q35 PPPE,DqDqPP ODOPPPPPlp OP13.65BqOp 5e00,6.66q4 0p9oz .16e33Be3v3 pqovelqopq pDoqepaebv v3qq6pp5e6 qp5oBb.E.TeE, p.6.616.2p6v6 oz 08s0z 533pQ6eq33 Dqe.665866 lwoftePoP 365365 6.1qq6q5p6e v.6.Bovvelo ozsoz 62.65e64e.a. 5665536 5.eoppqoBEIv 5ew.651.645 BpoD.Ecefieop e66Pp6.6-eql 09t0z 36.255eeeq.6 .6.1355q6e33 3q6q3333.63 Deqp.666ao qoqu.33qolq 00qOPODq0 ootoz opewepq opeqqe-Tqle .6q33q54o6.2 3.6v3e33q33 wBoopovqo q3336433.2 OPEOZ 33P1330q2 01qvv5Tee '4015eolqq 353.6,6q10 3.6o3535opb 5656655 cI

4PPqs6Q610 ov554005P0 qp1qqoqq.6. eP365.661qq. 335 D3353 3oBp3Bee36 OZZOZ 3000003303 533333333.6 56z .6q.poo6P.6 e6ep35v555 35qq03DP33 0910z qq3p3qe.6-23 peqpqooq.65 B&IDOPDOOD 353333533p 3353q3e04 03356vvP50 ooToz 6pbee6q5e5 35 5p333 .e..6.3Dqopv5e 3.1.666qq4q5 5e1P05P2P P35Pq6515q q35leeP3eP P.IDEq.65ep.6 p3eqeq.663 qBebp6vepe ebeopoe155 eP3q36u33e 01 pq.62.6qq3p3 P336P33qP.6 p3q6ePq010 e50eP5v5.6 poopqqopqo qPP333PP30 0z661 qe3.656q365 Pqe.EcepBeq 355 5e0655p55e 5eftp3p'1.6.q. po.6e6pq25p 6E.p.4.66eD ofteqoqeop TeopoqopEce peeqq6ege Bbqepeoupa Deb&eoebPq E6e6.6qq110 PP.65qe33e3 efiq33e64.1. elv3q5q2q1 qqopqqqqqq qqqqv5.6.633 0T7L61 eq.56q12365 q3q3333Eq3 q33.55133 36.6Ece.P.64.6 3000000300 333364.6.435 c goopopoqop e3eq3365et, 3E6q.555-ep5 v3e33be335 eplqpevlbe 5-45.666P064 0z961 oqopepoelv oppoppoqoo 65popeeDB6 Bqopoqqopq BeqoeeEpoD poBevp5.6qq 09561 3lqe13P36.6 55 356665 va61qqaq5o v3q.5p35qee 353665-e 00s61 pp3333e336 55epp55e55 15Te.655peq .6voo5eqopl 3q.6q33l3p3 463633 SZ
06612./66511/.13d LL,ZE/00 OM
-go-TOOZ ZEgZgEZO VD

Q5,02352532 2001-05-23 gggtggggtt cccgcgggcc aggagaggaa gcttgagtcc cagactctgc ctagccccgg 21660 gtgggatggg ggtctttcta ccctcgccgg acctatacag gacaaggcag tgtttccacc 21720 ttaaagggaa gggagtgtgg aacgaaagac ctgggactgg ttatggacgt acagtaagat 21780 ctactccttc cacccaaatg taaagcctgc gtgggctaga tagggtttct gaccctgacc 21840 tggccactga gtgtgatgtt gggctacgtg gttctctttt ggtacggtct tctttgtaaa 21900 atagggaccg gaactctgct gagattccaa ggattggggt accccgtgta gactggtgag 21960 agagaggaga acaggggagg ggttagggga gagattgtgg tgggcaaccg cctagaagaa 22020 gctgtttgtt ggctcccagc ctcgccgcct cagaggtttg gcttccccca ctccttcctc 22080 tcaaatctgc cttcaaatcc atatctggga tagggaaggc cagggtccga gagatggtgg 22140 aagggccaga aatcacactc ctggcccccc gaagagcagt gtcccgcccc caactgcctt 22200 gtcatattgt aaagggattt tctacacaac agtttaaggt cgttggagga aactgggctt 22260 gccagtcacc tcccatcctt gtcccttgcc aggacaccac ctcctgcctg ccacccacgg 22320 acacatttCt gtctagaaac agagcgtcgt cgtgctgtcc tctgagacag catatcttac 22380 attaaaaaga ataatacggg gggggggggc ggagggcgca agtgttatac atatgctgag 22440 aagctgtcag gcgccacagc accacccaca atctttttgt aaatcatttc cagacacctc 22500 ttactttctg tgtagatttt aattgttaaa aggggaggag agagagcgtt tgtaacagaa 22560 gcacatggag gggggggtag gggggttggg gctggtgagt ttggcgaact ttccatgtga 22620 gactcatcca caaagactga aagccgcgtt ttttttttta agagttcagt gacatattta 22680 ttttctcatt taagttattt atgccaacat ttttttcttg tagagaaagg cagtgttaat 22740 atcgctttgt gaagcacaag tgtgtgtggt tttttgtttt ttgttttttc cccgaccaga 22800 ggcattgtta ataaagacaa tgaatctcga gcaggaggct gtggtcttgt tttgtcaacc 22860 acacacaatg tctcgccact gtcatctcac tcccttccct tggtcacaag acccaaacct 22920 tgacaacacc tccgactgct ctctggtagc ccttgtggca atacgtgttt cctttgaaaa 22980 gtcacattca tcctttcctt tgcaaacctg gctctcattc cccagctggg tcatcgtcat 23040 accctcaccc cagcctccct ttagctgacc actctccaca ctgtcttcca aaagtgcacg 23100 tttcaccgag ccagttccct ggtccaggtc atcccattgc tcctccttgc tccagaccct 23160 tctcccacaa agatgttcat ctcccactcc atcaagcccc agtggccctg cggctatccc 23220 tgtctcttca gttagctgaa tctacttgct gacaccacat gaattccttc ccctgtctta 23280 aggttcatgg aactcttgcc tgcccctgaa ccttccagga ctgtcccagc gtctgatgtg 23340 tcctctctct tgtaaagccc caccccacta tttgattccc aattctagat cttcccttgt 23400 tcattccttc acgggatagt gtctcatctg gccaagtcct gcttgatatt gggataaatg 23460 caaagccaag tacaattgag gaccagttca tcattgggcc aagctttttc aaaatgtgaa 23520 ttttacacct atagaagtgt aaaagccttc caaagcagag gcaatgcctg gctcttcctt caacatcagg gctcctgctt tatgggtctg gtggggtagt acattcataa acccaacact aggggtgtga aagcaagatg attgggagtt cgaggccaat cttggctatg aggccctgtc 23700 tcaacctctc ctccctccct ccagggtttt gttttgtttt gtttttttga tttgaaactg 23760 -------oz6sz OPOTE5PP32 5vqqo3evoq qoqqeop361 61P2P1.600 6100610001 05ep555511 098SZ BP55101110 101115130P 15eP00140P 1012116P00 6BeBla.66q 6ee51-1qp6q gE
008cz 33oqzeq6.1.2 qp5E,B6vo6.6 .65qTqBpoop 1.6epaPP110 011P11015P
1010.6e0SP3 OT/LSZ le13623.6q5 qoop661v5o 3P 56z poolv5q6.66 ep565ploe5 PE,q6pepqb3 089s 3pq.1.6qpe5 qepoPEEpqv poqaelqqqo qqbeBeE,E.p EreopEe5BeD pEpbeevla6 onsz 6.4.6q311q31 q.65p5lovoq pooBeplopo o5vq1Dqobo BPODEPOPBP o6pbev5Boo ogssz qop3P55.1o3 evEreoppElBe qo546.65.1.5q. 530PDO6P61 ell6PDPBV0 611oP.65;oe OE
ooss eb5qoppvTe BeaTea6.54.e. 5pebPoqe5v 55v613D615 Eq.Do.6p.ep.e. qqvaEce-15.1.5 0T,D9z qoobqpqvqe ebqqa51111 0.6q5a61e1.6 2652111evl 11P6151115 1131513031 08ESz 615PPP613e 16E.P0v003q BeeP5651qo q.66.23qD15.6 e5qe3e35.65 eB3D3pop3D
ousz 111.664pD3e obqbe.6.61qo qq.epoo31:30 0566110e06 P5P61P56Q5 10P00v5100 09zsz l3q6q33pep oppol3paeo Do5v65epE, g3ooqq.66po epoqqpqooD vvPle15100 gz oonz p52535q5E. qolpevElloe DP6-eoppoBp 5.eopovE.155 510011056e 5v05q0q555 ovisz 526poe5pe5 55ea61354q op55q3E,6q pe-e51qq3;3 lopop3Da.a6 EpaepoBebe ogosz eu,ep33vvp5 .ea6qq5eevp p6p5Beepqe 3qopqapqqv poppeaea6v eloqqppeqq.
ozosz o2qppoo6p5 EreB3q0v030 lqpso3Deop p3popp5q2o POPOVOVDPO POPOPOPDPO
096t,z POPOPOPaPD eDPOPDPDPV ebBee5eoe6 e5v6pos6vo 5ee015p5e5 30.65P1PP11 OZ
0061/Z P16110656P 0P01eP11P1 611o55563e op6poobpoP 1555E11051 61.60P05v55 oT7817z beDE.63eq3; o'eq66q.65.6.5 v666;33s63 DE6POPPOPP POUPBPPP5P
aleopo5355 08LT7Z 1.055PP0P10 155661P001 00e10q3e1P '31Eceee651P 111e41q5P5 P1155epq56 oni7z el611peeop 355uPooDev PePPPVPOPP evoevptovp PPPPPPOPP e0PePOPPPO
099T7Z PPPPPVOPPP pev65.6pBee eoppovqqua poqp.61.6-43.6 664q65'210e BPSPB5PD54 cr 009Z e6P0P5e0e6 551D3q051 q3v6Erel33p peovpe000p peo335Te5q Bueop.3Be5p ov9T7z 6eDqeo.6.565 e5qe.666pop EcleopolvDD 011Q15q10p 0B1P05P111 3DTPD020pq 08T7T7Z P0005e556e 061P.663600 5555 p.61ofiBpovo PBPPPT2DPD 1D2001DP52 vpoftewlo qpv5epool6 65.5ea5p.6.6p e6400.61066 eft1051053 5P6P5PP5e0 555opqqvbqbvp p.6.6552opoo pE.3õ651e13.6 1ePTePPDPE Pft0560DBP 01 00Efiz 5Polqp5q53 DeeloPqbqp pq.61peq51.1 55appov6pe 646.6e3po.5.6 5qBe3p352.6 oy7n7z po.6qeqo5pD 115.43pe35 lnpq.651331 qBepTeBeep 011PV02PPP poop3l3o6v 08tTvZ
E.E.P11e55v 0;0e1555v0 P5e5105PP5 1PP0.611o.6. BpaSEE,E.Ble E.5e5q65E-e6 ornz p3.6155-4515 ea66Pvov33 po5poo5.63 oDe6p66Teo 36.6p65pD5p beg4Dq5lee 0901Z 6ePb3001e0 66P001pP55 BeolqqqovE, 5ve55epoqq v5eop1355q 5qoaD.6'355 c 000n 106.336q6p5 l'IppaelPpp pp.5.43q3q5q voTelepoTe qq.e.P53epeq o3poqvv6q OT/GEZ eP00010PP0 1115301130 55q3eo4o3o 5PDO3VQP;0 .4353.e.qoqo lo5epob.6.63 088u 36qoaeq5qq lo3666qopp p.6-41555564 aeDv3.61.63v q6.11533.3P.61 104e001106 OZ8EZ eelP1PP100 01-eq0l3Pp5 555e5q5051 1101P061.6e P016P001PP P11133uu3 LZ
06612/66511/.13d ELLZE/00 OM
-go-TOOZ ZEgZgEZO VO

0808Z e320061PDP Be0P000404 eQ1.66P3013 e3v033336E. 6656 33633e3336 0Z08Z Q1P116EeD0 T4evqvqq.pq Bevqe3.63.Pe 66-e6e6pEeE. pBepqqq3e1 35vee3o36.6 cc 096/2 33633e33e3 353.63.336.62 eeqqee5.633 636e.e33333 3633333633 OODOTePPER
006/2 333.ev63333 66136.6eppe 6=236333323 qove.661333 B335eqopor, 31e3,6333.33 017812 11656P3B6P 6311111E63 316111-651 1466331153 33531163.13 333ovqa6up 08a2 ppoe64333v 3656E 3353.36P301 1031316P10 306e301663 fiquP5lole6 0zu2 qp1p6,63336. 51v353661v s613636v66 66e6e36633 616e3336e3 331ov66636 . .
099/2 3333p366Te 31P6336631 365 D35 P33Be3v6v3 661E36336e opel6PB4DP
00912 EqoPl36Qpv D10636;43 eppep666P6 Bepv33v5P6 e36.6v63666 p5pbEeppbv vs/2 62316p56e6 .666P33.6612 vElp6v6p63 56e6v3v5eE, 651-e6e3e13 3431335Poo 081712 v66p36136.e. veoP633363 3633333E63 3ep3e66e6v 333e66ep5q e6vE.Bv666 oztLz e6ppPe6333 e6eep6pp36. 13361v5pze .616v315336e 066616P065 10.5e66100P SZ
09E1.2 pfte333613 3346333.36v 6333136666 331e33.6p3e 1003133331 3331s61P4P
00ELZ D01,6161633 3e53.13P626 qopae6.633P 3636.6.6p36 eq,3361e363 33333e3v33 0f7z12 63a6lee611 406P66.6PP6 10E0666361 36B6336ve.5 vp35.6vPe6e efiqopp3o6p 08112 3.3.ple55e4q eP36336361 e5ovv3lpeo Bvs663..eeP6 va633354e3 36e33.633e3 0z1/.2 p6p336p661 363.66366e3 Ere5ev6peee e336ep.6v3r, pP65513pov 33e333.6.6e3 oz 09012 6-P03301056 gee0631PD6 31366661pp 6333v3363.2 3.236365133 ep653v3v.ap 000/2 3613336E6v 3666ve66e3 66uSelpe51 v6v33ve63s, eu,66et666 3.4666vev6e 01769Z 3Pqe6P6B4P 1P55533055 e6311e.6.653 qoppvE,63Pe p6eevop5.6e 6e36ep636E, 0889z 3Te3333e5e .63133.6ep36 e5e636e666 6666 e633633366 vq333.3.6p3.6 0z89z -23.13e3.6333 335333336e .61PPO3ePE, 3e331.33v1E. peeqqe1333 lae1011e66 gi 09L9 5033PEPDO 00101e0130 63.3263es61 16633,ee36.6 ePo6333636 v63.31-26.e.33 00L9 3ep6331356 610136661.6 T21313333.6 6433e3e36e Bqpqe3e33.6 v3-233'26661 01799z 3636e13333 33613133v1 p36333v666 e333366p3.6 1336e43.663 0666136663 08S9Z 3er3361136 P663336631 2333133e36 663eopeqq3 3333=33= 33R6v3v333 0zs9z 13616e3656 e3133E.E.36 66ee336vp6 .236peo63e3 e36661v3.66 .2363.63v613 0-1 09179Z el6161633p peepvp.6-23p 3.63335ep66 v63evea6p3 p363.66163P
16266ee36u 00179z 66P3633e.63. e53u13663.6 3666336661 e3661e6e33 6613e6636.6 017E9z e3p3633.523 633E164E-21 3661p6ee6.6 3e464E6P33 ev5v333-23e 3p.65031P0 08z9z 3133613133 333166e3.6v vp.666333P6 ePPe30P6P1 111P306411 0003633331 0zz9z e3e33.ep33e 3.43.3e1.6333 336333636p 6P335PP3P'e VP25Q3eP23 PPE3PPPPe0 g 0919z PPVPE.PPPPQ BVPPlePPbe QPeDPPPPPD PPPPPPP501 3163333pee 5P6P3P3P30 0019Z 355P335P3e 6eP33116p6 3.6.266.3e331 663336336 e323335p63 533333.e563 01709z 36P3p6p5v3 66ps6pe33e 36P3333PPE. 33336313p3 6e356p663.6 6634511313 086SZ 333e3PE'562 e3P011106P 131PP33333 35cepe333.6p 3356E.316P0 5e61030111 SZ
066a/66Sna3d ELLWOO(Pa -go-TOOZ ZEgZgEZO VD

- , -of,z0E poq3up5Teo 1.6e5vavoeD POPOP3POPO PPeDPOPDVD epeq&e.24qo pvefiqe3w5 08T0E 6q1peqoqqo epqlpqopol qeopeBE6p1 loqopopvvo 613eqp6Eve Dopoq5p534 gE
ozToE polv36v.616 poqeqqopqp opu5qovepq opoqqqvleo 1q0015Q6BP 5sevpqD1Ze 0900E a6.1.6qpbppo qoqbpoowq 04VDOPP5D 03 633 61.6&e05P36 eeP161Thqop 0000E 16qopqoppe qa6v.e6qD6 q566.4B1poo POOODOOPPO 000qOPOS61 6TT6e3s6Pq ofq66z polfiu5elop pEceq3333o2 6equa66eqo P56Pe5w5q Balo.1.6515 Eileoppq.eq 0886Z 51PT63eq53 vq.6TelE'Deo Ec4005111Pq BeP1P45Tel PPeve11110 Tle0BlETP OE
OZ86Z eq01B2ePP4 qTevZ61B16 1515q.61516 16q66151.6 161516q65 q51BP1;051 09L6 oopeqp616E, leovq1.65pq p5p3D65pbq Depa6q55se pbeeepa6e5 ooL6z 6eqqapo6se Doqeo.3qqop Plva6Te6;5 EITepobeboe BPqoqBpebe Eq.q5551voo cg,96z 3v6p6.6v5Be oqBqq5voql 55B6p5epse POPPPD.104 40a2PeP55.6 TIBTe065.6 08s6z poneqe55e opp.E..evaft ovoeqolpbq opBepoBeee 3;q5P66.6qo qae36B4E5.4 5z ozs6z 6p5p6q3.6-ep eoqoqT134 Teze6ve151 .10.61P6z6.Ere qoloqbbso g000baqBeb 09f76Z 0400q1012 0E0115E403 30333313 400P1q62al 6.61POP3Oqq 0161E101qP
00f76Z 30 1DPP111D1q DE-10q36vq ovoopoD1le Dq066q110.6 eelPoeBeql 0T7E6Z DESZEIBPWe qqP41De3fie 5P6q010eq0 PelPPOPP01 3416PT43p3 ob000lppq 08z6z qlpTerieTev q.E,Bqp6peo 61e62Tepee geov.6.6woe poD.qa6.61.6q olooplqw3 oz 0ZZ6Z 3qqp55qq6q. popTeq.E.pe ov556qpqop pq.lo5opE,D5 evE6q56651 o5ft3loa6 0916z ep613.6.6e.63 SpoBqoqopo wlaeoEloo 5oop.66eool 3.6.6q5qpleo q6vve6poqg 00/6z Tee5poqwq 31.6.36466 Ecelollo3'q5 ep6qopp62o q351PqeDu1 De6P06e051 opo6z .1pe6epE6eo Teo6.2w5qp 51qq.651D15 43q6a6pueq 3qqqqq63v 3qa664.6q0 0868z 6.6115WP3P ODVPOPPPO 6.626.2.6poo 356leePope Eve5qq&e,Eq pqpoo5q401 gI
OZ68Z Ppq064e0eD qB1P5W4DP 6q515154Ecl qe.6.q.66q1E, pepq3eq5P 3el6e36e6 0988z e633.1eppve 6PP56ePPPP Pe5PQP0eD0 P3PO20e0P3 VOPOPOPOPD P3P020P3P0 0088Z 20P3P3P3e3 V3POP1PD00 615P310PPD 00136P62.6 e3P102046E, pe6p6.6q06 of7L8z eq33.61eq65 ev5we35ep gelp,25q.Eq 3p56ve5epo ep1.6.65Eqpq 56.6vEcqpqp 0898Z E'303656lee Q15qP5BaPD lq.61v2pq.61 .65epqbe63.6 5.1.66vE6v63 pooloolBq OI
oz98z 36.613.6.6e6 6v65qopp5.6 p6,w6p3e3 E.355a6eqq. epps6ppepp 6e6p.le330q 09s8 5pep5leep 5e1D1Bleeq eo5Tleopq5 Tqoec,w6.6 P.6610061q0 e5e3ePe665 008Z 1Pe33B323 Tea5q55.6q D.660.5poe ofteeplEoq 51ov1T6110 qm3Deppqqv 0T7v8z pe0p6vBe3l 3ee6eo3-3Do vo5lwov3o v333e3w65 lopoqlo355 Pe6eP3bego 36eef.66q1 333.5Q33e5 lopeoBqpq op.6.136q11 qoaeppqppo De6p5e6.66q c ozEsz 3p665.65v6P 6p651voelD 65geelpow be5p351q3 DqE,BuBpbq qqe6661T1D
09z8z 3.6p5v36e65 qqqeo6poq5 3Be3oeqlq ovpup&Ipbe e55.6.13el5e vw.611e0q0 pozsz 1.6es6-43.4.6e Bpo6.661qq q6peelowq 5p3e3z6e32 Eca5pTeepbe voeloavepo or,T8z q545weee'4 3qeeq6e33 polgoopqoP BEqeDlowq eppboP1D41 543OPP3PPe 066LZ/66S11/1Dd ELL Z100 OM-EZ-g0-100Z ZEgZgEZO VD

Q5,02352532 2001-05-23 caaaagcatg catgtacacc attcttatta gactatgctt tgctaaaaga ctttcctaga 30300 tactttaaaa catcacttct gccttttggt gggcaggttc caagattggt actggcgtac 30360 tggaaactga acaaggtaga gatctagaaa tcacagcagg tcagaagggc cagcctgtac 30420 aagagagagt tccacacctt ccaggaacac tgagcagggg gctgggacct tgcctctcag 30480 cccaagaaac tagtgcgttt cctgtatgca tgcctctcag agattccata agatctgcct 30540 tctgccataa gatctcctgc atccagacaa gcctagggga agttgagagg ctgcctgagt 30600 ctctcccaca ggccccttct tgcctggcag tattttttta tctggaggag aggaatcagg 30660 gtgggaatga tcaaatacaa ttatcaagga aaaagtaaaa aacatatata tatatatatt 30720 aactgatcta gggagctggc tcagcagtta agagttctgg ctgcccttgc ttcagatctt 30780 gctttgattc ccagcaccca catgatggct ttcaactgta tctctgcttc caggggatcc 30840 aacagcctct tctgacctcc atagacaaga cctagtcctc tgcaagagca ccaaatgctc 30900 ttatctgttg atccatctct ctagcctcat gccagatcat ttaaaactac tggacactgt 30960 cccattttac gaagatgtca ctgcccagtc atttgccatg agtggatatt tcgattcttt 31020 ctatgttctc acccttgcaa tttataagaa agatatctgc atttgtctcc tgagagaaca 31080 15 aagggtggag ggctactgag atggctctag gggtaaaggt gcttgccaca aaatctgaca 31140 acttaagttt ggtcttggaa tccacatggt ggagagagag aagagattcc cgtaagttgt 31200 cctcaaaCtt cccacacatg tgctgtggct tatgtgtaac cccaataagt aaagatagtt 31260 ttaaacacta cataaggtag ggtttcttca tgaccccaag gaatgatgcc cctgatagag 31320 cttatgctga aaccccatct ccattgtgcc atctggaaag agacaattgc atcccggaaa 31380 cagaatcttc atgaatggat taatgagcta ttaagaaagt ggcttggtta ttgcacatgc 31440 tggcggcgta atgacctcca ccatgatgtt atccagcatg aaggtcctca ccagaagtca 31500 tacaaatctt cttaggcttc cagagtcgtg agcaaaaaaa gcacacctct aaataaatta 31560 actagcctca ggtagttaac caccgaaaat gaaccaaggc agttctaata caaaaccact 31620 tcccttccct gttcaaacca cagtgcccta ttatctaaaa gataaacttc aagccaagct 31680 25 tttaggttgc cagtatttat gtaacaacaa ggcccgttga cacacatctg taactcctag 31740 tactgggcct caggggcaga gacaggtgga gccctggagt ttgaattcca ggttctgtga 31800 gaaactctgt ctgaaaagac aatatggtga gtgacccggg aggatatctg atattgactt 31860 ctggccaaca cacagccatc tctgcacatc tgtagttgca agccttttgc actaagtttg 31920 gccagagtca gagtttgcaa gtgtttgtgg actgaatgca cgtgttgctg gtgatctaca 31980 30 aagtcaccct ccttctcaag ctagcagcac tggcttcggc cagctgctca ttcaagcctc 32040 tttgcagagt catcacgggg atgggggagc agggcccctc cctagaacac caagcctgtg 32100 gttgtttatt caggacatta ttgagggcca agatgacaga taactctatc acttggccaa 32160 cagtcgggtg ttgcggtgtt aggttatttc tgtgtctgca gaaaacagtg caacctggac 32220 aaaagaaata aatgatatca tttttcattc aggcaactag attccgtggt acaaaaggct ccctggggaa cgaggccggg acagcgcggc tcctgagtcg ctatttccgt ctgtcaactt 32340 ctctaatctc ttgatttcct ccctctgtct gtttccttcc tcttgctggg gcccagtgga _ gtctgtgtac tcacagggag gagggtggca aagccctggt cctctacggg ctgggggaag 32460 gggggaagct gtcggcccag tgactttttc ccctttctct ttttcttaga aaccagtctc 32520 aatttaagat aatgagtctc ctcattcacg tgtgctcact attcataggg acttatccac 32580 ccccgccctg tcaatctggc taagtaagac aagtcaaatt taaaagggaa cgtttttcta 32640 aaaatgtggc tggaccgtgt gccggcacga aaccagggat ggcggtctaa gttacatgct 32700 ctctgccagc cccggtgcct tttcctttcg gaaaggagac ccggaggtaa aacgaagttg 32760 ccaacttttg atgatggtgt gcgccgggtg actctttaaa atgtcatcca tacctgggat 32820 agggaaggct cttcagggag tcatctagcc ctcccttcag gaaaagattc cacttccggt 32880 ttagttagct tccacctggt cccttatccg ctgtctctgc ccactagtcc tcatccatcc 32940 ggtttccgcc ctcatccacc ttgccctttt agttcctaga aagcagcacc gtagtcttgg 33000 caggtgggcc attggtcact ccgctaccac tgttaccatg gccaccaagg tgtcatttaa 33060 atatgagctC actgagtcct gcgggatggc ttggttggta atatgcttgc tgcaaaatcg 33120 tgagaactgg agttcaattc ccagcacatg gatgtatttc cagcacctgg aaggcaggga 33180 gcagagatct taaagctcct ggccagacag cccagcctaa ttagtaatca gtgagagacc 33240 ctgtctcaag aaacaagatg gaacatcaaa ggtcaacctc ttgtctccac acacacaaat 33300 acacacatgc acatacatcc acacacaggc aaacacatgc acacacctga acaccctcca 33360 caaatacata cataaaaaaa taaatacata cacacataca tacatacacc aacattccct 33420 ctccttagtc tcctggctac gctcttgtca cccccactaa ggcttcaact tcttctattt 33480 cttcatcttg actcctctgt actttgcatg ccttttccag caaaggcttt tctttaaatc 33540 tccgtcattc ataaactccc tctaaatttc ttcccctgcc cttttctttc tctctaggga 33600 gataaagaca cacactacaa agtcaccgtg ggaccagttt attcacccac ccacccctgc 33660 ttctgttcat ccggccagct aagtagtcca acctctctgg tgctgtaccc tggaccctgg 33720 cttcaccaca gctcctccat gctacccagc cctgcaaacc ttcagcctag cctctggttc 33780 tccaaccagc acaggcccag tctggcttct atgtcctaga aatctccttc attctctcca 33840 tttccctcct gaatctacca ccttctttct cccttctcct gacctctaat gtcttggtca 33900 aacgattaca aggaagccaa tgaaattagc agtttggggt acctcagagt cagcagggga 33960 gctgggatga attcacattt ccaggccttt gctttgctcc ccggattctg acaggcagtt 34020 ccgaagctga gtccaggaag ctgaatttaa aatcacactc cagctgggtt ctgaggcagc 34080 cctaccacat cagctggccc tgactgagct gtgtctgggt ggcagtggtg ctggtggtgc 34140 tggtggtgct ggtggtggtg gtggtggtgg tggtggtggt ggtggtggtg tgtgtgtgtg 34200 ttttctgctt ttacaaaact tttctaattc ttatacaaag gacaaatctg cctcatatag 34260 gcagaaagat gacttatgcc tatataagat ataaagatga ctttatgcca cttattagca 34320 atagttactg tcaaaagtaa ttctatttat acacccttat acatggtatt gcttttgttg 34380 gagactctaa aatccagatt atgtatttaa aaaaaaattc cccagtcctt aaaaggtgaa 34440 gaatggaccc agatagaagg tcacggcaca agtatggagt cggagtgtgg agtcctgcca 34500 atggtctgga cagaagcatc cagagagggt ccaagacaaa tgcctcgcct cctaaggaac 34560 On. eElTqopoov oopoopoqoo 6535.66p5.63 ea6p56o656 3ovv5TeopQ bvuove3pe6 09E p65q35265o opoolDo5e5 3poovq6e62 B53lobpboo oolvolpea6 Bo2opEcT26.4 gE
00E p2Bpvoqq6o bBt.o.5.6.6B6 fivoobB5P.65 ;5v154.6pol "4035P3PDPD
pq55qo6q3p Of7Z 5o1Bo'qoq6 qb000.acao eooDqp6E,D5 qpopeq66ao 6.6.1opoqop 36q6o66q6.6 081 peb6laeqo6 E.woB.G.E,po op5v65q0B5 0Q01536002 6055PPPPP1 qq06.55,6PBv ot 6eobpp61vo 65.4o6Bqqpo loD15qoo5P opoo6l0005 poop566Teo pev555.25q3 09 56653pqopo lopollqopo DPoso5v6go qofqq.466.6e bE.56.1qqp.6 p25e5BeEpq IDE
81 <00T7>
uaTdps owoH <ETZ>
NQ <Z1Z>
10E6 <TTZ>

81 <01Z>
8Z8SE p&15.1.6op OZ8SE 460e10506 q16.6q5635 lpoqq&Teop 3e656161.6 6e.66qoqopo paeoppoqbe 09LsE BqopqBepv.E. BOV36PP313 oolqopqppq P5VPOPPOP 6eP1643lop PODBUPWOD OZ
OOLSE qEreae005q0 1.6cql5Evoqo v1q01-aloB q1.611oe.55q oqo5q433po qqalopopql 0f,9SE DP110qq6lo 35.613poqo5 Bqoolpolofi eqqe6oq0Be 1300qOPOD3 5PEO2e5DD3 08SSE .666'400'416v ooa5epTe.5 p3o6b613ol q6PD016P'e3 Te5000,6560 4De6lopplP
ozssE 5qopq356.6q 3365661330 oe31303eP Deq56-400P3 DBP50-140 5106106P31 09E e666eT4e3p q5TeoQopoe oo556olqqe pa6qq56q5p 03qP54.65T2 elloqlpoop 51 00f7sE 33pe433365 pooBoepopo D55555pEp4qe.65.6D DqV2PBPDD peBoopE61 ol'EsE o6e3o26p; .6343oPooe e.66qopEc43 65qo345eq6 464D13436 MuovElpbq 08ZSE 41115134 6qq14113q1 POOD3PP'10 o1q01P0.65q epq.PoeoT34 11Q0DPP0P0 ozzsE p535PeeP.63 oq2q.6.3p33 45.43Teo6Te 3.61P16peov 66epe3ooPD oop65353o5 09TsE pbeoeppPPq oqvq11e5q1 .64o&a6peo.6 epeem6o5q o535opEcep BPODDOPOBP of 001sE 3356116656 poo6pon.64t, qv.65pPlee .6.6v111.PD QP,66136261 303D4P435P
otosE 3335 65P3 p6s,o3513.66 Eceo64ea6vo ol6qool056 poDoEreeD5 63.61.6e6P6I
086E 56e0PD3e5 510qqpl3op po5leo5651 peqooBebe EcepbbeEpev vooTepeoeq (:)6y7E 33le64.6303 p6epoqo5eo 3eo.6.6.101.6 op000Poppo op5qov3eo 33335833.64 098f7E 135E643336 pgp333436.6 qoppeolvoo 56.66eaeeep 23PvE.61.6p .62.611.6.63p g 0061,E uoueogglo 5qe6Bee33o obvpaieopE, v561535615 ql;e556515 5a655eqq46 OT7L.E qq-eoppBBE, 3333e335e5 64.13163263 66pEre54u3e 335e56.615P 050615EPP
08917E 3335365536 5p3343B6E5 p.643533433 36u35p1613 p51535veP3 33.6.655.e561 0Z9T/E P333PE.P012 56Q0P1Pe56 P6535ep436 11p6p5v33p 1.6.6.e5412633 3o5re35613p ZE
0661ZUWIL13d EUZCVOCVA
-go-TOOZ ZEgZgEZO VO

08SZ lopEoqoppq Bvpqqopopq oopoqvBqop .1.600qq-es5 Tevqp162.23 111paqqa6q OZSZ 1333.6qpeov aeBEqq6B65 6ebqpele51 Tepqa3qpaB uploo55610 6613q31.1TI gE
09VZ oqq6ee3666 411loobpDo oft.351p5p5 6003Be5BqE. vt,555e00D3 P66610.64E6 00tZ evvpoo5v56 DobqloqDpo D1Do6p6poB opv6p.aEcqu 56-avoopqo3 5U0306P33 OtEZ po3epolo5p oqopp5pobv eptreq5pov6 aevq6eva65 vq.6.6qt,433o obb5volDbp 08ZZ 6eo-456.611q 45.6E,36qoop E.54o5vD55.4 51445qepoq 65voo5e6e6 vo6e3po5q5 OZZZ lvEv6pepep 6eoppeP4e6 eDlpo3D6o 5526qpoDvD qeqbeEBBeo 035 6o 09TZ 0weo3362.6 BolDoEbepo 65olp6.66.6 pa6z6q5q6E. pvp16.6.4voe 5-GB6v1poop OOTZ polBepqaTe 033ep3o35 eveepqq1eq Dfto3vE3P6 oPoupqopq6 6qoe661666 OtOZ 65q1loopv.e, 51poopaqeq oPe56q1voe 3154Qpq-qqo qe3qqqqq.e. 13.6evaqol 0861 E.61165loq Dq0000151D 1D1q3365a6 66666-46655 616p5sollo q36q13.6q6q.
0Z61 p6q3p30l0l e0vo366q6.2 36o6p opooBeo6vp vBeDaeBpop 333 30 gz 0981 D5le56P6q6 5P6P05q000 5P0013eoPo poeovoopEq. pooppopt, ep5.65qp301 008T qpopoo5Tee eP05005vee eB0101.13 6DE.565e.aD3 66q30646pe 5.6pBeD5101 Ott:I p6Teol5ppe opppvpqabq e3p3352e56 v5qpoop6eo Poo55eEce6o p3p33e6.64 0891 Daqqoppoog EqUPPPOPDe ppoDoe.6.6p eq.61a656Pe 613D513.26o 6eopq3666q 0Z91 36v3q56ee3 65eloTloo3 oq3.6v3v333 oDDqDo5p6fi 5evae.6655 oz 09S1 53opope56 Po36p3365e 666 353p vo555ePpep 1D5q65.66.4e qopE6ETT63 00ST opeeppolon 600polDoo6 e000pp.6.6qD 353335 13PD0'452P6 P.6E056166E' OttT 3olvq1p33.6 Ec43';o336To 33q36e5p.e. wv6eDllov 3p5qpee643 q536.66.66qp 08E1 Do65P6.4331 eeB5PD55ql. ppq.213356 loqv36q6B eoP6q.56e3p oBee6v5eop OZET 36q33.66p3q 56qoo.aq666 qopoev55p6 ve6Beu6v56 6v5566epoq Teogo3610 gI
09ZT opPopoqBeo DopeopBE.D5 enloogbqp.4 poe5,65e6v 6.6ep5.6q3q 33qe3.6.235e 0OZT 5e5556p55.1 3poE6po3l5 Q6q3.62a6.6. .65zepe6qD3 po6Pqqa5p peoBfolqq 6e56.64ee55 pe5qqppo55 qo5v5E05poo 06.3D5.6Teo 30.61qe0615 16P060000 0801 ea65P6eBeD DE5ep.66v6E. .66.q5e36eE.E. 3p=q3126e De65656161 6665E6541 OZOT .6q56l33D6e 1P 53P p33qe.6qq-4.6 .6q33ep.66; 6EIv.6q36e6e P6v6q6P4.56 01 096 qq3pp36e3q peo156epolq 65epecre5ep .65.66Pp5ow 5pooppove6 -465p55e000 006 3qvp.66.Te3q coepoovElvo POPDOPOD= '150651.66 5.61D61P403 3Q3P6500e0 0t8 56551.6q3pe 35p3epEpDe poqopowo Ep665pe;66 epoppEBBE.E. 5653e33pq 08G 6.6q35q36e3 pq6oppo5p6 6elpTep6.65 looloogoqv efipvp31.1E. 5PPPv5eeue OZG 6e34356.66e plo55eope5 p34p3666P3 pa55Tel.E.E. 65q55166.6 510.60e55P6 099 .66.e3BE6poo 36p6p6q6.61 obopEpoveD 56Pfilvo65p 6v351.66eal 3PPP03q00B
009 v35.4.6353 q366TepEpe .6555515.6e6 .6q33.64.13q5 epeR55e5o1 66q0e05e3o OtS
6lDqo3.6610 6.4qu5pe646 popae6Pq56 6513P6st,55 llo66e6664 410103'10PP
08t e6eqqqE.5e 5656q33qe5 pvevq&eqqo 14veEce6e56 P66.a5.6661v 366vpp3326 EE
066a/66V4L3d ELLVDTIO OAt-EZ-g0-TOOZ ZEgZgEZO VD

---------NJ/ 30.203v330.6 30qq336ope pv6'2611330 003P0e060 DP6Ppe010e ve0PO4P006 0891, E.35P566v50 DDE6P06'qq0 06630e0P66 ee31366pe 6q6pp6e6ep 633qq3Depq gE
OZ917 6PPPTTaqbq u336pqppoo eBoo303q00 Dqvp6e35e6 PPPDODT4OV PDODDBODqP
095V pe3vvv6eq p6.6.6p65.6.16 .6q6.66e6001 .666-eBe66PP ev6pq666.6.4 ;v3pqv0.4e3 005t pvweeEceev 0qoqp0oloo opPopqopeq 366q.q16qe6 600o0q1066 3005e00013-OVVV 35q36.1v10.6 ep6pp.6603 qopppopEcqo 66aeqqqv.qe 0a6q366q.eq qqe.qpqBe6p 08Et ePq6vE,PeeP eppv6voepe opev2e61P.6 evv30.6v025 .43qq3q3qo6 Te646qq6v6 0c OZEt vaTTee6v03 qq0.201662v 036q.6v3q.qv 64T206qq5v 06qeeblepE pEe5p5pepb 09E17 e5e6e5p6p6 p6p6;56e6v 0.6661ps511 q1036q6vo3 p.e..66.E,P033 Te61v1e066 00Z17 13eq0v6v5.6 E.63q3qe.6.6,2 qp.e65.6q65E, 66q5.66p6qp pePq6;qqou, opq4.120 OVTV 3PE6601q03 3033eppeop q.nve63.403v pq.6q6q35.1q 0P2q3e00.63 3306630036 080V ploqlq661,5 q.p6E1.613ue 06.6666.10e5 penu06p6Pn 306q6D66e6.
qop6pep6p3 cz OZOV le30354363 oovo6q33 poplebee4 p.66qq6eve6 pex46.6q0p5 66E00.46ppe 096E 66636e6663 616605P665 ePlqqq6656 q33363aeoq qqq.uo6epE6 lppv6p066e 006E 66E.B.G.31qoP opoq..66q3.6q. Tqopv40063 p66661.3q03 .503306e350 P00.6630v0 Ot8E p620e0.q6p6 p64qva66.6.2 6666e066.66 opoo0q666.6 e6qp.6pD36.3 33.6v310333 08GE 30.66ve36.63 063666003q pp65p6q003 66P30qqope BP6q06.66.66 e366.6s0ooe OZLE PD6q005qtre Pq611P0331 elPT11613.e. 6qq466z63.6 3.61003q5q 3.1.1.paepoo 099E 3.606030ep.6 .1=3660003 6o66535.603 v303013333 636=5003 6e6vo2133 009E 60pebp66q3 6p 6306.6e00 E.P0oBeevoo 63.6p66333.6 36=33.66o6 336pe663D6 Ot5E 66pe6p06o3 663q3.633.66 P603P6663q pe.65E.e0.13 5p66oqbpoo evoeopTI.D5 08VE 303p0q0360 6e-e06.6veo 6q5pq3D661 6610060636 6PE0606060 606006065P CI
OZVE 6165q66300 153630Ecq36 e0E.a.60.636p 363636o3pq 3.633a60030 Te05q36.001 09EE q3v633366.6 q6e303p606 6.16.6q6pp06 .636036.60Te 30.6Dep3005 -q061006060 00EE 6E0006636'3 6u006603q3 633e3q6.603 6ev3050.6e0 5036q60366 OtZE 61P600P636 0P306300P0 340P36qoBe 6oBoo6q3.6p ovq.6e6D0q6 16o2Eepp3o 08TE q33q.30.6DE. 36 D33 0366366636 Ece336.6-15.66 6p0666p004 55635 01 OZTE e6v6P66166 6630666P06 ePeP06.6qq0 66e3e6p6.6q 3q336epe0 555:
090E e6pqve3610 3q33E,66.6 3q-206p5eq.3 qp6e33e0pq q;35v6p55e 3606q6.66e0 000E e6,666v.6001 065q1qe01.6 P06e0000ee Baqq6q6Bqo 3o5.2.66Te63 Be5q.6p0613 0V6Z 3e3e6q26vv 303.6;q3pp5 62pqq66v6t, 5p 5p55 e3P5v36331 le00066066 088Z .66qq3Bee0 6.310eelqp6 6Tep16q516 3613P0610q ql4P36.6.1Pe p006q.6.4v1p g OZ8Z qoBvplqp.6 op6036e0pe TepegPvTew gevqv6.4vvp 6p6ev6p3o3 3v5peBevqp 09L 3P355q6.6e.e0voE. 506q303e.66 6v336.6e300 6q000e3102 36P6D6q61 00GZ 66PP&03552 006e6P006P e6e666131.2 .255eeee.65q 055q6ve033 10033pp3q.6 0V9Z .13330qe303 3q.333330e4 0100pelTeu 3q3001qpq q1e6q33o6q 36v36p0v30 VE
066a/660412d ELLZE/00 OM
-go-TOOZ ZEgZgEZO VO

0069 p6.6E,q65qop epEeqlofiqp 6v6qoepl3 DoovoopSq q33.116eDqo 655q1peqvo DEleoeopp5q 'lq;.61Peelq 555.6ePoppo Ecev5qopBeb 6e5qozevEcq qq6.36.655 gE
08L9 PDOODBPODO PDPOOPOOEre 51E066popq qp.65.6q35.1.6 P'ePOOD23D6 50'100.6'4035 OZL9 qp4e63Ep23 lpop.6Tqa1.0 PP50401BPq 066P3056q1 Bleopeolqg 666-2 0999 1P5q1.1qq4 5qqqqqepqo B5qop5oPoo PD061605q6 5e0PTIP565 105v45pEcep 0099 oqop.Bogoo .6.1powqqP.6 .4.6.ve3qqe65 pooqqop.6q3 TIDPPoBlDe qoBboqoqv 09 povoq6q6Po 6qEu.66;p56 eoppE.qqsp qa6010q6e5 PouBP5TIT4 Blq164q161 0E
06179 qq6qq.6q5q q5Broq56.6; ow.65.6oppv 556 popq66o6a5 po5fiv6v6qo 0Z19 Doqpooqwq vo.6v6Pope E.5.6v2.65vo.6 p6P6.4PE,P.6 epoo555e3q D4.61.6e6qeD
09E9 qP05e0Dq2 PPPBE06qop o35616PDq op.6p.65e5o opoo6pooqo 6665;op;q3 00E9 Do;66po66q 5p3 p555 5e5q6-3.65pq u6;413Q.5ee oqeolo3po weTweBql 017Z9 OP33OPP2VR opE.qouTTI 5poqBqqap6 qpBqoolopp qB5e6o52e p&evtovopo gz 0819 3op333vp66 5.65q133uqo DBPopqae-e; wepq1135P eP564DeeDq 651PE.56q5.6 0Z19 PPqn005116 Pqloqoell Bppeqopope 1.5p5ee6qp.6 volwqeDae, qqbe6q06P4 0909 oBar.6535q BpTevgq.3.1.6 4.65vpopooq 33Bp'3D1.6ev PO1D40;q0P
obloo.wofip 0009 op.655q.E.ppp eoqqolebBe lbapepbelB 55;30q0061 P.611T2565 45ee50'1101 01769 laTeoqqaep pESp3s3q35 vDovEZpoll ee015Pe00 BeeepPDPPD PfieqP355q OZ
068S pq5e-4w6qo 3167eD36.6 op5eopP.65e BE.wo.4qeol 5pae61qqvo OZ8S evqopq55q qqoeweopp Dvqp.15.431q opooBeopqq 65.254o1Do'a oloqoDolp6 09LS lvopowTee 3D 66 eDolqoovvq popoglooq eo1.641oPql Poe011v66P
OOLS 1.02q1D'ID 0qPPDPV1P PODOODQDBQ 4qOP3P33Te 0061qQ5eMq 33-40.6000 01,9S
1v5PDB;Ppq 56qeve33qD v5qop33lq. 36ep3Dpopq oPpooloovo poqoP.614oE. 51 06SS q3'IDT4D06.6 e6E6qP3e6 ogepoogq.q 5eo.6-a5vpoq p65epoo5.41 p000pqobvq OZSS popaqopBee PPD13.6q300 olDp3qe1.61 leow;15qo BWPPPODT4 065q05T200 0917S qpoopqoaBe 35pmqlqopq ez6q1.335po qp3t-eqvel.6 .6qP066.6PD0 DepTqDqqqP
00VS 0600P5EODD PPPOD1q1= lopogpqqq 5'1Q0q5UPPe 6T4100qq0 5qP"4WE'055 4.61qD3D6.6 5.643.33qoBq 3e6qqqDopo PDE,56qloop vopqobpSuq 0,901561q0q OI
08Z5 po33PoP6 qq5PPPD5q D'111eDq5p 6PDPDVDOP poq.644qpo qpq.561.6qv6 OZZS 6PeeboopEca epTepEiqueo v5t)epTepq .641voe6e5p ooqBP.Elepoq 13q610q6v 091S pePqwebqv Tqlobqwqe pll5Tepov5 pp5p5p5 5pp pv3ble1q1P
OOTS gq&euTIDE.D q.paqq.qpqqq PqPDP'115PP 11.6P5PveT4 qqq6.6q6PQ6 'E'e5qee5Pe DPDD15q4v 555150P30q 43Tqu'e0.654 q414q1E06 Eclon.E.Evo.6q DoBvE.Pbqeq. g 06617 Q3v64p3eo6 epEreopevqq qq6peppp.ev eev,p11.6q1e p.33qqq6Pq..6 q60qqq.DP;

PD3JP3P eppETTTI.qo TePPPPDODP op5;a6eTee 3P05P6eD.Eq.
09817 3E.Pp5p6lo5 TelvopElqo EqBETopqoe pe.e.e5.6,565q qp.31eqpv5v veeweopqq 00817 DPPD6EPBqE.t.pelq.D qo5q3egqoq qo6voeeppb pq33.633q1 P3PDP553eD
SE
066a/66WILLNI MUM) OM
-go-TOOZ ZEgZgEZO VO

0v1.0611P0 01Te311Ppq lvoTe535.6.6 pEoe6ee66a6 sEbqeb6515q 656e5e.65eu 22p5.36pp65 pE.P55.6o666 6-4.26e565.6p p5.6265355e vlqa6p35e Do5qeal56q gf 01,68 356.6v6;op; loveovEloo 61D000lDoe peoobebvp 655662.6e6 6ee56v6v5 v6PP6E,516 E663 3Dv155pqa6 e5-266p.q65e o5qa62D5p5 o6v3.65qa.6.2 5P00655P59 PopeD00Bo 11beP43o6 E.5.6.6qpq6a .luvovqopoq qqvpoopEae 2D01300510 B50P1-10113 op3peblopq 665511op qqpoqvolTe eeqop6u6P5 q.665.1P3Q46 .0354vo5q5 PPP6qD035P 61Doee600p Be5ellqDEI 311.6ov5qvu. 0E

EclooD6.6B.5 vp.6.3De15.66 leo.456.6gDE. aebebofiEcee s6Beo61Doq opEopeo5v3 551.6eqep3e 3q6e6qo665 Be6p6ue6E.E, Bp5lep.61e q65epooPe6 pe66ppEcqoP

oqepplEqDe 1.255epeve5 ppeepoqpo Tgos6epobe 6PpeeD651D opBroolopo 6loop651 le6e.ep36p6 3.6.2DE6E2 6ea56eB6o 3pep6T1o56 levbpa6pop 5s61D55pB6 boqoeqB5eo opqepbloo Po&Teo651B EqeD6EPP06 eqqPPPePoP gZ
017E8 weErepqap loq.Doppe vp.e.o.66eDE v=6610365 lae5P5PE, e50PD155PB

.00epTe5Bq 556a66epoq 5ovE551.13 elBeopoqse q5loovo2D-4 a6535B.abob B6q36140PP 354Da'BeP1 qP1pEqeopo 516.6.4v1vqq p2Plao566 101PP0DDP.6 lpeppeeloq DE.1D1qoppo plaapopeqq P41PQ1606e Q0056P16qD aeoPqqq5D.4 0018 p165qE6Blea o3614q6Blo 3516.1eDDPE o6lePol6E,q p55.6.6po6a. oz 55Q061P6P6 BDEIBlEcIqq loop552.61 DEpopeo3-25 qqqa623.655 l'Iv1D5P010 65Bet.36.6e3 66.6evoo.351 qoopwl1D4 6loqoppB1D DevE61.33.4 PODDDPDODD

qppeop5qo3 qloolBoopo 51DDE565qo qobeleqqp6 qleolvBeop o;o563paep ovooqD61fiq pooBvaeopEl lqpoqoBloo PDODQ0DP3D DEODOPOVOD 016Te00615 EqD6Qeppoo 3P D36 ebeol-qbevo 35Q6636 D3poo62e.6 pobloo6v6t, gi Ot,LL 655E5666 q35235oplq 3p55p355eo qe.2.6q1poqq BqD33-1.1e PPPOODOPE5 epy3o5e66e 651.163p5e pep66-eppq5 5eE6e20eae qqqepeop.65 eplePv0eqE, pe3o3o5Teq qoelo6655 egBlo336oP 3P0'eD2OPOP 5POP3POPO2 opaeologol 3DDD oqoqoalol 3.1poqlqop 3q6a.wev6 Bee6PqP.656 00SL e5epe6povE. ppq5p6eBD
5T4D5.6.6poq DpEceopEce,os q5555.6qaqq 01 5qepeoB6EE. oBBEBBeqBq 3.6'a11qP36e 3.6656v66.6q obvqopebeo PPOPRPOPP5 6eppeB2Dle po36DB5loo 6eeTeloB5 551vaeqp3e 1013P12T4 ePv6.61-2q11 OZEL
elqB5e5e1 66.5356eq5 lveeloBbe vB4Doeppve ep6B5.66p5D epoplepoo 09ZL et,o636.6e apobpoqvBE. qp.66e3pqe5 2v6100P666 101q1D610q 55.6Pqo3PeP
00ZL 33-15qppoBe epol.lpeBbe opP455.6553 e3p.e36.6vpo 6q6Te33.61 opq.62.63ovq g OVEL Sqvopeoqop qlpopepo.qp e5p6v.e5e16 66qpoq35-40 656vu DEqDvEvvvD
080L 6ev3D35212 a6bvP5e36E. Qq3;13.6666 BPPE.E.E6155 eop111.4e5e .6-eqq36T46q OZOL 56.6.4eveop Z.113.6p2e66 6v36615vq PBB5e64e56 6.6vE6q.E.qo 36qzeP3.6e 0969 66PqP5P23D leP66Bgell EcT6Peop'15 .1411.5qoppe 3155eo3usq eea55.61voP
9f.
M56a/66011.3d ILLZE/00 OM
EZ-go-TOOZ ZEgnEZO VD

gactggatgc cagctgtgag ccaggcacca ccctagctct gggcatgtgg ttgtaatctt ggagcctcat ggagctcaca gggagtgctg gcaaggagat ggataatgga cggataacaa ataaacattt agtacaatgt ccgggaatgg aaagttctcg aaagaaaaat aaagctggtg agcatataga cagccctgaa ggcggccagg ccaggcattt ctgaggaggt ggcatttgag c <210> 19 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 19 ccggagctgg agaacaacaa g <210> 20 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> PRimer for PCR
<400> 20 gcactggccg gagcacacc <210> 21 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
, , _ <400> 21 aggccaaccg cgagaagatg acc <210> 22 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 22 gaagtccagg gcgacgtagc a <210> 23 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 23 aagcttggta ccatgcagct cccac <210> 24 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 24 aagcttctac ttgtcatcgt cgtccttgta gtcgtaggcg ttctccagct 50 ¨ ¨

<210> 25 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
=
<400> 25 gcactggccg gagcacacc 19 <210> 26 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 26 gtcgtcggat ccatggggtg gcaggcgttc aagaatgat <210> 27 <211> 57 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 27 gtcgtcaagc ttctacttgt catcgtcctt gtagtcgtag gcgttctcca gctcggc <210> 28 <211>29 <212> DNA
=

<213> Artificial Sequence <220>
<223> Primer for PCR

<400> 28 gacttggatc ccaggggtgg caggcgttc =
<210> 29 10 <211> 29 <212> DNA
<213> Artificial Sequence <220>
15 <223> Primer for PCR
<400> 29 agcataagct tctagtaggc gttctccag 20 <210> 30 <211> 29 <212> DNA
<213> Artificial Sequence 25 <220>
<223> Primer for PCR
<400> 30 gacttggatc cgaagggaaa aagaaaggg <210> 31 <211> 29 <212> DNA
<213> Artificial Sequence <220>
- , _ <223> Primer for PCR
<400> 31 agcataagct tttaatccaa atcgatgga <210> 32 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 32 actacgagct cggccccacc acccatcaac aag 33 <210> 33 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 33 acttagaagc tttcagtcct cagccccctc ttcc <210> 34 <211> 66 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 34 aatctggatc cataacttcg tatagcatac attatacgaa gttatctgca ggattcgagg gcccct <210> 35 <211> 82 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 35 aatctgaatt ccaccggtgt taattaaata acttcgtata atgtatgcta tacgaagtta tagatctaga gtcagcttct ga <210> 36 <211> 62 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 36 atttaggtga cactatagaa ctcgagcagc tgaagcttaa ccacatggtg gctcacaacc 60 at <210> 37 <211> 54 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 37 _ _ aacgacggcc agtgaatccg taatcatggt catgctgcca ggtggaggag ggca <210> 38 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 38 attaccaccg gtgacacccg cttcctgaca g <210> 39 <211> 61 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 39 attacttaat taaacatggc gcgccatatg gccggcccct aattgcggcg catcgttaat <210> 40 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 40 attacggccg gccgcaaagg aattcaagat ctga <210> 41 <211> 34 <212> DNA
<213> Artificial= Sequence <220>
<223> Primer for PCR
<400> 41 attacggcgc gcccctcaca ggccgcaccc agct =

Claims (135)

CLAIMS:

1. An isolated nucleic acid molecule selected from the group consisting of:
(a) an isolated nucleic acid molecule comprising any one of SEQ ID Nos., 1, 5, 9, 11, 13, or 15 or the complement thereof;
(b) an isolated nucleic acid molecule that specifically hybridizes to any one of SEQ ID Nos., 1, 5, 9, 11, 13, or 15 or the complement thereof under conditions of high stringency comprising 5XSSPE, 5XDenhardt's solution and 0.5% SDS at 55-60°C, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content;
(c) an isolated nucleic acid molecule comprising a sequence at least 85%
identical to any one of SEQ ID NOs: 1, 5, 9, 11, 13, or, 15, or the complement thereof, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content;
(d) an isolated nucleic acid molecule comprising a sequence at least 85%
identical to nucleotides 48-689 of SEQ ID NO: 1 or the complement thereof, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content;
(e) an isolated nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 2, 6, 10, 12, 14 and 16, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content;
(f) an isolated nucleic acid molecule comprising nucleotides 119-686 of SEQ ID

NO: 1 or the complement thereof;
(g) an isolated nucleic acid molecule comprising a sequence at least 85%
identical to nucleotides 119-686 of SEQ ID NO: 1 or the complement thereof and encodes a protein that retains the ability of BEER protein to decrease bone mineral content;

(h) an isolated nucleic acid molecule comprising a sequence that encodes amino acids 25-213 of SEQ ID NO: 2; and (i) an isolated nucleic acid molecule comprising a sequence that encodes a polypeptide at least 85% identical to amino acids 25-213 of SEQ ID NO: 2 that retains the ability of BEER
protein to decrease bone mineral content.

2. The isolated nucleic acid molecule according to claim 1 that encodes a polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID
NO: 2 that retains the ability of BEER protein to decrease bone mineral content.

3. The isolated nucleic acid molecule according to claim 1, that encodes a polypeptide comprising amino acids 25-213 of SEQ ID NO: 2.

4. The isolated nucleic acid molecule according to claim 1, comprising any one of SEQ ID NOs., 1, 5, 9, 11, 13, or 15 or the complement thereof.

5. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule that specifically hybridizes to any one of SEQ
ID Nos., 1, 5, 9, 11, 13, or 15 or the complement thereof under conditions of high stringency comprising 5XSSPE, 5XDenhardt's solution and 0.5% SDS at 55-60°C, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content.

6. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule comprising a sequence at least 85% identical to any one of SEQ
ID Nos, 1, 5, 9, 11, 13, or, 15, or the complement thereof, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content.

7. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule comprising a sequence at least 85% identical to nucleotides 48-689 of SEQ ID NO: 1 or the complement thereof, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER protein to decrease bone mineral content.

8. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 2, 6, 10, 12, 14 and 16, wherein the isolated nucleic acid molecule encodes a polypeptide that retains the ability of BEER
protein to decrease bone mineral content.

9. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule comprising nucleotides 119-686 of SEQ ID NO: 1 or the complement thereof.

10. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule comprising a sequence at least 85% identical to nucleotides 119-686 of SEQ ID NO: 1 or the complement thereof and encodes a protein that retains the ability of BEER protein to decrease bone mineral content.

11. The isolated nucleic acid molecule according to claim 1, comprising an isolated nucleic acid molecule comprising a sequence that encodes a polypeptide at least 85%
identical to amino acids 25-213 of SEQ ID NO: 2 that retains the ability of BEER protein to decrease bone mineral content.

12. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO: 2.

13. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO: 6.

14. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO:
10.

15. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO:
12.

16. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO:
14.

17. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a protein comprising the amino acid sequence of SEQ ID NO:
16.

18. An expression vector, comprising a regulatory sequence that controls transcriptional expression operably linked to a nucleic acid molecule according to any one of claims 1-7.

19. The expression vector according to claim 18 wherein said promoter is selected from the group consisting of cytomegalovirus (CMV) major immediate early (I-E) promoter, simian virus 40 (SV40) early promoter and murine leukemia virus (MuLV) long terminal repeat (LTR).

20. The expression vector according to claim 18 wherein said promoter is a tissue-specific promoter.

21. A method of producing a protein that decreases bone mineral content comprising, culturing a cell which contains a vector according to any one of claims 8 to 20 under conditions and for a time sufficient to produce said protein and optionally purifying or isolating said protein.

22. A viral vector comprising the nucleic acid molecule according to any one of claims 1-17.

23. The viral vector according to claim 22 wherein said vector is selected from the group consisting of a herpes simplex viral vector, an adenoviral vector, an adenovirus-associated viral vector and a retroviral vector.

24. A host cell carrying a vector according to any one of claims 18-20, 22 and 23.

25. A host cell comprising a nucleic acid molecule operably linked to a heterologous regulatory sequence that controls transcriptional expression, wherein said nucleic acid molecule encodes a polypeptide comprising amino acids 25-213 of SEQ ID
NO: 2.

26. The host cell according to claim 24 or 25, wherein said cell is selected from the group consisting of a human cell, dog cell, monkey cell, rat cell and mouse cell.

27. A method of making a polypeptide that decreases bone mineral content comprising (a) culturing the host cell according to claim 25 under conditions and for a time sufficient to produce said polypeptide, and optionally (b) isolating said polypeptide.

28. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence encoded by a nucleic acid molecule according to any one of claims 1-17.

29. An isolated protein comprising SEQ ID NO: 2.

30. An isolated protein comprising amino acids 25-213 of SEQ ID NO: 2.

31. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence at least 85% identical to SEQ ID NO:
2.

32. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence at least 85% identical to amino acids 25-213 of SEQ ID NO: 2.

33. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence encoded by a nucleic acid of claim 5.

34. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence encoded by a nucleic acid of claim 6.

35. An isolated protein that retains the ability of BEER protein to decrease bone mineral content, comprising an amino acid sequence encoded by a nucleic acid of claim 10.

36. An isolated polypeptide comprising a fragment of a protein of SEQ ID
NO:
2 that is at least 50 amino acids in length.

37. An isolated polypeptide comprising a fragment of a protein of SEQ ID
NO:
2 that is at least 100 amino acids in length.

38. The polypeptide or protein of any one of claims 29-37 which comprises a detectable label.

39. An antibody that binds to a protein encoded by the nucleic acid molecule according to any one of claims 1-38.

40. An antibody or fragment thereof which binds to a protein encoded by any one of SEQ ID NO: 1, 5, 7, 9, 11, 13 or 15, with a Ka of greater than or equal to 10 6M-1.

41. An antibody which binds to the secreted protein of SEQ ID NO: 2 with a Ka of greater than or equal to 10 6M-1.

42. An antibody or fragment thereof that binds to a protein encoded by SEQ
ID NO: 1 with a Ka of greater than or equal to 10 6M-1.

43. The antibody or fragment according to claim 40, wherein the encoded protein is selected from the group consisting of SEQ ID NOs: 2, 6, 8, 10, 12, 14 and 16.

44. The antibody or fragment according to claim 40, wherein the antibody or fragment binds the protein obtainable by expressing any one of SEQ ID NO: 1, 5, 7, 9, 11, 13 or 15 in insect cells, with a Ka of greater than or equal to 10 6M-1.

45. The antibody or fragment according to claim 44, wherein the antibody or fragment binds the protein obtainable by expressing any one of SEQ ID NO: 1, 5, 7, 9, 11, 13 or 15 using a baculovirus system in Sf9 cells, with a Ka of greater than or equal to 10 6M-1.

46. The antibody or fragment according to claim 42, wherein the antibody or fragment binds the protein obtainable by expressing any one of SEQ ID NO: 1, 5, 7, 9, 11, 13 or 15 in human embryonic kidney cells, with a Ka of greater than or equal to 10 6M-1.

47. The antibody or fragment of any one of claims 40-46, which binds to said protein with a Ka of greater than or equal to 10 7M-1.

48. The antibody according to any one of claims 39-47, wherein said antibody is a monoclonal antibody.

49. The antibody according to claim 42 wherein said monoclonal antibody is selected from the group consisting of a murine antibody and a human antibody.

50. The antibody of claim 41 that is an IgG antibody.

51. The antibody of claim 49, wherein the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

52. The antibody according to any one of claims 39-47, wherein said antibody is an antibody fragment.

53. The antibody according to any one of claims 39-47 which is a polyclonal antibody.

54. The antibody according to any one of claims 39-47, wherein said antibody is an antibody fragment.

55. An isolated antibody that binds the protein obtainable by expressing SEQ
ID NO: 1 in human embryonic kidney cells, with a Ka of greater than or equal to 10 6M-1.

56. The antibody or fragment according to claim 55, wherein the antibody or fragment is conjugated to a polymer.

57. The antibody or fragment according to claim 56, wherein the antibody or fragment is conjugated to polyethylene glycol.

58. The antibody or fragment of any one of claims 39-57, wherein the antibody or fragment is isolated.

59. The antibody of any one of claims 39-57 which increases bone density or bone mineral content.

60. A polypeptide comprising the antibody of any one of claims 39-59 and a reporter or effector molecule.

61. The polypeptide of claim 60 wherein the effector or reporter molecule is selected from the group consisting of antineoplastic agents, toxins, biologically active proteins and fragments thereof, enzymes and fragments thereof, nucleic acids and fragments thereof, naturally occurring and synthetic polymers and derivatives thereof, radionuclides, chelated metals, fluorescent compounds and compounds which may be detected by NMR or ESR
spectroscopy.

62. A polypeptide comprising the antibody of any one of claims 39-59, wherein the polypeptide binds a polypeptide of SEQ ID NO: 2 with a Ka of greater than or equal to 10 6M-1.

63. Use of an antibody according to any one of claims 39-47or a polypeptide according to claim 56 for the manufacture of a medicament for increasing bone mineral content in a warm-blooded animal.

64. The use according to claim 63, wherein the medicament is for treatment of osteopenia.

65. The use according to 64, wherein the osteopenia is caused by an anemic state, steroids, heparin, a bone marrow disorder, scurvy, malnutrition, calcium deficiency, idiopathic osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, senility, post menstrual state, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, transient regional osteoporosis or osteomalacia.

66. The use according to claim 63, wherein the medicament is for treatment of osteoporosis.

67. The use according to claim 63, wherein the animal has achondroplasia, cleidrocranial dysostosise, echondromatosis, fibrous dysplasia, Gaucher's hypophoshatemic rickets, Marfan's, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis sclerotic lesions, fractures, periodontal disease, pseudoarthosis or pyogenic osteolmyelitis.

68. The use according to claim 63, wherein the medicament is for treatment of bone fracture.

69. The use of claim 63 wherein the medicament is for treatment of dysplasias associated with abnormal growth or development of bone.

70. The use of the antibody according to any one of claims 39-59 or the polypeptide according to claim 58 for preparation of a medicament for co-administration with an inhibitor of bone resorption.

71. The use of claim 70 wherein the inhibitor of bone resorption is selected from the group consisting of calcitonin, estrogen, bisphosphonates, growth factors having anti-resorptive activity, and tamoxifen.

72. A pharmaceutical composition, comprising the antibody according to any one of claims 39-59 or the polypeptide according to claim 62 and a pharmaceutically acceptable carrier or diluent.

73. The pharmaceutical composition of claim 72 further comprising an inhibitor of bone resorption.

74. A nucleic acid molecule encoding the antibody or fragment according to any one of claims 39-42.

75. A hybridoma which produces the antibody according to any one of claims 39-59.

76. A fusion protein, comprising a polypeptide encoded by the nucleic acid molecule according to any one of claims 1 to 17 and a heterologous protein.

77. An isolated oligonucleotide which hybridizes to a nucleic acid molecule according to claim 4, under conditions of high stringency comprising 5XSSPE, 5XDenhardt's solution and 0.5% SDS at 55-60°C.

78. An isolated oligonucleotide which hybridizes to RNA that is fully complementary to at least 100 nucleotides of nucleic acid molecule according to claim 4, under conditions of high stringency comprising 5XSSPE, 5XDenhardt's solution and 0.5% SDS at 55-60°C.

79. An isolated oligonucleotide according to claim 77 or 78, wherein said oligonucleotide is at least 20 nucleotides in length.

80. An isolated oligonucleotide according to claim 77 or 78, wherein said oligonucleotide is at least 30 nucleotides in length.

81. An isolated oligonucleotide according to claim 77 or 78, wherein said oligonucleotide is at least 50 nucleotides in length.

82. An isolated oligonucleotide according to claim 77 or 78, wherein said oligonucleotide is at least 50 to 100 nucleotides in length.

83. A pair of primers which specifically amplifies all or a portion of a nucleic acid molecule according to any one of claims 1 to 17, wherein the primers are selected from the group consisting of the primers of SEQ ID NOs: 19 and 20;
(ii) the primers of SEQ ID NOs: 23 and 24 (iii) the primers of SEQ ID NOs: 26 and 27; and (iv) the primers of SEQ ID NOs: 28 and 29.

84. A ribozyme comprising an antisense sequence that specifically recognizes RNA fully complementary to the nucleic acid molecule of any of SEQ
ID NOs: 1, 5, 7, 8, 11, 13 or 15.

85. The ribozyme according to claim 84, wherein said ribozyme is composed of ribonucleic acids.

86. The ribozyme according to claim 85, wherein one or more of said ribonucleic acids are 2'-0-methyl ribonucleic acids.

87. The ribozyme according to claim 85 wherein said ribozyme is composed of a mixture of deoxyribonucleic acids and ribonucleic acids.

88. The ribozyme according to claim 84 wherein said ribozyme is composed of nucleic acids having phosphothioate linkages.

89. A nucleic acid molecule comprising a nucleic acid sequence which encodes a ribozyme according to claim 83 or an oligonucleotide according to claim 77 or 78.

90. The nucleic acid molecule of claim 89, wherein the nucleic acid is DNA
or cDNA.

91. The nucleic acid molecule of claim 89, under the control of a promoter to transcribe the nucleic acid.

92. A use for nucleic acid molecule of claim 89 for the manufacture of a medicament for increasing bone mineral content in a warm-blooded animal.

93. The use according to claim 92 wherein the medicament is for treatment of osteopenia.

94. The use according to 93, wherein the osteopenia is caused by an anaemic state, steroids, heparin, a bone morrow disorder, scurvy, malnutrition, calcium deficiency, idiopathic osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, senility, post menstrual state, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, transient regional osteoporosis or osteomalacia.

95. The use according to claim 92, wherein the medicament is for treatment of osteoporosis.

96. The use according to claim 92, wherein the animal has achondroplasia, cleidrocranial dysostosise, echondromatosis, fibrous dysplasia, Gaucher's hypophoshatemic rickets, Marfan's, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis sclerotic lesions, fractures, periodontal disease, pseudoarthosis or pyrogenic osteolmyelitis.

97. The use according to claim 92, wherein the medicament is for treatment of bone fracture.

98. The use of claim 92, wherein the medicament is for treatment of dysplasias associated with abnormal growth or development of bone.

99. The use of a nucleic acid molecule of claim 89, for preparation of a medicament for co-administration with an inhibitor of bone resorption.

100. The use of claim 99, wherein the inhibitor of bone resorption is selected from the group consisting of calcitonin, estrogen, bisphosphonates, growth factors having anti-resorptive activity, and tamoxifen.

101. A pharmaceutical composition, comprising the nucleic acid molecule of claim 84 and a pharmaceutically acceptable carrier or diluent.

102. The pharmaceutical composition of claim 101, further comprising an inhibitor of bone resorption.

103. A host cell comprising the ribozyme of claim 84.

104. A vector, comprising the nucleic acid molecule of any one of claims 89-91.

105. The vector of claim 104, wherein the vector is a plasmid, a virus, retrotransposon or a cosmid.

106. The vector of claim 105, wherein said virus is selected from the group consisting of a retrovirus, an adenovirus, and an adeno-associated virus.

107. A host cell containing the vector according to any one of claims 104 or 105.

108. The host cell according to claim 107, wherein said host cell is stably transformed with said vector.

109. The host cell according to claim 107, wherein the host cell is a human cell.

110. A method for producing a ribozyme, comprising providing DNA encoding the ribozyme according to claim 84 under the transcriptional control of a promoter, transcribing the DNA to produce the ribozyme and purifying the ribozyme.

111. The method of claim 110, wherein the ribozyme is produced in vitro.

112. A pharmaceutical composition, comprising the ribozyme according to any one of claims 84-88, and a pharmaceutically-acceptable carrier or diluent.

113. A method for detecting a nucleic acid molecule which encodes a polypeptide that decreases bone mineral content comprising hybridizing an isolated oligonucleotide according to claim 77 under conditions of high stringency comprising 5XSSPE, 5XDenhardt's solution and 0.5% SDS at 55-60°C, and detecting hybridization of said nucleic acid molecule and said isolated oligonucleotide.

114. The method according to claim 113 wherein said oligonucleotide is labeled.

115. The method according to claim 113 or 114 wherein said oligonucleotide is bound to a solid support.

116. A method for detecting a polypeptide encoded by SEQ ID NO: 1, comprising incubating an antibody according to any one of claims 39-59 under conditions and for a time sufficient to permit said antibody to bind to said polypeptide, and detecting said binding.

117. A method of detecting binding of a polypeptide encoded by SEQ ID NO: 1 to an antibody, comprising the step of incubating an antibody according to any one of claims 39-59 with said polypeptide under conditions and for a time sufficient to permit said antibody to bind to said polypeptide, and detecting said binding.

118. A method of detecting binding of a fusion protein according to claim 76 to an antibody, comprising the step of incubating an antibody according to any one of claims 39-59 with said fusion protein under conditions and for a time sufficient to permit said antibody to bind to said fusion protein, and detecting said binding.

119. A method of detecting binding of SEQ ID NO: 2 minus the secretion signal sequence to an antibody, comprising the step of incubating an antibody according to any one of claims 39-59 with said polypeptide under conditions and for a time sufficient to permit said antibody to bind to said polypeptide, and detecting said binding.

120. The method according to any one of claims 116-119 wherein said antibody is bound to a solid support.

121. The method according to any one of claims 116-120 wherein said antibody is labeled.

122. The method according to claim 121, wherein said antibody is labeled with a marker selected from the group consisting of an enzyme, a fluorescent protein, and a radioisotope.

123. A method for determining whether a candidate molecule is capable of increasing bone mineral content, comprising: determining whether a candidate molecule inhibits the binding of a protein according to claim 28 to bone, or an analogue thereof.

124. The method according to claim 123, wherein said analogue of bone is hydroxyapatite.

125. A kit for detection of BEER gene expression, comprising a container that comprises a nucleic acid molecule and a pharmaceutically acceptable carrier or excipient, wherein said nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,5,7,9,11,13, or 15; (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of (a); and (c) a nucleic acid molecule that is a fragment of (a) or (b) of at least 20 nucleotides in length.

126. A kit for detection of a mature protein of SEQ ID NO: 2 without the signal sequence, comprising a container that comprises an antibody according to any one of claims 39-59.

127. An antisense oligonucleotide, comprising a nucleic acid molecule which hybridizes to a nucleic acid molecule according to SEQ ID NOs: 1, 5, 7, 9, 11, 13, or 15, or the complement thereto, and wherein said oligonucleotide inhibits the expression of a protein according to claim 28.

128. The oligonucleotide according to claim 127, wherein said oligonucleotide is 15 nucleotides in length.

129. The oligonucleotide according to claim 127, wherein said oligonucleotide is 20 nucleotides in length.

130. The oligonucleotide according to claim 127, wherein said oligonucleotide is 50 nucleotides in length.

131. The oligonucleotide according to claim 127, wherein said oligonucleotide is comprised of one or more nucleic acid analogs.

132. The oligonucleotide according to claim 127, wherein said oligonucleotide is comprised of one or more ribonucleic acids.

133. The oligonucleotide according to claim 127, wherein said oligonucleotide is comprised of one or more deoxyribonucleic acids.

134. The oligonucleotide according to claim 127, wherein said oligonucleotide sequence comprises one or more modified covalent linkages.

135. The oligonucleotide according to claim 134, wherein said modified covalent linkage is selected from the group consisting of a phosphorothioate linkage, a phosphotriester linkage, a methyl phosphonate linkage, a methylene (methylimino) linkage, a morpholino linkage, an amide linkage, a polyamide linkage, a short chain alkyl intersugar linkage, a cycloalkyl intersugar linkage, a short chain heteroatomic intersugar linkage and a heterocyclic intersugar linkage.