WO1993008831A1

WO1993008831A1 - Expression of recombinant hemoglobin and hemoglobin variants in yeast

Info

Publication number: WO1993008831A1
Application number: PCT/US1991/008108
Authority: WO
Inventors: Joseph De Angelo; Nalini M. Motwani; Wajeeh Bajwa
Original assignee: Strohtech, Inc.
Priority date: 1991-10-30
Filing date: 1991-10-30
Publication date: 1993-05-13

Abstract

The invention is directed to certain substantially pure hemoglobins comprising certain globin chains. The globin chain may be an alpha-like globin chain or a beta-like globin chain, or variants thereof. The invention is further directed to an expression vector which specifically comprises DNA sequences encoding a certain globin chain or heme-binding fragment thereof operably linked to a yeast promoter. The invention is also directed to methods for producing certain hemoglobins in yeast. The substantially pure hemoglobins of the present invention and the hemoglobins produced by methods of the present invention may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander.

Description

EXPRESSION OF RECOMBINANT HEMOGLOBIN

AND HEMOGLOBIN VARIANTS IN YEAST 1. FIELD OF THE INVENTION

The invention is directed to certain substantially pure hemoglobins comprising certain globin chains. The globin chain may be an alpha-like globin chain or a beta-like globin chain, or variants thereof. The invention is further directed to an expression vector which specifically comprises a DNA sequences encoding a certain globin chain or heme-binding fragment thereof operably linked to a yeast promoter. The invention is also directed to methods for producing certain hemoglobins in yeast. The substantially pure hemoglobins of the present invention and the hemoglobins produced by methods of the present

invention may be used in applications requiring

physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander.

2. BACKGROUND OF THE INVENTION

2.1. USE OF HEMOGLOBIN AS A BLOOD SUBSTITUTE

Transfusion of a patient with donated blood has a number of disadvantages. Firstly, there may be a

shortage of a patient's blood type. Secondly, there is a danger that the donated blood may be contaminated with infectious agents such as hepatitis viruses,

cytomegalovirus, Epstein-Barr virus, serum parvoviruses, syphilis, malaria, filariasis, trypanosomiasis, babsiosis, pathogenic bacteria, and HIV (Bove, 1986, Progr. Hematol. 14:123-145). Thirdly, donated blood has a limited shelf life.

An alternative to transfusion involves the use of a blood substitute. A blood substitute is an oxygen carrying solution that also provides the oncotic pressure necessary to maintain blood volume. Two types of substitutes have recently been studied, fluorocarbon emulsions and hemoglobin solutions.

Hemoglobin as it exists within the red blood cell is composed of two alpha-like globin chains and two beta-like globin chains, each with a heme residue. One alpha-like globin chain and one beta-like globin chain combine to form a dimer which is very stable. Alpha-like and beta-like globin genes are each a family of related globin genes which are expressed at different stages of development and regulated by oxygen tension, pH, and the development from embryo to fetus to newborn. Two dimers then line up in antiparallel fashion to form tetramers. The binding of dimers to form the tetramers is not as strong as in the case of monomers binding to associate into dimers. The tetramers, therefore, have a tendency to fall apart to form dimers and there is always an equilibrium between tetramers, dimers, and monomers. At high

concentrations of globin, the predominant form is the tetramer; with dilution, the dimer becomes the predominant form. This equilibrium is also affected by solvent, salts, pH and other factors as the forces binding the monomers together are primarily electrostatic.

The alpha-like globin genes are clustered together on chromosome 16 and include genes encoding the embryonic zeta-globin chain and the adult alpha-globin chain, present in both the fetus and newborn. The beta-like globin genes reside on chromosome 11 and include genes encoding the embryonic epsilon-globin chain, the fetal gamma-globin chain, and the adult delta-globin and adult beta-globin chains. Two types of gamma-globin chains have been identified, ^Ggamma and ^Agamma, which differ by the presence of a single glycine or alanine residue,

respectively, at amino acid 135 (Schroeder et al., 1968, Proc. Natl. Acad. Sci. U.S.A. 60: 537-544). The gamma chain has been found to contain a polymorphic site at position 75, which also can be occupied either by

isoleucine or threonine. A variety of hemoglobins may be formed (reviewed in Kutlar et al., 1989, Hemoglobin 13:671-683 and Honig and Adams, Human Hemoglobin Genetics,

Springer Verlag, New York pp. 29-33). Examples include HbA (alpha₂beta₂), HbA₂ (alpha₂delta2), HbF (alpha₂gamma₂),

HbBarts (gamma₄), HbH (beta₄), and Hb Portland I

(zeta₂gamma₂), Hb Portland II (zeta₂beta₂), Hb Portland III

(zeta₂delta₂) Hb Gower I (zeta₂epsilon₂), and Hb Gower II (alpha₂epsilon₂).

There are obstacles however to using native hemoglobin as a blood substitute. Firstly, large dosages are required (Walder, 1988, Biotech '88, San Francisco, Nov. 14-16, 1988). A single unit (450 ml) of a 10%

hemoglobin solution contains 45 g of protein. It is estimated that at least 12 million units of blood are used in the U.S. per year. Therefore, the production of 450,000 kg of hemoglobin per year would be required. Secondly, it is important to obtain hemoglobin that is free from

infectious agents and toxic substances. Thirdly, although hemoglobin is normally a tetramer of 64,000 molecular weight, it can dissociate to form alpha-beta dimers. The dimers are rapidly cleared by the kidneys and the residence time is much too short for cell-free hemoglobin to be useful as a blood substitute. Fourthly, cell-free

hemoglobin has too high an oxygen affinity to effectively release oxygen to the tissues due to the absence of 2,3-diphosphoglycerate (2,3-DPG). Efforts to restore 2,3-DPG have been unsuccessful since 2,3-DPG is rapidly eliminated from the circulation.

Several approaches have been taken to

circumvent these difficulties. These include the

expression of hemoglobin via recombinant DNA systems, chemical modification of hemoglobin, and the production of hemoglobin variants .

2. 1 . 1 . EXPRESSION OF RECOMBINANT HEMOGLOBIN

Human embryonic zeta-globin (Cohen-Sohal, 1982, DNA 1:355-363), human embryonic epsilon-globin (Baralle et al., 1980, Cell 21:621-630), human fetal gamma-globin

(Slightom et al., 1980, Cell 21:627-630), human adult deltaglobin (Spritz et al., 1980, Cell 21:639-645), human adult alpha-globin genomic DNA (Liebhaber et al., 1980, Proc.

Natl. Acad. Sci. U.S.A. 77:7054-7058) and human adult betaglobin cDNA (Marotta et al., 1977, J. Biol. Chem. 252: 50405053) have been cloned and sequenced.

Both human adult alpha- and beta-globins have been expressed in bacterial systems. Nagai et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:7252-7255 and 1984, Nature (London) 309:810-812) expressed adult beta-globin in E.

coli as a hybrid protein consisting of the 31 aminoterminal residues of the lambda ell protein, an Ile-Glu-GlyArg linker, and the complete human adult beta-globin chain. The hybrid was cleaved at the single arginine with blood coagulation factor Xa, resulting in the liberation of the beta-globin chain. PCT Application No. PCT/US88/01534

(Publication No. WO 88/091799, published December 1, 1988) discloses the expression of a DNA sequence encoding the adult alpha-globin gene and the N-terminal 20 amino acid sequence of beta-globin in which the alpha- and beta-globin sequences are separated by spacer DNA encoding a Factor Xa cleavage site.

Efforts have also been made to secrete beta-globin into the periplasm of E. coli, in which the beta-globin gene was inserted behind an OmpA secretion signal sequence (Brimgar et al., 1988, Symposium on Oxygen

Binding Heme Proteins-Structure, Dynamics, Function and Genetics). However, it was found that though the fusion was correctly processed, the beta-globin was not secreted. Nagai et al. (1985, Proc. Natl. Acad. Sci.

U.S.A. 82:7252-7255) have also reported the reconstitution of adult beta-globin expressed in E. coli and adult alpha-globin obtained by conventional sources along with a heme source to obtain hemoglobin. However, it would not be possible in E. coli to produce recombinant hemoglobin that has the same functional properties as normal human

hemoglobin because of E. coli's inability to remove the N-formyl-methionine by post-translational processing. The amino terminus is known to be critical in determining the oxygen binding properties of human hemoglobin as has been shown in the case of Hb Raleigh (Moo-Penn, et al., 1977, Biochemistry, 16:4872-4879). Furthermore, the hemoglobin produced in bacteria can contain E. coli endotoxins.

Attempts have also been made to express

hemoglobin in yeast. Reports from two groups indicate that yeast cells were unable to excise the intervening sequences in both alpha- and beta-globin precursor mRNA (Langford et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1496-1500 and Beggs et al., 1980, Nature (London) 283:835-840). An attempt was also made to secrete beta-globin in

Streptomyces by constructing a plasmid having a GalK-FX-beta-globin sequence behind a beta-galactosidase secretion signal sequence (Brinigar et al., 1988, Symposium on Oxygen Binding Heme Proteins Structure, Dynamics, Function and Genetics). GalK-FX-beta-globin however remained within the cells under conditions where galactokinase was secreted.

Recently, the construction of two yeast

plasmids containing adult beta-globin was reported

(Brinigar et al., 1988, Symposium on Oxygen Binding Heme Proteins Structure, Dynamics, Function and Genetics). One contained a constitutive promoter, glyceraldehyde-3-phosphate dehydrogenase and ubiquitin fused directly to adult beta-globin, and the other contained metallothionein, an inducible promoter, and ubiquitin fused directly to beta- globin. It was reported that in both instances, both intracellular soluble and intracellular insoluble adult beta-globin was obtained. No further details were

disclosed regarding the construction of the plasmids or the quantity of adult beta-globin obtained.

The expression of globin in mammalian cells has also been reported. The construction of recombinant herpes simplex virus, adenovirus, SV-40, and retrovirus vectors containing a DNA sequence encoding the human adult betaglobin gene has been disclosed (Dobson et al., 1989, J. Virol. 63:3844-3851; Yanagi et al., 1989, Gene 76:19-26; Miller et al., 1988, J. Virol. 62:4337-4345; and Karlsson et al., 1985, EMBO J 5: 2377-2386). The expression of human adult alpha-globin genes in Chinese hamster ovary cells which involved introducing a recombinant DNA molecule containing the normal human adult alpha-globin gene and a hybrid gene containing the 5' promoter-regulator region of the mouse metallothionein gene linked to a SV2-cDNA

dihydrofolate reductase gene has also been disclosed (Lau et al., 1984, Mol. Cell Biol. 4:1469-1475). However, the expression of the globin genes was found to be rather low due to low efficiency of gene transfer.

2.1.2. CHEMICAL MODIFICATION OF HEMOGLOBIN

One approach that has been taken to circumvent the problem of dissociation of the hemoglobin tetramer to a dimer has been to chemically modify the hemoglobin by either intramolecular or intermolecular crosslinking.

Examples of such modification include crosslinking with polyalkylene glycol (Iwashita, U.S. Patent No. 4,412, 989 and 4,301,144); with polyalkylene oxide (Iwasake, U.S.

Patent No. 4,670,417); with a polysaccharide (Nicolau, U.S. Patent Nos. 4,321,259 and 4,473,563); with inositol

phosphate (Wong, U.S. Patent Nos. 4,710,488 and 4,650,786); with a bifunctional crosslinking agent (Morris et al., U.S. Patent No. 4,061, 736); with insulin (Ajisaka, U.S. Patent No. 4,377,512); and with a crosslinking agent so that the hemoglobin composition is intramolecularly crosslinked between lys 99 alpha₁ and lys 99 alpha₂ (Walder, U.S.

Patent No. 4,598,064).

Hemoglobin has also been chemically modified to decrease the oxygen affinity of isolated hemoglobin. One approach has involved polymerization with pyridoxal

phosphate (Sehgal et al., 1984, Surgery, 95:433-438).

Another approach has involved the use of reagents that mimic 2,3-DPG (Bucci et al., U.S. Patent No. 4,584,130). Although these compounds do lower the oxygen affinity of hemoglobin, the affinity is still relatively high.

2.1.3. HEMOGLOBIN VARIANTS

Categories of naturally occurring hemoglobin variants include: variants which autopolymerize, variants which prevent the dissociation of the tetramer, variants with lowered intrinsic oxygen affinity, and variants that are stable in alkali. Examples of autopolymerizing

hemoglobin variants include Hb Porto Alegre, Hb

Mississippi, and Hb Ta-Li.

Hb Porto Alegre is a beta chain variant first reported by Tondo et al. (1974, Biochem. Biophys. Acta 342: 15-20; 1963, Am. J. Human Genet. 15:265-279). The beta-9 serine is replaced by cysteine which is able to form disulfide bonds with other cysteine residues. Through these crosslinks, Hb Porto Alegre forms poly-tetramers. These polymers however do not form in the blood of Hb Porto Alegre carriers. It has been shown that Hb Porto Alegre carriers have a two-fold elevated level of glutathione and three-fold elevated level of glutathione reductase which prevents the polymerization of the Hb Porto Alegre within the red blood cells (Tondo et al., 1982, Biochem. Biophys. Res. Commun. 105:1381-1388). The exact structure of these polymers is not known.

Hb Mississippi is a recently isolated

polymerizing variant of hemoglobin. The new variant was first reported by Adams et al. (1987, Hemoglobin 11:435- 452). The beta-44 serine is replaced by cysteine in this variant resulting in inter-tetramer disulfide bonds. This variant is believed to form polymers with as many as ten tetramers.

Hb Ta-Li is another known polymerizing beta variant. The beta-83 glycine is replaced by cysteine.

This variant was first reported in 1971 (Blackwell et al., 1971, Biochem. Biophys. Acta 243:467-474). This variant also forms inter-tetramer crosslinks.

Another group of variants include those with nondissociating tetramers. One example is Hb Rainier, a well characterized variant of the beta chain (Greer and Perutz, 1971, Nature New Biology 230:261 and

Statoyannopoulos et al., 1968, Science 159:741). The beta- 145 tyrosine is replaced by cysteine. This cysteine is able to form disulfide crosslinks with beta-93 cysteine which is present in natural beta-globin. This disulfide bond is intra-tetramer, i.e, it is formed between the two beta subunits within a tetramer. This covalent disulfide bond stabilizes the tetramer form and prevents the

dissociation of the tetramer into its constituent dimers. Hb Rainier has also been found to have a high affinity for oxygen, a reduced Hill coefficient, and only half the alkaline Bohr effect of normal hemoglobin.

Another group of variants includes those that are stable in alkali. Hb Motown/Hacettepe is a variant reported to be stable in alkali (Gibb and Rucknagel, 1981, Clinical Research 29:795A and Altay et al., 1976, Biochem. Biophys. Acta 434:1-3). The beta-127 glutamine is replaced by glutamic acid in this variant. This portion of the beta chain is involved in the alphaibeta₁ interface between the monomers forming a dimer. The substituted glutamic acid forms an ionic bond with alpha-31 arginine. This is a stronger bond than that formed between the alpha-31 arginine and the normal beta-127 glutamine and is believed to be responsible for the increased stability of Hb

Motown/Hacettepe. HbF (fetal hemoglobin) and bovine hemoglobin are also in this group of alkali stable variants

(Perutz, 1974, Nature 247:341).

There are also over 30 naturally occurring hemoglobin variants which exhibit lowered oxygen affinity.

Several examples of such variants are disclosed in PCT

Application No. PCT/US88/01534 (Publication No. WO

88/091799, published December 1, 1988), Bonaventura and

Bonaventura, 1980, In: Abnormal Human Hemoglobins and Red

Cell Enzymes, Huisman, T., Ed., Marcel Dekker, NY,

Hemoglobin 4 (3 & 4) : 275-289 and Bonaventura and

Bonaventura, 1978, in Biochemical and Clinical Aspects of

Hemoglobin Abnormalities, Caughey, W.S., Ed., Academic

Press, NY, pp. 647-663. There seems in a group of these low oxygen affinity mutants to be a generalizable

relationship between the intrinsic oxygen affinity of an alpha₂beta₂ hemoglobin and the cluster of positively charged residues that are involved in the binding of 2,3-DPG and other anionic allosteric cofactors of hemoglobin function (Bonaventura and Bonaventura, 1980, Amer. Zool. 20:131-138).

One example of a low oxygen affinity mutant is Hb Chico where the beta-66 lysine is replaced by threonine (Shih et al., 1987, Hemoglobin 11: 453-464). The P50 of Hb Chico's red blood cells is 38 mm Hg compared with normal red blood cell controls with P50 of 27 mm Hg. All other properties, i.e, Hill coefficient and alkaline Bohr effect are normal.

Another low oxygen affinity variant is Hb

Raleigh, a beta chain variant in which beta-1 valine is replaced by alanine (Moo-Penn et al., 1977, Biochemistry 16:4873). A post-translational modification of the amino- terminal alanine results in the formation of acetylalanine. Because the positively charged amino group of valine is involved in 2,3-DPG binding, the acetylation results in a decreased charge cluster in the DPG binding site. This charge difference acts to decrease the oxygen affinity of Hb Raleigh and to lessen the effect of DPG which lowers the oxygen affinity of normal HbA. The Hill coefficient

(cooperativity) and alkaline Bohr effect (pH dependent oxygen binding) are unaffected by this change.

Hb Titusville (alpha-94 aspartate to asparagine) is one of a group of low affinity hemoglobin variants with altered alphaιbeta₂ contacts (Schneider et al., 1975, Biochem. Biophys. Acta 400:365). The

alphaιbeta₂ interface is stabilized by two different sets of hydrogen bonds between the alpha and beta subunits. One set stabilizes the T-structure which is the low-affinity form and the other stabilizes the R-state which is the high affinity form. It is the shifting back and forth between these two sets of bonds and alternating between the T- and R-states which is responsible for the positive

cooperativity. The deoxyhemoglobin is primarily in the T-state. For hemoglobin with one oxygen bound, the amount of R-state molecules increases and therefore binds oxygen with a higher affinity. In hemoglobin with two oxygens bound, there is an even higher proportion of R state molecules. In Hb Titusville, the R-state bonds are disrupted. The alpha-94 aspartate would normally form a non-covalent bond with beta-102 asparagine. Because this bond is disrupted, the equilibrium is pushed in the direction of the T-state and Hb Titusville's oxygen affinity is very low.

Hb Beth Israel is another variant affecting the alpha₁beta₂ interface which destabilizes the high oxygen affinity R-state (Nagel et al., 1976, New Eng. J. Med. 295:125-130). The beta-102 asparagine is replaced by serine. The whole blood of an Hb Beth Israel patient has a P50 of 88 mm Hg as compared with the normal value of 27. The Hill coefficient is biphasic with a value of 1.0 at the high end and 1.8 at the low end. The Bohr effect is normal. A hemolysate of Hb Beth Israel has a P50 of 17 mm Hg and a Hill coefficient of 1.65 at the bottom and 1.29 at the top of the curve as compared to a P50 of 5.6 and a Hill coefficient of 2.72 for normal hemoglobin.

Another example of a low affinity human hemoglobin mutant is Hb Kansas (PCT Application No.

PCT/US88/01534, Publication No. WO 88/091799, published December 1, 1988 and Bonaventura and Riggs, 1968, J. Biol. Chem. 243: 980-991). The beta-102 asparagine is replaced by threonine. It has been shown that isolated Hb Kansas' heme-containing beta-globin chains have lowered oxygen affinity (Riggs and Gibson, 1973, Proc. Natl. Acad. Sci. U.S.A. 70:1718-1720).

2.2. EXPRESSION QF HETEROLOGOUS DNA IN YEAST

With the advent of recombinant DNA technology, efforts have been made to express heterologous DNA in a variety of prokaryotic and eukaryotic systems. One such system is yeast.

Yeast has a number of advantages over bacteria and other eukaryotes as a system for the production of polypeptides or proteins encoded by recombinant DNA. Yeast has been used in large scale fermentations for centuries, so the technology for fermenting yeast is well known and a number of yeast hosts are commercially available.

Additionally, yeast can be grown to higher densities than bacteria and many other types of eukaryotic cells, and is readily adaptable to continuous fermentation processing. Since yeast is a eukaryotic organism, yeast may be capable of glycosylating expression products, may exhibit the same codon preferences as higher organisms, and may remove the amino terminal methionine during post-translational processing.

A number of heterologous proteins have been expressed in yeast. Examples include interferon (Hitzeman and Leung, U.S. Patent No. 4,775,622, issued October 4, 1988; Hitzeman et al., Canadian Patent No. 1,205,026, issued May 27, 1986; Hitzeman et al., 1981, Nature (London) 293: 717); platelet derived growth factor (Murray et al., U.S. Patent No. 4,801,542, issued January 31, 1989);

glucagon (Norris et al., U.S. Patent No. 4,826,763, issued May 2, 1989).

Heterologous proteins expressed in yeast have been linked to a wide variety of promoters. Examples include operably linking heterologous proteins to SV40 and RSV promoters (Gelfand et al., U.S. Patent No. 4,8710,013, issued September 26, 1989). Additionally, DNA sequences encoding heterologous proteins have been linked to yeast promoters, which are inducible. European Patent

Application-Publication No. 132, 309, published January 30, 1985 discloses the construction of a plasmid containing the yeast galactose-induced promoters for galactokinase (GAL1) and UDP-galactose epimerase (GAL10), hereinafter referred to as the GAL1-10 promoter, which is bidirectional.

Another example of a bidirectional yeast promoter is the YPT1/TUB2 intergene sequence which contains overlapping binding sites for the transcription factor BAF1 (Halfter et al., 1989, EMBO J. 8:3029-3037). Broach et al.

(Manipulation of Gene Expression, ed. Inouye, 1983)

disclose a plasmid containing a GAL10 upstream activator sequence which promotes transcription and an alcohol dehydrogenase transcription (ADH1) terminator sequence to prevent run through transcription derived from YEp51.

Kingsman et al., U.S. Patent No. 4,615,974, issued October 7, 1986 disclose the use of the 5' regions of the yeast phosphoglycerate kinase genes as a promoter of the

transcription of interferon. Hitzeman et al., Canadian Patent No. 1,205,026, issued May 27, 1986 disclose the use of the 5' flanking sequence of the ADH1 structural gene to promote the transcription of interferon. Burke et al., U.S. Patent No. 4,876,197, issued October 24, 1989 disclose a DNA construct comprising a first transcription regulatory region obtained from the yeast alcohol dehydrogenase II gene (ADH2). the regulatory region of acid phosphatase (PHO5) or the regions regulated by GAL4, which provides for inducible transcriptional regulation and a second

transcriptional initiation region from the yeast

glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) and a terminator region.

3. SUMMARY OF THE INVENTION

The invention is directed to certain substantially pure mammalian hemoglobins. "Substantially pure" as defined herein refers to a globin chain that is free of erythrocyte membrane components and E. coli

endotoxins. Such hemoglobins include but are not limited to hemoglobin variants having a lowered oxygen affinity (e.g. HbF Chico (gamma-66 lysine is replaced by threonine); Hb Portland Titusville (zeta-94 aspartate to asparagine); and Hb BovII (N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu)); a high oxygen affinity variants (e.g. HbA Deer Lodge (beta-2 histidine is replaced with

arginine); HbA Abruzzo (beta-143 histidine is replaced with arginine); and HbA McKees Rock (beta-145 tyrosine is replaced with a termination sequence); alkali stable variants (e.g. HbA Motown/Hacettepe and a variant in which serine replaces the alpha-104 or zeta-104 cysteine);

variants which have a lowered oxygen affinity and are stable in alkali (e.g. a variant which combines the

mutations of Hb Titusville where the alpha-94 aspartic acid is replaced with asparagine and the alpha-104 cysteine residue is replaced with serine).

The term "hemoglobin variant" as defined herein is a hemoglobin comprising a least one variant globin chain. The term "variant globin chain" as defined herein refers to a globin whose nucleotide sequence has been altered in such a fashion so as to result in the alteration of the structure or function of the globin, but so that the globin still remains functionally active as defined by the ability to reversibly bind to oxygen. The variant may be naturally occurring or non-naturally occurring.

The invention is further directed to yeast cells capable of producing the foregoing hemoglobins.

These yeast cells contain recombinant DNA vectors which are capable of expressing certain globin chains.

The above hemoglobins may be used in

applications requiring physiological oxygen carriers such as in blood substitute solutions, or as in a plasma

expander.

The invention is also directed to a

recombinant DNA vector capable of expressing a globin chain or heme-binding fragment thereof selected from the group including but not limited to a zeta globin, an epsilon globin, a variant globin chain substantially homologous to a human embryonic zeta-globin chain and comprising a serine at the zeta-104 position, a variant globin chain

substantially homologous to a human fetal gamma-globin chain and comprising a glutamic acid at the gamma-127 position in a yeast cell comprising:

(a) a DNA sequence encoding a globin chain or heme-binding fragment thereof;

(b) a yeast inducible transcriptional promoter which promotes the transcription of the DNA sequence encoding the globin chain or heme-binding fragment thereof;

(c) a DNA sequence encoding a yeast selectable marker or functionally active portion thereof; and

(d) a yeast replication origin.

The invention is further directed to making Hb Portland I (zeta₂gamma₂) or II (zeta₂beta₂) comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a yeast inducible transcriptional promoter regulated by galactose; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human zeta-globin chain; (iii) a URA3 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located

'downstream from the DNA sequence encoding the human embryonic zeta-globin chain, which comprises the

transcription termination region of the CYC1 gene; and in which the second recombinant vector comprises: (i) an inducible promoter or hybrid promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human fetal gamma-globin chain or a human adult beta-globin chain; (iii) a LEU2 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the fetal gamma-globin or adult beta-globin chain, which comprises the transcription termination region of the alcohol dehydrogenase I gene; and

(b) growing the yeast cell in an appropriate medium such that the zeta-globin chain or heme-binding fragment and the gamma-globin or heme-binding fragment or beta-globin or heme-binding fragment thereof are expressed and assembled together with heme in the yeast cell to form hemoglobin. Other specific embodiments of the invention include methods for making the hemoglobin variants, Hb Mississippi (beta-44 serine is replaced with cysteine), Hb Motown (beta-127 glutamine is replaced with glutamic acid), and Hb Titusville (alpha-94 aspartate is replaced with asparagine), HbF Porto Alegre (gamma-9 alanine is replaced with cysteine) HbA Porto Alegre (beta-9 alanine is replaced with cysteine) comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a DNA sequence encoding an alpha like- globin chain or variant thereof; (ii) a yeast

transcriptional promoter which promotes the transcription of the DNA sequence encoding the alpha like-globin chain or variant thereof; and (iii) a DNA sequence encoding at least one yeast selectable marker or functionally active portion thereof; and (iv) a yeast replication origin and in which the second recombinant DNA vector comprises (i) a DNA sequence encoding a beta like-globin chain or variant thereof; (ii) a yeast transcriptional promoter which

promotes the transcription of the DNA sequence encoding the beta like-globin chain or variant thereof; and (iii) a DNA sequence encoding at least one yeast selectable marker or functionally active portion thereof; and (iv) a yeast replication origin; and

(b) growing the yeast cell in an

appropriate medium such that the alpha and beta like-globin chains or variants, thereof are expressed and assembled together with heme in the yeast cell to form a hemoglobin variant.

The certain hemoglobins produced by the above methods may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander. 4. BRIEF DESCRIPTION OF THE FTGURES

Figure 1 shows the nucleotide sequences of the embryonic zeta (1A), embryonic epsilon (IB), fetal gamma (1C), adult delta (1D), adult alpha (IE) and adult beta (1F) chains of human hemoglobin. The deduced amino acid sequences are shown underneath. The AUG start codon and the corresponding amino-terminal methionine which is removed by methionine aminopeptidase in a post-translational modification are not shown in the figures.

Figure 2 shows a partial restriction map of the plasmid pSPβC. The complete insert of the beta-globin gene is shown by the double line and the plasmid sequences are shown by a single line. Restriction sites shown above the line are A=AccI; E=EcoRI; F=SfaNI; H=HindIII; N=NcoI; and B=BamHI. The line above the restriction sites

represents the coding region of the beta-globin gene.

Numbers below the lines represent length in base pairs from an EcoRI site present in the vector.

Figure 3A shows the strategy used to clone the adult beta-globin gene into YEp51. Figure 3B shows the strategy used to clone the Porto Alegre beta-globin gene.

Figure 4 shows the restriction map of the plasmid YEpWB51/NAT.

Figure 5 shows the strategy for cloning ADH1-terminator into YEpWB51/NAT.

Figure 6 shows a map of AAH5.

Figure 7 shows the restriction map of the plasmid YEp51T/NAT.

Figure 8 shows an autoradiograph of total RNA extracted from yeast strain Sc340 transformed with YEp51 (340g2C) and YEp51T/NAT (340g2B). Total RNA was subjected to electrophoresis on a 1.1% agarose gel, transferred to the Hybond paper and probed with an ApaLI-HindIII fragment

(600 bp) of the beta-globin gene from plasmid mp18βHS. The level of a control RNA (CYH2) was determined with the plasmid mpl9CYH22 (9.0 kb) which carries the coding region of the CYH2 gene. 20 μg of the total RNA was loaded into each lane. Sample in each lane is as follows : Lane 1 :

340g2C, Lane 2: 340g2B, and Lane 3: 340g2P. β marks the beta-globin mRNA. The CYH2 mRNA is marked with Cl

(precursor form) and C2 (mature mRNA). Numbers on the side indicate the length in nucleotides.

Figure 9 shows the results of scanning an autoradiograph containing both beta-globin and CYH2 mRNA obtained from a Northern Blot using an LKB gel scanner. The large peak in A (340g2B) represents the beta-globin mRNA and two small peaks at either side of the large peak represent the CYH2 mRNA. Figure 6B shows the results of scanning an autoradiograph containing both Porto Alegre beta-globin mRNA and CYH2 mRNA obtained from a Northern blot using an LKB scanner. The large peak in B (340g2P) represents the Porto Alegre beta-globin mRNA and the two small peaks at either side of the large peak represent the CYH2 mRNA.

Figure 10 shows the sequences of and restriction sites present on 51-A-1 (5'-end primer) and 519-A-3 (3'-end primer). These primers were used to synthesize alpha-globin DNA.

Figure 11 shows the restriction map of pUT/2A.

Figure 12 shows the construction of YEp51T/G. Figure 13 shows the DNA sequence of the gamma globin gene.

Figure 14 shows the sequences of and restriction sites present on GAM-5-S (5'-end primer) GAM-3-H (3'-end primer). These primers were used to synthesize gamma-globin DNA.

Figure 15 shows the restriction map of plasmid YEp51T/G.

Figure 16 shows the strategy used for isolating epsilon cDNA from genomic epsilon DNA.

Figure 17 shows the sequences of the primers used to construct the epsilon cDNA:5EPSL-1, INPE-1, INPE-2,

INPE-3, INPE-4, and 3EPH.

Figure 18 shows the restriction map of plasmid

YEp51T/ε3.

Figure 19 show the sequence of and restriction sites present on 5ZETASAC (5'-end primer) and ZETA3HSLS (3'-end primer). These primers were used to synthesize zetaglobin cDNA.

Figure 20 shows the restriction map of plasmid

YES2-ζ2.

Figure 21 shows the sequences of Mu-145Cy, Mu-66Th, and Mu-9Cy.

Figure 22 shows a restriction map of YEp51NTl.

Figure 23 shows the sequences of and the restriction sites on 5'-end primer, G-5-9CY and the 3'-end primer, GAM-3-H. Site specific mutations are shaded.

Figure 24 shows the sequences of and the restriction sites on 5'-end primer, B-G127-5 and the 3'-end primer, Beta-3-H.

Figure 25 shows the sequences of and the restriction sites on the 5'-end primer, A-Tit-5 and the 3'-end primer, G10T3H.

Figure 26 shows the sequences of and the restriction sites on the 5'-end primer, 51-A3-SL and the 3'-end primer, A-Hin3-3.

Figure 27 shows the sequences of and the restriction sites on the 5', B-44C-5, and 3' primers, Beta- 3-H used to synthesize by PCR the Mississippi β-globin gene. Site specific mutations are shaded.

Figure 28 shows the sequences of and the restriction sites on 5'-end primer, A104Ser and the 3'-end primer, G10T3H. Figure 29 shows the sequences of and the restriction sites on 5'-end primer, Z-5-SAL and the 3'-end primer, Z-104S-B.

Figure 30 shows the sequences of and the restriction sites on 5'-end primer, Z-BST-5 and the 3'-end primer, Z2-3-H.

Figure 31 shows the sequences of and the restriction sites on 5'-end primer, Z-5-SAL and the 3'-end primer, Z-A95-3.

Figure 32 shows the sequences of and the restriction sites on 5'-end primer, G2-Mot-5 and the 3'-end primer, GAM-3-H.

Figure 33 shows the sequences of and the restriction sites on 5'-end primer, B-Bov2-5 and the 3'-end primer, Beta-3-H.

Figure 34 shows the sequences of and the restriction sites on 5'-end primer, B-2ARG-5 and the 3'-end primer, Beta-3-H.

Figure 35 shows the sequences of and the restriction sites on 5'-end primer, BN-5-SAL and the 3'-end primer, B-143A-3.

Figure 36 shows the sequences of and the restriction sites on 5'-end primer, BN-5-SAL and the 3'-end primer, B-145T-3.

Figure 37 shows the sequences of and the restriction sites on 5'-end primer, GAM-5-S and the 3'-end primer, G66T-3.

Figure 38 shows a map of mplδβHS.

Figure 39 shows a map of L19βAt.

Figure 40 shows the sequence of TDH3-5' and

TDH3-3'.

Figure 41 shows the restriction map of plasmid pUC19-HβAt.

Figure 42 shows part of the GAL1-10 promoter sequence .

Figure 43 shows the sequences of the primers, GAL1-10-5' and GAL1-10-3'.

Figure 44 shows the restriction map of plasmid pUC19-GHβAt.

Figure 45 shows the restriction map of plasmid pNML-V-G-1.

Figure 46 shows the restriction map of plasmid YEpWB51/PORT.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to certain substantially pure hemoglobins comprising an alpha-like globin chain or variant thereof and a beta-like globin chain or variant thereof. The alpha-like globin chain may be selected from the group including but not limited to an embryonic zeta-globin chain and an adult alpha-globin chain. The beta-like globin chain may be selected from the group including but not limited to an embryonic epsilon-globin chain, a fetal gamma-globin chain, an adult delta-globin chain, and an adult beta-globin chain. Alpha-like globin and beta-like globin may be mixed with a source of heme to obtain hemoglobin comprising alpha-like globin and beta-like globin. Hemoglobin produced by methods of the present invention may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or in a plasma expander.

The invention is also directed to recombinant vectors capable of expressing certain globin chains or heme binding fragments thereof in yeast. The invention also relates to methods for expressing the foregoing hemoglobins in yeast where the heme which is produced by the yeast or obtained from an exogenous source is ligated to the globin to form functional hemoglobins in vivo. 5.1. ISOLATION AND CLONING OF GLOBIN

The nucleotide sequence of the genes encoding the human embryonic zeta-globin, the human embryonic epsilon-globin, the human fetal gamma-globin, the human adult delta-globin, the human adult alpha-globin and the human adult beta-globin chains and their derived amino acid sequences are depicted in Figures 1A-F respectively, and are described in the Sequence Description as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6. These include but are not limited to nucleotide sequences comprising all or portions of the nucleotide sequence depicted in Figures 1A-F which are altered by the substitution of different codons that encode the same or a functionally equivalent amino acid residue thus producing a silent change as well as amino acid sequences comprising all or portions of the amino sequence depicted in Figures 1A, 1B, 1C, 1D, 1E, or 1F which are altered by the

substitution of functionally equivalent amino acid residues within thejsequence thus producing a silent change and derivatives thereof which are modified or processed.

The genes encoding alpha-like globin and beta-like globin chains may be isolated from hemoglobin

containing cells using procedures known in the art. The DNA encoding alpha-like globin and/or beta-like globin may be obtained by standard procedures known in the art from cloned DNA (e.g. a DNA "library"), by chemical synthesis, by cDNA cloning or by the cloning of genomic DNA, or fragments thereof, purified from for example human

reticulocytes (see for example, Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). DNA encoding alpha-like or beta-like globin DNA may also be obtained using polymerase chain reaction (PCR) technology (see for example Mullis et al., 1989, U.S. Patent No. 4,800,159). Clones derived from genomic DNA may contain regulatory and intron DNA regions, in addition to coding regions; clones derived from cDNA will contain only exon sequences.

Whatever the source, a globin gene should be molecularly cloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired globin gene. The DNA may be cleaved at specific sites using various restriction enzymes.

Alternatively, one may use DNAse in the presence of

manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column

chromatography.

Once the DNA fragments are generated,

identification of the specific DNA fragment containing the globin may be accomplished in a number of ways. For example if an amount of a globin gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labelled probe (Benton and Davis, 1977, Science 196:180 and Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. If a purified globin-specific probe is

unavailable, nucleic acid fractions enriched in globin sequences may be used as a probe, as an initial selection procedure. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection on the basis of the properties of the gene, or the physical or chemical properties of its expressed product, as described infra, can be employed after the initial selection.

The globin gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization, In vitro translation products of the isolated mRNAs identifies the mRNA, and therefore the complementary DNA fragments that contain the globin sequences.

Alternatives to isolating the globin genomic DNA include, but are not limited to, chemically

synthesizing the gene sequence itself from the known sequence or making cDNA to the mRNA which encodes the globin gene.

The identified and isolated gene or cDNA can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322, pUC, pGEM1^®, or Bluescript^® plasmid derivatives.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc.

In an alternative embodiment, the gene may be identified and isolated after insertion into a suitable cloning vector, in a "shot gun" approach. Enrichment for a globin gene, for example, by size fractionation or

subfractionation of cDNA, can be done before insertion into the cloning vector.

The globin gene is inserted into a cloning vector which can be used to transform, or infect

appropriate host cells so that many copies of the gene sequences are generated. This can be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the

complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease

recognition sequences. In an alternative method, the cleaved vector and globin gene may be modified by

homopolymeric tailing.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate an isolated globin gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

After the globin-containing clone has been identified, grown, and harvested, its DNA insert may be characterized using procedures known in the art. The cloned DNA or cDNA corresponding to the globin gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), restriction endonuclease mapping (Maniatis et al., 1989,

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor

Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. DNA sequence analysis can be performed by any techniques known in the art, including but not limited to chemical methods (Maxam and Gilbert, 1980, Meth. Enzymol.

65:499-560), enzymatic methods (see e.g. Innes, 1988, Proc.

Natl. Acad. Sci. U.S.A. 85:9436; Tabor and Richardson,

1987, Proc. Natl. Acad. Sci. U.S.A. 84:4767; and Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), or the use of an automated DNA sequenator (see for example Martin et al., 1985, Biotechnology 3:911-915).

5.2. HEMOGLOBIN VARIANTS

The invention is directed to the following categories of hemoglobin variants: variants which

autopolymerize; variants in which the tetramer does not dissociate under physiological conditions in vivo; variants with lowered intrinsic oxygen affinity, i.e. an oxygen affinity having a P₅₀ of at least about 10 mm Hg under physiological conditions; variants that are stable in alkali, variants with a higher intrinsic oxygen affinity, i.e. an oxygen affinity having a P₅₀ of at most about 1 mm

Hg under physiological conditions. In a specific

embodiment, poly alpha-like globin or poly beta-like globin may result.

An example of a hemoglobin variant which autopolymerizes is Hb Mississippi (beta-44 serine is replaced with cysteine). Another example includes Hb Porto Alegre. The beta-9 or gamma-9 serine is replaced by cysteine which is able to form disulfide bonds with other cysteine residues.

Alkali stable hemoglobin variants are those in which the dimers do not dissociate into monomers in the presence of alkali. An example of a naturally occurring alkali stable mutant is HbA Motown/Hacettepe where beta-127 glutamine is replaced with glutamic acid or alternatively HbF Motown, where gamma-127 glutamine is replaced with glutamic acid. It has been shown that the alpha-104 cysteine cause the hemoglobin to be susceptible to alkali denaturation (Perutz, 1974, Nature 247:371). Examples of non-naturally occurring alkali stable variant include a variant in which serine replaces the alpha-104 or zeta-104 cysteine. Examples of naturally occurring variants which have a lowered oxygen affinity include but are not limited to HbF Chico (gamma-66 lysine is replaced by threonine); HbA Titusville (alpha-94 aspartate to asparagine); Hb

Portland Titusville (zeta-94 aspartate to asparagine).

It has additionally been shown that bovine hemoglobin has a lower oxygen affinity than human HbA

(Perutz, 1974, Nature 247:341). This is thought to be due to the N-terminal amino acid sequence of beta-globin, Met Leu Thr Ala Glu Glu. Therefore, one hemoglobin variant may comprise a (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian adult beta-globin chain, and (ii) comprises an N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme, and which variant has the ability to bind to oxygen at a low oxygen affinity and is free of erythrocyte membrane components and E. coli

endotoxins. The term "substantially homologous" as used herein refers to the ability of a DNA sequence encoding a first globin chain to hybridize to a DNA sequence encoding a second globin chain under stringent conditions, for example, at about 0.1X SSC at a temperature of about 65°C. For example, if a globin variant is substantially

homologous to an adult beta-globin chain, a DNA sequence encoding the globin variant is capable of hybridizing to a DNA sequence encoding the adult beta-globin chain under stringent conditions.

In yet another embodiment, the hemoglobin variant may be a a variant having an increased oxygen affinity, a high oxygen affinity variant. Examples include but are not limited to HbA Deer Lodge (beta-2 histidine is replaced with arginine) (Labossiere et al., 1972, Clin. Biochem. 5:46-50); HbA Abruzzo (beta-143 histidine is replaced with arginine) (Tentori et al., 1972, Clin. Chim. Acta 38:258-262); and HbA McKees Rock (beta-145 tyrosine is replaced with a termination sequence) (Winslow et al., 1976, J. Clin. Invest. 57:772-781).

In a further embodiment, a variant may be constructed which combines the mutations of HbA Titusville and replacement of the alpha-104 cysteine residue with serine. This may result in the formation of a tetramer with the desirable properties of lowered oxygen affinity and stability in alkali.

The globin variants may be produced by various methods known in the art . The manipulations which result in their production can occur at the gene or protein level. The globin may.be altered at the gene level by sitespecific mutagenesis using procedures known in the art . One approach which may be taken involves the use of

synthetic oligonucleotides to construct variant globins with base substitutions. In one embodiment, a short oligonucleotide containing the mutation is synthesized and annealed to the single stranded form of the wild-type globin gene (Zoller and Smith, 1984, DNA 3:479-488). The resulting short heteroduplex can serve as primer for second strand synthesis by DNA polymerase. At the 5' end, a single stranded nick is formed which is closed by DNA ligase. In another embodiment, two complementary

oligonucleotides are synthesized, each containing the mutant sequence. The duplex that forms after annealing these complementary oligonucleotides, can be joined to a larger DNA molecule by DNA ligase provided that the ends of both molecules have complementary single-stranded "sticky" ends. Another approach which may be taken involves

introducing a small smgle-stranded gap m the DNA molecule followed by mis-repair DNA synthesis i.e., the

misincorporation of a non-complementary nucleotide in the gap (Botstein and Shortle, 1985, Science 229:1193). The incorporation of a thiol nucleotide into the gap may minimize the excision of the non-complementary nucleotide. Alternatively, a globin variant may be prepared by

chemically synthesizing the DNA encoding the globin variant using procedures known in the art (see for example

Froehler, 1986, Nucl. Acids Res.14:5399-5407 and Caruthers et al., 1982, Genetic Engineering, J.K. Setlow and A.

Hollaender eds., Plenum Press, New York, vol. 4, pp. 1-17). In a preferred embodiment, fragments of the variant globin are chemically synthesized and these fragments are

subsequently ligated together. The resulting variant globin strands may be amplified using procedures known in the art, e.g. PCR technology and subsequently inserted into a cloning vector as described in Section 5.1., supra. In a specific embodiment, site specific mutants may be created by introducing mismatches into the oligonucleotides used to prime the PCR amplification (Jones and Howard, 1990,

Biotechniques 8:178-180).

Manipulations of the globin sequence may be carried out at the protein level. Any of numerous chemical modifications may be carried out by known techniques including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8

protease, NaBH₄; acetylation, formylation, oxidation, reduction; etc. Alternatively, the variant globin protein may be chemically synthesized using procedures known in the art, such as commercially available peptide synthesizers and the like. Such standard techniques of polypeptide synthesis can be found described in such publications as Merrifield, 1963, J. Chem. Soc. 85:2149-2154 and

Hunkapillar et al., 1984, Nature (London) 310:105-111.

5.3. EXPRESSION OF HEMOGLOBIN

The nucleotide sequence coding for a globin chain is inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence. A variety of host-vector systems may be utilized to express the DNA sequence encoding the globin chain. These include but are not limited to mammalian cell systems infected with virus (e.g. vaccinia virus,

adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); yeast containing yeast vectors, and bacteria transformed with plasmid DNA, cosmid DNA, or bacteriophage DNA. In a preferred aspect, the host cell is a yeast cell.

5.3.1. EXPRESSION OF HEMOGLOBIN IN YEAST

Special considerations however have to be taken into account when expressing globin chains in yeast. Different signals regulating the expression of sequences encoding globin chains in yeast are required than when expressing such sequences in procaryotic systems or

mammalian systems. For example, a yeast replication origin is required in a recombinant DNA vector capable of

expressing a globin sequence in order for there to be replication of such a vector and thus significant

expression. The nucleotide sequence coding for the alphalike and/or beta-like chain of globin is inserted into a vector which may be expressed in yeast. In one embodiment, one DNA sequence encoding one globin chain or variant thereof is inserted into the recombinant DNA vector.

In a further embodiment, the yeast cell is a member of the species Saccharomyces cerevisiae. Such a vector comprises in addition to the DNA sequence encoding the globin: (a) a yeast transcriptional promoter which promotes the transcription of the DNA sequence encoding the globin chain; (b) a DNA sequence encoding a yeast

selectable marker or functionally active portion thereof; and (c) a yeast replication origin.

The first component of the vector, a yeast transcriptional promoter comprises two components: (a) a transcriptional regulatory region which contains a

structural gene distal region, or activator sequence which provides for regulated (inducible) or constitutive

transcription and (b) the transcriptional initiation region which includes the transcription initiation site, the

"TATA" sequence, capping sequence as appropriate, and an RNA polymerase binding sequence, which includes nucleotides upstream from the initiation site for directing the

initiation of synthesis of the messenger RNA. In a

preferred embodiment, the activator sequence is an upstream activator sequence. The transcriptional regulatory region will preferably be at least 100 base pairs (bp) and will not exceed 3000 base pairs. The regulatory region may begin at least about 200 bp from the initiation codon, usually at least about 300 bp and may begin at 400 bp or farther upstream from the initiation codon. The transcriptional initiation region will be at least about 150 bp, more usually at least about 200 bp, usually not more than about 600 bp, and preferably about 400 bp. The sequence may extend in the downstream direction of transcription from about bp -10 to about bp -25 (relative to transcription initiation at +1).

In one embodiment, the yeast transcriptional promoter is an inducible promoter. Inducible promoters may be unidirectional or bidirectional. Unidirectional

inducible promoters in a preferred embodiment are located upstream from the DNA sequence encoding the globin chain. Unidirectional inducible promoters may include but are not limited to promoters which are regulated by galactose (e.g. UDP-galactose epimerase (GAL10), galactokinase (GAL1)), glucose (e.g. alcohol dehydrogenase II (ADH2 ), and phosphate (e.g. acid phosphatase (PHO5)). In another embodiment, the inducible promoter may be a bidirectional promoter. A bidirectional promoter may be located upstream (5' of the ATG start codon) from the DNA sequence encoding a globin chain on the plus strand at one of its ends and upstream from the DNA sequence encoding a globin chain on the minus strand at its other end; and thereby, control the transcription of both. In a specific embodiment, such a bidirectional promoter is GAL1-10.

The promoter may also be a constitutive promoter. In a specific embodiment, the constitutive promoter is a promoter of glyceraldehyde-3-phosphate dehydrogenase III (TDH3) transcription and is herein after referred to as the TDH3 promoter. Other constitutive promoters include but are not limited to glyceraldehyde-3- phosphate dehydrogenase II (TDH2), glyceraldehyde-3- phosphate dehydrogenase I (TDH1) , alcohol dehydrogenase I (ADH1), phosphoglycerate kinase (PGK), pyruvate kinase (PYK) , enolase (ENO), and triose phosphate isomerase (TPI). Such promoter sequences will be at least about 200 bp and will not exceed about 5000 base pairs.

In another embodiment, the promoter can be a hybrid promoter, in which the sequence containing the transcriptional regulatory region is obtained from one source and the sequence containing the transcription initiation region is obtained from a second source. In one embodiment, the sequence containing the transcriptional regulatory region is an upstream activating sequence of a yeast inducible promoter. The inducible promoter can be a unidirectional or a bidirectional promoter. The sequence containing the transcriptional initiation region may be obtained from the transcriptional initiation region of a constitutive promoter. In a specific embodiment, the hybrid promoter comprises a transcriptional regulatory region which is the upstream activation sequence of the

GAL10 promoter and a transcription initiation region which contains the transcription initiation region of the TDH3 promoter. In another specific embodiment, the hybrid promoter can regulate the expression of two separate DNA sequences in opposite orientations if the hybrid promoter comprises an upstream activating sequence with

transcription initiation sites located on both sides, thereby forming a bidirectional promoter. In a very specific embodiment, a GAL1-10 upstream activating sequence may be flanked on either side by the initiation region of the TDH3 promoter. DNA encoding a globin chain is located downstream from each TDH3 sequence. In another embodiment, the ADH2 UAS may be used in place of the GAL1-10 UAS . In still other embodiments, the transcriptional initiation region of the TDH3 promoter can be substituted by TDH1 . TDH2 , PGK, ENO, TPI, CYC1, or PYK.

Another component of the recombinant DNA vector is a sequence encoding a yeast selectable marker. The recombinant DNA vector in one embodiment may contain more than one such sequence. A yeast selectable marker provides for selective pressure for survival of yeast cells expressing the marker. In a preferred aspect, the

selectable marker complements a genetic defect in the host strain. For example, URA3 can be used as a selectable marker in a yeast strain which is deficient in the URA3 gene product. Such sequences may include but are not limited to the LEU2 gene, the URA3 gene, the HIS3 gene, the LYS2 gene, the HIS4 gene, the APE8 gene, the CUP1 gene, and the TRP1 gene . Another example of such a sequence includes the leu2d gene which is a promoter defective LEU2 gene. In a preferred embodiment, the leu2d gene is inserted into a multicopy recombinant DNA vector. A yeast cell transformed by a vector comprising the LEU2 or leu2d gene may grow in leucine free media; a yeast cell transformed by a vector comprising the URA3 gene may grow in uracil free media; a yeast cell transformed by a vector comprising the LYS3 gene may grow in lysine free media, a yeast cell transformed by a vector comprising the HIS3 gene may grow in histidine free media, a yeast cell transformed by a vector comprising the ADE8 gene may grow in adenine free media, a yeast cell transformed by a vector comprising the HIS4 gene may grow in histidine free media, a yeast cell transformed by a vector comprising the CUP1 gene may grow in media

containing levels of copper inhibitory to the host strain without plasmid; and a yeast cell transformed by a vector comprising the TRP1 gene may grow in tryptophan free media. The recombinant DNA vector may also comprise a DNA sequence encoding a functionally active portion of a yeast

selectable marker. The term "functionally active portion" as defined herein is a portion of the sequence that encodes a portion of the marker which provides an effective amount of selective pressure for the survival of yeast cells expressing the portion of the marker.

The recombinant vector also comprises a yeast replication origin or functionally active portion of the replication origin which effects replication of the vector. Any replication origin useful in yeast may be employed which provides for efficient replication and maintenance (reviewed for example in Kingsman and Kingsman, U.S. Patent No. 4,615,974, issued October 7, 1986). Examples of such replication origins include but are not limited to the 2μ plasmid replication system, or a functionally active portion thereof and autonomous replicating sequences (ARS). Examples of ARS include but are not limited to ARS1 or ARS3. The replication origins may be of high or low copy number, depending on the effect of the construct on the viability of the host. The vector may further comprise centromeric sequences (CEN) which may provide meiotic and mitotic stability. Examples of CEN sequences include but are not limited to CEN3, CEN4, and CEN11.

The expression vector may further comprise but does not always require a transcription termination

sequence. A transcription termination sequence may include the necessary transcription signals for termination and polyadenylation and may be derived from any yeast sequence. In a specific embodiment, the transcription termination sequence is the alcohol dehydrogenase I (ADH1) termination sequence. Other termination sequences suitable for use include but are not limited to those of iso-1-cytochrome c (CYC1), UDP-glucose-4-epimerase (GAL10), phosphoglycerate kinase (PGK). acid phosphatase (PHO5), enolase (ENO), and triose phosphate isomerase (TPI). The transcription

termination sequence is at least about 100 bp and should not exceed about 1500 bp. In a preferred embodiment, the transcription termination sequence ranges from about 150 bp to about 1200 bp.

The expression vectors of the present

invention may be constructed using recombinant DNA

procedures known in the art. Such procedures were

disclosed in detail in Section 5.1., supra. Specific examples of yeast expression vectors and their

construction, comprising sequences encoding adult beta-globin under the control of the hybrid promoter containing the GAL1-10 promoter, and the TDH3 promoter, are disclosed in Section 19. A specific example of a yeast expression vector and its construction, comprising sequences encoding the adult alpha-globin chain under the control of a hybrid promoter are disclosed in Section 7. A specific example of a yeast expression vector and its construction, comprising sequences encoding the gamma-globin chain under the control of the GAL10 inducible promoter is disclosed in Section 8. Specific examples of globin variants are disclosed in

Section 11. A specific example of a yeast expression vector and its construction, comprising sequences encoding the zeta-globin chain under the control of a the GAL10 promoter is disclosed in Section 10. Specific examples of the expression of hemoglobin by coexpression of plasmids comprising sequences encoding alpha like- and beta like- globin are disclosed in Sections 12-18, 20-24, and 26-28.

Specific examples of the construction and expression of globin variants are disclosed in Sections 11 and 25.

The expression vectors of the present

invention may be propagated in yeast using procedures known in the art. The expression vectors may be propagated in yeast which may or may not be capable of producing heme. The yeast can be transformed with one or more of the expression vectors using procedures known in the art (e.g. the spheroplast method, (Hinnen et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933) or the lithium acetate method (Ito et al., 1983, J. Bact. 153:163-168) or through electroporation. Transformants may be selected by the presence of the marker (selectable) gene function in the transformant. For example, a leu2-yeast cell transformed with an expression vector comprising a LEU2 marker gene is selected by virtue of its ability to grow in leucine free media. The transformed yeast cells may be grown in media comprising a nitrogen and carbon source as well as

essential vitamins, minerals, and trace elements (Hinnen et alt, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933). If the vector comprises an inducible promoter, the media should also comprise the inducer.

If the expression vector comprises DNA

sequences encoding both an alpha-like globin chain and betalike globin chain or a beta-like globin chain (e.g. gammaglobin chain), hemoglobin may be expressed in the yeast cell transformed with the vector. In one embodiment, the heme is produced by the yeast and ligated to the globin to form functional hemoglobins in vivo. In another

embodiment, the yeast cell may be deficient in components required for heme production, for example 5-aminolevulinic acid. Hemoglobin may still be expressed in such a cell if the required component is added.

The protein product of the expressed globin gene may be isolated and purified using standard methods including but not limited to chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. If one globin chain is expressed, the expressed globin chain may be combined with another globin chain and a source of heme to form hemoglobin. If hemoglobin is expressed in the yeast cell, no further steps are necessary.

The expressed gene and its product may be analyzed at the genomic level or the protein level using procedures known in the art. For example, hemoglobin gene expression may be analyzed by Southern or Northern

hybridization. The expressed hemoglobin protein may for example be analyzed by Western Blot procedures known in the art and also described herein in Section 6.6., infra.

5.4. USES FOR EXPRESSED RECOMBINANT HEMOGLOBINS

Hemoglobin of large quantity and high purity may be obtained using the methods of the present invention. Examples of hemoglobin which may be obtained include but are not limited to HbA (alpha₂beta₂), HbA₂ (alpha₂delta₂),

HbF (alpha₂gamma₂), HbBarts (gamma₄) , HbH (beta₄), and Hb

Portland I (zeta₂gamma₂), Hb Portland II (zeta₂beta₂), Hb Portland III (zeta₂delta₂₎ Hb Gower I (zeta₂epsilon₂), and

Hb Gower II (alpha₂epsilon₂). The hemoglobin will be free of cellular material and other contaminants. Such

hemoglobins and especially hemoglobin variants which autopolymerize; variants which prevent the dissociation of the tetramer, variants with lowered intrinsic oxygen affinity, variants that are stable in alkali, variants that are stable in acid, variants which do not autooxidize, and/or variants which do not bind to haptoglobin through the use of variant alpha and/or beta-globin genes described in Section 5.2., supra are of value for use in blood substitutes.

In another embodiment, alpha-like and/or beta- like globin may be chemically modified using procedures known in the art to increase tetramer stability and/or lower oxygen affinity (see Section 2.1.2., supra for examples of such procedures). A wild-type or variant alpha- like or beta-like globin may be modified. Such chemically modified hemoglobins may also be used in blood substitutes.

The hemoglobin compositions, in addition to being used in blood substitutes, may be used in a blood plasma expander, in a pharmaceutical composition with an acceptable carrier, and with other plasma expanders, or in any application where a physiological oxygen carrier is needed. The pharmaceutical carriers may be such

physiologically compatible buffers as Hank's or Ringer's solution, physiological saline, a mixture consisting of saline and glucose, and heparinized sodium-citrate-citric acid-dextrose solution. The hemoglobin produced by the methods of the present invention can be mixed with

colloidal-like plasma substitutes and plasma expanders such as linear polysaccharides (e.g. dextran), hydroxyethyl starch, balanced fluid gelatin, and other plasma proteins. Additionally, the hemoglobin may be mixed with water soluble, physiologically acceptable, polymeric plasma substitutes, examples of which include polyvinyl alcohol, poly(ethylene oxide), polyvinylpyrrolidone, and ethylene oxide-polypropylene glycol condensates. Techniques and formulations for administering the compositions comprising the hemoglobin generally may be found in Remington's

Pharmaceutical Sciences. Meade Publishing Col., Easton, PA, latest edition.

The following examples are presented by way of illustration not by way of limitation. 6. EXAMPLE 1: EXPRESSION OF NATURAL BETA-GLOBIN IN A

YEAST EXPRESSION VECTOR CONTAINING GAL10 PROMOTER AND ADH1 TERMINATOR

The beta-globin gene from plasmid pSPβC was modified and cloned into the yeast expression vector, YEp51. ADH1-transcription termination sequences were placed at the end of the beta-globin gene in this plasmid. The modified plasmid was called YEp51T/NAT (for the natural beta-globin gene). Yeast strain Sc340 was transformed with plasmids YEp51T/NAT and YEp51 (control). Total RNA was isolated from yeast strain Sc340 transformed with YEp51 (340g2C), YEp51T/NAT (340g2B). Quantitation of RNA by scanning the autoradiograph showed that mRNA for the natural beta-globin is around 3.0% of total yeast RNA.

Western blot analysis indicated that natural beta-globin was expressed.

6.1. MATERIALS

The restriction enzymes, Klenow enzyme and T4-DNA ligase were obtained from New England Biolabs

(Biolabs), Bethesda Research Laboratories (BRL) or

Boerhinger Mannheim (BM). All enzymes were used according to the suppliers specifications. Plasmid DNA was isolated from a one liter culture of the transformed cells and purified by CsCl gradient centrifugation.

6.2. CLONING OF THE BETA-GLOBIN GENE INTO THE YEAST

EXPRESSION VECTOR YEp51

The general procedure used to clone the beta-globin gene into the yeast expression vector YEp51 is shown in Figure 3. The plasmid pSPβC (see Figure 2 for

restriction map of pSBβC) was digested with NcoI and

HindIII. Digestion with this combination of enzymes generated two fragments, a 600 base pair DNA containing the beta-globin gene and a 2700 bp fragment from the plasmid. The 600 bp fragment was isolated from a 0.6% agarose gel. After the band was excised from the gel, the DNA was electroeluted, and ethanol precipitated. The precipitated DNA was spun in an Eppendorf Centrifuge, the supernatant was removed and the DNA pellet was dried under vacuum.

The 600 bp fragment was modified by adapter addition before cloning into the plasmid YEp51. The DNA fragment carrying the beta-globin gene isolated from pSPBC was Ncol compatible at the 5'-end while the 3'-end was

HindIII compatible. These ends had to be modified so that they could be compatible with the restriction sites present in YEp51. To modify the 5'-end of the isolated fragment, a synthetic adapter was used. This adapter had a NcoI compatible end at its 3'-end and a SalI compatible end at its 5'-end (see Figure 2). The 3' -end of the isolated fragment did not receive any adapter as the HindIII site was compatible with the HindIII site introduced into the YEp51.

The recipient plasmid YEp51 was cleaved with SalI and HindIII restriction enzymes. To insert the isolated fragment containing the beta-globin gene, a three-way ligation was set up (see Figure 3) . The ligation reaction was carried out according to the standard ligation procedures (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The ligation mixture was

transformed into the E. coli HB101 cells using standard transformation procedure. Cells were spread on plates containing LB-media with 100 mg/L ampicillin. Plates were incubated overnight at 37°C. Forty eight colonies from the ampicillin plates were picked and a 5 ml culture was inoculated with individual transformants. Cultures were grown overnight at 37°C with vigorous shaking. The plasmid DNA was isolated from 1.5 ml of the overnight culture using quick plasmid isolation procedure. The plasmid from each transformant was digested with EcoRI to confirm the presence of a DNA fragment containing natural beta-globin gene. The plasmid carrying the natural beta-globin gene was called YEpWB51/NAT. The map of the plasmid YEpWB51/NAT is shown in Figure 4.

6.3. CLONING OF THE ADH1-TERMINATOR SEQUENCES

INTO YEPWB51/NAT

The strategy used to insert ADH1 terminator sequences into YEpWB51/NAT is shown in Figure 5. The plasmids YEpWB51/NATd- (d-=dam- and dcm-, i.e. methylation minus) was digested with restriction enzymes BelI and

HindIII. After the digestion, a 6.8 kb DNA fragment containing the beta-globin gene and vector was isolated from a 0.6% agarose gel (in IX TBE, 0.1 M Tris, pH 8.0, 0.09 M boric acid, 1 mM EDTA) . DNA was electroeluted from the gel slice and precipitated with ethanol at -20°C. The precipitated DNA was spun in an Eppendorf Centrifuge for 15 min and the pellet was dried under vacuum. The DNA was suspended in 20 μl H₂O.

The ADH1-transcription termination sequences were isolated from plasmid AAH5 (Ammerer, G., 1983, Methods in Enzymology, 101, pp. 192-201). AAH5 was obtained from Dr. Ben Hall at the University of Washington, Seattle. The plasmid AAH5 was digested with BamHI and HindIII (see

Figure 6 for a map of plasmid AAH5) . Digestion with this combination of enzymes generated three fragments. A 450 base pair (bp) DNA fragment containing the ADH1-transcription termination sequence was isolated from the 0.6% agarose gel. DNA was electroeluted from the gel slice and precipitated with ethanol at -20°C. The precipitated DNA was spun in an Eppendorf Centrifuge for 15 min and the pellet was dried under vacuum. The DNA was suspended in 20 μl H₂O. The DNA fragment carrying the ADH1- transcription terminator isolated from AAH5 was BamHI compatible at the 3'-end while the 5'-end was HindIII compatible. These ends were compatible with the

restriction sites present in YEpWB51/NAT.

The recipient plasmid YEpWB51/NATd^_ was cleaved with BclI and HindIII restriction enzymes. As shown in Figure 5, a two-way ligation was set up to insert the isolated fragment. The ligation mixture was transformed into E. coli HB101 cells using standard transformation procedures. Cells were spread on plates containing LB- media with 100 mg/L ampicillin. Plates were incubated overnight at 37°C. Twenty four colonies from the

ampicillin plates were picked and a 5 ml culture was inoculated with individual transformants. Cultures were grown overnight at 37°C with vigorous shaking. The plasmid DNA was isolated from 1.5 ml of the overnight culture using standard alkaline miniprep procedures (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The plasmid from each transformant was digested with PstI and HindIII restriction enzyme to confirm the presence of a DNA fragment containing the ADHl-terminator. The plasmid carrying the natural beta-globin gene with the ADH1-terminator was called YEp51T/NAT and is shown in Figure 7.

6.4. TRANSFORMATION OF YEAST STRAIN Sc340 WITH YEo51T/NAT

.The yeast strain Sc340 was obtained from Dr.

J.E. Hopper of Hershey Medical Center. The genotype of this strain is:

MATa ura3-52 , leu2, ade1, his3 : : GAL10uas-GAL4- URA3+ , MEL+ .

Sc340 was transformed with the plasmids

YEp51T/NAT and YEp51 (control). The spheroplast method of transformation was performed according to the published procedure (Hinnen et al., 1978, Proc. Natl. Acad. Sci.

U.S.A. 75:1929-1933). The transformants were selected by plating out on minimal media containing 0.67% Bacto yeast nitrogen base without amino acids, 2% glucose, 20 mg/L adenine sulfate, 20 mg/L histidine, and 20 mg/L uracil.

The plates were incubated at 28ºC for three days and were examined for colony formation.

Colonies were picked from these plates

following incubation and were precultured in yeast minimal media (0.67% yeast nitrogen base without amino acids) containing 0.5% glucose plus 20 mg/L each of adenine, uracil, and histidine. The overnight culture was then used to inoculate 1000 ml of the yeast minimal media containing 2% lactic acid, 3% glycerol and appropriate amino acids. The cultures were inoculated to OD₆₀₀ of 0.02. Cultures were grown at 30°C until they reached OD₆₀₀ of 0.20 (usually after 48 hours). Induction was initiated by the addition of galactose to a final concentration of 2% in the media.

After four hours, cultures were harvested by centrifugation and the pellet was washed with 150 mM NaCl. The pellet was divided into two parts. One part was used for RNA

isolation and the other was kept at -70°C for Western blot analysis. 6.5. QUANTITATION OF RNA FROM SC340 CELLS TRANSFORMED WITH

PLASMIDS YEp51 and YEp51T/NAT

RNA was isolated using published procedures (Meyhack et al., 1982, The EMBO Journal 1:675-680 or

Carlson and Botstein, 1982, Cell 28:145). Yeast cells were washed with 150 mM NaCl and the pellet was resuspended in RNA buffer (0.5 M NaCl, 0.2 M Tris-HCl, pH 7.6, 0.1 M EDTA and 1% SDS). Approximately 0.5 g of glass beads (0.45-0.5 mm) were added to the tubes. An equal volume of phenol mixture (phenol: chloroform: isoamyl alcohol 25:24:1, equilibrated with RNA buffer without SDS) was added. Yeast cells were broken by vortexing at maximum speed for 2.5 minutes and the sample was placed on ice for 3 minutes.

The above step was repeated twice more. Equal volumes of

RNA buffer and phenol mixture were added to the cells and tubes were centrifuged. Aqueous phase was transferred to a clean Corex tube and 2.5 volumes of ethanol were added to each tube. RNA was allowed to precipitate at -20°C for 4 to 6 hours. RNA was pelleted by centrifugation and dried under vacuum. RNA pellet was suspended in sterile water.

Total RNA was denatured using the glyoxal method (Thomas, P., 1983, in "Methods in Enzymology",

Colowhich, S. P. and Kaplan, N. O. eds. Vol.

100: pp. 255-266, Academic Press, New York). RNA was electrophoresed on 1.1% agarose gel in 10 mM NaPO₄ for approximately 4 hours at 75 volts (constant). After the electrophoresis was complete, RNA was transferred to

Amersham Hybond-N paper (Thomas, P., 1983, in "Methods in Enzymology" Colowhick, S. P. and Kaplan, N. O. eds. Vol. 100: pp. 255-266, Academic Press, New York).

Total yeast RNA bound to the filter paper was hybridized to the radioactive labelled beta-globin DNA. Hybridizations were carried out at 42°C overnight in 50% (v/v) formamide with 5X SSC (SSC : 3 .0 M NaCl, 0 .3 M Na citrate, pH 7.5); 50 mM NaPO₄, pH 6.5; 250 μg/ml salmon sperm DNA; and 1X Denhardt's solution; (Denhardt's

solution: 0.02% Ficoll, 0.02% polyvmylcarbonate, and 0.02% BSA, fraction V). The CYH2 mRNA which codes for yeast ribosomal protein L19 was used as control. The probe was plasmid mplOCYH22 which carries the yeast CYH2 gene. After the hybridizations, filters were washed three times at room temperature in 2X SSC and 0.1% SDS and four times at 50°C in 0.1X SSC and 0.1% SDS. Filters were exposed to X-ray films for 1 hour to overnight depending on the

radioactivity. X-ray films were developed in a Konica automated film developer.The results from these RNA blot hybridizations are shown in Figure 8. The results indicate that the mRNA samples from all sources were intact and no degradation was detected. It was also observed that no beta-globin mRNA could be detected in lane 1, which

contains the parent plasmid only. These results indicate that nonspecific hybridization of the beta-globin probe is minimal.

Autoradiographs containing bands corresponding to both beta-globin and CYH2 mRNA were scanned using the LKB gel scanner. Results obtained from the scanner are shown in Figure 9. It can be clearly seen that the

abundance of CYH2 mRNA in all three lanes is approximately the same while the abundance of the beta-globin mRNA was higher.

6.6.WESTERN BLOT ANALYSIS OF EXPRESSED BETA-GLOBIN

Four major steps were involved in the analysis of the expressed beta-globin:

(1) Sample preparation via yeast cell

disruption using glass beads followed by protein

solubilization using SDS-containing buffer.

(2) Extracted protein separation via

polyacrylamide gel electrophoresis.

(3) Protein transfer to nitrocellulose paper by application of a transverse electrical field.

(4) Globin protein detection via a three-stage antibody procedure. The primary antibody is specific for hemoglobin. The secondary antibody is a conjugate of biotin and antibody against IgG of the animal in which the first antibody was raised. Additionally, strepavidin conjugated to horseradish peroxidase was utilized.

The nitrocellulose membranes wee immersed in Enhanced Chemiluminescent developer ([ECL], Amersham) according to manufacturer's instructions and generated light detected by exposure to X-ray film for 15-60 seconds. Phosphate-buffered saline (PBS; 0.9 % NaCl (w/v), 0.01 M phosphate, pH 7.6) solution (2 ml) was added to thawed yeast samples (0.02 g wet weight). The samples were centrifuged at 4°C for 10 min. and decanted. Cold disruption buffer (50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0) prepared immediately before use (0.2 ml) was added, followed by enough glass beads to just reach the top surface of the liquid. After vortexing for 30 seconds at maximum speed the samples were placed on ice for 5 min.; this step was repeated twice more. Ice-cold disruption buffer (1 ml) was added to each sample and the homogenate was transferred to an Eppendorf tube. In another Eppendorf tube, 200 μl of homogenate was combined with 200 μl of freshly prepared standard discontinuous 2X sample buffer (Laemli, 1970, Nature 227:680-685) and the sample was boiled for 10 min.

After centrifugation for 10 min., the samples were loaded onto a discontinuous denaturing gel in which the stacking gel was 3.75% acrylamide and the separating gel was 12%-15%. The stacking gel was run at a constant current of 25 mA/cm² and the separating gel was run at a current of 33 mA/cm².

After the electrophoresis was complete and the dye band had reached the bottom of the separating gel, the gels were removed from the electrophoresis unit and the plates were pried apart under running deionized water. The stacking gel was discarded and the lower gel was separated from the plate. The transfer unit was filled with transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine, pH 8.30, in 10L distilled water), 2L of the transfer buffer was put into a shallow pan. The transfer sandwich

consisting in sequence of large pore gauze, 3M blotting paper, the gel, a piece of nitrocellulose paper precut to just cover the gel, 3M blotting paper, and another piece of large pore gauze was assembled under the buffer in the shallow pan.

Protein was then transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose sheet was removed and placed into a small, covered shallow pan with 50 ml blocking solution (200 g dried milk per liter PBS) and gently agitated for 1 hr. The blocking solution was discarded and the nitrocellulose was washed three times with PBS containing 0.1% Tween 20. The duration of the washes were 15, 5, and 5 minutes

respectively. After discard of the third wash, 25 μl of primary antibody in 25 ml of PBS was added to the pan and the sheet agitated for 2 hrs. The washing of the above was repeated and the nitrocellulose incubated, with agitation, for 1 hour in 5 μl of secondary antibody in 25 ml PBS. The washing procedure of above was carried out and then 2 μl of streptavidin-horseradish peroxidase conjugate in 25 ml of PBS containing 0.1% Tween 20 was added. Following a 20 minute incubation with agitation, the nitrocellulose was washed as above and the presence of human globin chains detected using enhanced chemiluminescence (ECL).

The nitrocellulose was immersed in ECL

developer reagents and incubated according to the

manufacturer's (Amersham) instructions. After incubation, the sheet was wrapped in clear polyethylene wrap and exposed to x-ray film for the appropriate length of time (10 to 60 seconds). The film was developed and then scanned with a laser densitometer. The quantity of globin in each sample was estimated using the hemoglobin

regression line. The standard was apo-human beta-globin purified from red blood cell lysate on reverse phase HPLC . The detection limit was less than 1 ng.

Total soluble protein was determined with the Bio-Rad Protein Assay Kit according to the manufacturer ' s instructions . Hemoglobin isolated from red blood cell lysate was used as a standard. Insoluble proteins were removed by centrifugation prior to analysis.

7. EXAMPLE 2: CLONING OF ALPHA-GLOBIN INTO A

YEAST EXPRESSION VECTOR

The alpha-globin gene was isolated using Polymerase Chain Reaction (PCR). The DNA Sequence of this alpha-globin gene was confirmed by sequencing. Results showed that the alpha-globin gene was complete without any deletions or mutations. The resulting plasmid was called pUT/2A.

7.1. MATERIALS

Restriction and DNA modifying enzymes were obtained from Boehringer-Mannheim, Bethesda Research

Laboratories, Perkin-Elmer or New England Biolabs. All enzymes were used according to the supplier's

specifications.

The E. coli strain used for all bacterial transformations was DH5α. The genotype of this strain is as follows:

F-_o80dlacZΔM15Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 (r_k- ,m_k ⁺) supE44λ- thi-1 gyrA relA1.

Oligonucleotides were synthesized on an

Applied Biosystems., DNA synthesizer 380B using cyanoethyl chemistry. The Polymerase Chain Reaction (PCR or PC reaction) was carried out in a Thermal Cycler obtained from Cetus, Inc.

7.2. CLONING OF THE ALPHA-GLOBIN IN A YEAST

EXPRESSION VECTOR 7.2.1. ISOLATION OF THE ALPHA-GLOBIN GENE

The alpha-globin gene was isolated by PCR from plasmid pJW101 (Wilson et al., 1978, Nucleic Acid Research 5: 563-580). The primers used for the PCR, 51-A-1 and 519-A-3, are shown in Figure 10 and are described in the

Sequence Description as SEQ ID NO: 7 and SEQ ID NO: 8

respectively. The PCR product was purified by

electrophoresis on 1.0 % agarose gel in IX TBE,

electroelution and ethanol precipitation. Purified PCR product was digested with restriction enzymes SalI and

BamHI. Digested DNA was cleaned by phenol extraction and ethanol precipitation.

7.2.2. PREPARATION OF THE YEAST EXPRESSION VECTOR

The yeast expression vector used to clone the alpha-globin gene was prepared by digesting plasmid

YEp51UT/NAT with SalI and BamHI YEp51UT/NAT was prepared in the following manner. Specifically, YEpWB51T/NAT was digested with the restriction enzyme KpnI. The linearized plasmid was treated with T4-DNA polymerase to make it a blunt-ended molecule. The URA3 gene was isolated as a 1300 bp SmaI-ClaI fragment from plasmid YEp24. This fragment was also treated with the T4-DNA polymerase to make it blunt-ended. This 1300 bp fragment containing the URA3 gene was ligated to the YEpWB5IT/NAT which was cleaved with KpnI and made blunt-ended. The ligation reaction was carried out according to published procedures (see Section 7.2.3., infra). The ligation mixture was transformed into the E. coli DH5α cells using standard transformation procedures (see Section 7.2.3., infra). The cells were spread on plates containing LB-media with 100 mg/l

ampicillin. Plates were incubated overnight at 37°C.

Twelve colonies from the Ampicillin plates were picked and a 5 ml culture was inoculated with individual transformant. Cultures were grown overnight at 37°C with vigorous shaking. The plasmid DNA was isolated from a 1.5 ml culture and the DNA was digested with EcoRI to confirm the presence of 1300 bp fragment. Twelve plasmids were analyzed for the insert and all of them had the 1300 bp fragment inserted in the plasmid.

A 7200 bp fragment containing the GAL10 promoter and ADH terminator was gel purified (0.6 % agarose gel in 1X TBE).

7.2.3. LIGATION AND TRANSFORMATION

YEpUT51/NAT digested with SalI and BamHI was ligated to purified PCR product described in Section

7.2.1., supra to obtain pUT/2A. The structure of pUT/2A is shown in Figure 11. DNA ligation was carried out using .standard ligation procedures (Ligation Reactions in

"Laboratory Cloning: A Laboratory Manual", Sambrook, J., Fritsch, E. F. and Maniatis, T. eds., Cold Spring Harbor Laboratory Press, 1989, Second Edition pp: 1.63 - 1.71) and E. coli transformation was also carried out using standard transformation procedures (Preparation and Transformation of Competent E. coli in "Laboratory Cloning: A Laboratory Manual" (Sambrook, J., Fritsch, E. F. and Maniatis, T.

eds., Cold Spring Harbor Laboratory Press, 1989, Second Edition pp. 1.74 - 1-84). Transformed cells were plated on LB-media with 100 mg/l ampicillin. Plates were incubated at 37°C overnight. Colonies appearing on these plates were used to inoculate 5.0 ml LB media with 100 mg/l ampicillin and cultures were grown at 37°C overnight. DNA isolated from these cultures was analyzed using restriction enzyme HindII.

7.2.4. DNA SEQUENCING

The reagent kit for DNA sequencing was

purchased from United States Biochemical Corporation (USBC) and it is based on the dideoxy method as developed by Sanger et al. (1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467). The radioisotope used comes with "Sequetide kit" (NEN Research Products). Sequetide is a ³⁵S-labeled nucleotide premix used during the labeling step of the sequencing reaction. The forward primer (GAL10KG) was synthesized on a 380B DNA synthesizer from Applied

Biosystem. The primer sequence was 5' CTT CTT TGC GTC CAT CCA 3' and is described in the Sequence Listing as SEQ ID NO :9. The 5' and the 3' ends of the primer were checked for optimal hybridization to ensure minimal non-specific annealing to the template using the HIBIO DNASIS program (Hitachi America, LTD). The sequencing gels were 6.0% and were prepared with Gel-Mix 6 (GIBCO BRL). The sequencing protocol was provided with the Sequetide S-labeled Premix (NEN Research Products).

The only deviations from this protocol were during the termination reaction. A supplement of 1 μl of a 1:14 dilution of the Sequenase 2.0 enzyme was used, and incubation was for ten minutes instead of five.

The sequencing gels were fixed in 2 liters fixing solution containing 10% acetic acid and 5% methanol, and were dried for 1 hour at 80°C in a slab dryer by Bio-Rad.

Sequences of the alpha-globin gene as read from the gels and was entered into the computer using the DNASIS program. The sequences of the new alpha-globin gene was compared to the sequence of natural alpha-globin gene (Wilson et al., 1978, Nucleic Acids Research 5:563-580).

Sequencing of the alpha-globin gene in plasmid pUT/2A showed two silent mutations. These silent mutations were both in the wobble position of the mRNA codon and they did not affect the translation of the globin protein. The first mutation was carried over from plasmid pJW101 which was used to create pUT/2A. The second mutation occurred within the plasmid and was two amino acids away from the first one. These mutations might have occurred either during the PCR or was present in the original gene.

8. EXAMPLE 3: EXPRESSION OF NATURAL GAMMA-GLOBIN IN A

YEAST EXPRESSION VECTOR CONTAINING GAL10 PROMOTER

AND ADH1 TERMINATOR

The gamma-globin gene was obtained from plasmid pJW151 using PCR. The gamma-globin gene was modified by PCR to have a SalI site at the 5'-end and a HindIII site at the 3'-end. The modified gamma-globin gene was cloned into the yeast expression vector YEp5IT/NAT, which contains the ADH1 transcription termination sequence, the GAL10 promoter, and the DNA sequence encoding the beta- globin gene. YEp51T/NAT had been cut with SalI and HindIII to remove the beta-globin gene. The plasmid containing the gamma-globin gene was called YEp51T/G. Yeast strain Sc340 was transformed with YEp51T/G and the transformant was called 340g2G. Following growth of 340g2G and induction by galactose, expressed proteins were analyzed by Western blot analysis. The results from Western blot analysis indicated that gamma-globin was expressed.

8.1. MATERIALS

Laboratories, Perkin-Elmer or New England Biolabs. All enzymes were used according to the suppliers

specifications.

The E. coli strain used for all bacterial transformations was DH5α.

Oligonucleotides were synthesized on the

Applied Biosystem Inc.'s DNA synthesizer 380B using

Cyanoethyl chemistry. Polymerase Chain Reaction (PCR or PC reaction) was carried out in a DNA thermal cycler obtained from Cetus, Inc. 8.2. CLONING OF THE GAMMA-GLOBIN GENE INTO THE YEAST EXPRESSION VECTOR YEp5IT/NAT

The general procedure used to clone the gamma-globin gene into the yeast expression vector YEp51T/NAT (see Section 6, supra) resulting in the construction of YEp51T/G is shown in Figure 12.

The gamma-globin gene was synthesized by PCR using appropriate primers and plasmid pJW151 DNA (Wilson, J.T., et al., Nucleic Acid Research 5:563-581, 1978) as template. The sequence of gamma-globin DNA is shown in Figure 13, and is described as SEQ ID NO: 10. The 5' and 3' primers used for synthesizing the gene, GAM-5-S and GAM-3-H respectively, are shown in Figure 14 and are described as SEQ ID NO: 11 and SEQ ID NO: 12. The PCR product was

analyzed by electrophoresis in a 1.5% agarose gel (in IX TBE). The 530 bp PCR product was removed from the gel by electroelution and the DNA was precipitated with ethanol. The purified PCR product was then digested with restriction enzymes SalI (5'-end) and HindIII (3'-end). The digested PCR product was phenol extracted and ethanol precipitated.

Plasmid YEp51T/NAT which contains the human beta-globin gene was digested with SalI and HindIII to remove the beta-globin gene. The digested plasmid was electrophoresed in 0.6% agarose gel (in IX TBE). A 7000 bp fragment was electroeluted and ethanol precipitated.

A ligation reaction mixture was set up between the gamma-globin obtained by digestion of the PCR product described above and YEp51T/NAT cut with Sail and HindIII (7000 bp). The ligation mixture was used to transform E. coli DH5GC cells using standard transformation procedure and plated on LB plates containing Ampicillin (100 mg/L).

Plasmid DNA was isolated from 20 clones and digested with restriction enzyme Pstl. The resulting plasmid was called YEp51T/G (Figure 15). 8.3. TRANSFORMATION AND GROWTH OF YEAST STRAIN Sc340 CELLS WITH PLASMID YEP51T/G

Yeast strain Sc340 cells were transformed with plasmid YEp51T/G (Rose, et al., 1989, Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring

Harbor, N.Y., pp. 112-115). The starter culture was grown in SD supplemented with adenine and histidine, and 3% glycerol and 2% lactate as carbon source. The preculture was used to inoculate 2 L of the above media in a Braun Biostat E fermentor. The pH was maintained at 5.5 using a 5% ammonium hydroxide solution. The pO₂ was maintained at

80% until the culture was induced with galactose at which point it was lowered to 10% . The stirrer speed was set at 500 rpm and then reduced to 100 rpm at galactose induction. The culture was incubated at 30°C and grown to an O.D. 600 of 30.4 at which time it was induced with galactose added at the rate of 5g/L/hour. Samples were collected from 0 to 74 hours after induction for globin analysis.

8.4. WESTERN BLOT ANALYSTS OF EXPRESSED GAMMA-GLOBIN

The expressed gamma-globin was quantitated by Western Blot analysis using procedures described in Section 6.6., supra. The results indicated that up to 0.05% of the total yeast protein in yeast cell line 340g2G was gamma-globin.

9. EXAMPLE 4. CLONING OF EPSILON GLOBIN cDNA IN A

YEAST EXPRESSION VECTOR AND EXPRESSION OF EPSILON GLOBIN IN YEAST

9.1 MATERIALS

Laboratories, New England Biolabs or Perkin-Elmer. All enzymes were used according to the supplier's

specifications.

Oligonucleotides were synthesized on the

Applied Biosystem Inc.'s DNA synthesizer 380B using

cyanoethyl chemistry. PCR was carried out using DNA thermal cycler obtained from Cetus and according to the methods described by Cetus.

The genomic clone for human epsilon gene pNEVll was obtained from the Beatson Institute for Cancer Research. The recombinant bacteriophage clones containing beta-type globin genes and flanking sequences (Fritsch et al., 1980, Cell 19:959-972) were used by scientists at the Beatson Cancer Research Institute. The EcoRI fragment containing epsilon genomic sequences from one of these clones was recloned in pBR322 based plasmid (Montague, Ph.D. Thesis entitled "The Behaviour of Human Globin Gene Recombinants in Mammalian Cells"). This plasmid was labeled pNEVll. The DNA from plasmid pNEV11 was isolated and used as a template for PCR.

The E. coli strains DH5α and NM522 (Invitrogen, Inc.) were used for bacterial transformations,

9.2. SYNTHESIS OF EPSILON GLOBIN cDNA

Figure 16 shows the structure of the epsilon globin genome, as well as the strategy used for the

synthesis of epsilon globin cDNA. The epsilon gene

contains three exons and two introns. The first 92 base exon (exon A) is separated from the second exon (exon B) by 121 bases intron. The second exon is 221 bases and the third exon (exon C) is 120 bases. The second and third exons are separated by 855 bases of intron.

The six primers used for the synthesis of epsilon globin cDNA, 5EPSL, INPE-1, INPE-2, INPE-3, INPE-4, and 3-EPH, are described in the Sequence Description as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, and SEQ ID NO: 18, respectively, and are also shown in Figure 17 with pertinent restriction sites. As shown in Figure 16, 5EPSL contains the 5' sequences of epsilon cDNA, INPE-1 contains a 23 base sequences at the 3' end of the exon A joined to a 15 base sequences at 5' end of the exon 2, INPE-2 contains the complementary sequences present in INPE-1, INPE-3 contains 3' sequences at exon B joined together with 5* sequences of exon C, INPE-4 contains complementary sequences of INPE-3, and 3-EPH contains 3' end sequences complementary to the coding strand of exon 3 with HindIII site at the 3' end.

The genomic clone was PCRed using two outside primers, 5EPSL and 3-EPH. The entire genomic DNA fragment containing 2 kb fragment was obtained with these two primers. This confirmed that the plasmid pNEV11 contains epsilon-globin genomic sequences.

In one reaction, all six primers were used with the template DNA. This reaction was carried out at 45°C, using 35 cycles. Equal concentrations of all primers were used in this reaction. The entire epsilon-cDNA (440 bp) was isolated using this method. The cDNA also

contained appropriate cloning sites.

9.3. CLONING OF EPSILON GLOBIN cDNA WITH YEAST

EXPRESSION VECTOR YEo51NTl

The epsilon globin cDNA was cut with Sall/HindIII. Ligation was set between Sall/HindIII cut YEp5iNTl (see Section 11.4., infra for a description of the construction of YEp51NTl). The ligation mixture was transformed in competent E. coli NM522 cells and the DNA was isolated from 24 transformants by alkaline digestion. The DNA samples from the clones were analyzed by

SalI/HindIII enzymes and the resulting plasmid was labeled

YEp51T/ε3. The map of this plasmid is shown in Figure 18. 9.4. TRANSFORMATION OF YEAST STRAIN Sc1041 WITH pYEp51T/ε3

Yeast strain Scl041 was transformed with plasmid pYEp51T/ε3 (supra, 9.3.) using electroporation. BioRad (Richmond, CA) Gene Pulser with Pulse Controller was used for electroporation. The 0.2-cm cuvettes were obtained from Bio Rad. 40 μl of yeast cells were

transferred to a sterile Eppendorf tube. DNA (1-100 ng) in 5 μl TE was added to the cells. The mixture was incubated on ice for 5 min., transferred to a 0.2 cm cuvette, and pulsed at 1.5 kV, 25 uF, 200 ohms for 5 msec. 250 μl cold IM sorbitol was immediately added to the cuvette, the contents were gently mixed and the cells were plated on appropriate plates.

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final

concentration of 2%. At induction, the pH was adjusted to 6.91 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between two and 24 hours after induction.

9.5. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section

6.6, supra. Samples taken after induction had detectable levels of globin (0.004%). 10. EXAMPLE 5: CLONING OF ZETA GLOBIN cDNA INTO A YEAST EXPRESSION VECTOR AND EXPRESSION OF ZETA GLOBIN IN YEAST

10.1. MATERIALS

specifications.

Oligonucleotides were synthesized on the

Applied Biosystem Inc.'s DNA synthesizer 380B using

The plasmid p4-7-7 containing zeta globin cDNA was obtained from Dr. Forget's laboratory (Cohen-Solal et al., 1982, DNA 1:255). The yeast expression vector pYES2 was obtained from Invitrogen Corp. (San Diego, CA). The vector contains the GAL1 portion of the divergent

GAL1/GAL10 promoter region, polylinker for cloning genes, the CYC1 transcription terminator and the URA3 gene for selection in yeast.

E. coli strain NM522 was obtained from

Invitrogen. Competent cells were prepared according to the protocol provided by Invitrogen.

10.2. CLONING OF ZETA GLOBIN CDNA IN YEAST

EXPRESSION VECTOR pYES2

The zeta globin cDNA was PCRed using appropriate primers. These primers, 5ZETASAC and

ZETA3HSLS, are described in the Sequence Description as SEQ ID NO: 19 and SEQ ID NO:20, and are shown in Figure 19 with restriction sites. The pcred DNA was cut with SacI/SphI and cloned into SacI/SphI cut DNA from plasmid pYES2. The DNA was isolated from 24 transformants by alkaline

digestion. The DNA samples from the clones were analyzed by restriction digestion with SacI/SphI. The plasmid containing zeta globin cDNA was labeled pYES2-ζ2 and is shown in Figure 20.

10.3.TRANSFORMATION OF YEAST STRAIN Sol 041 WITH pYES2-ζ2

Yeast strain Scl041 was transformed with plasmid pYES2-ζ2 (supra, Section 10.2) using

electroporation (see Section 9.4., supra).

concentration of 2%. At induction, the pH was adjusted to 7.00 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between two and 48 hours after induction.

10.4. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Samples taken after induction had detectable levels of globin (0.13%).

11. EXAMPLE 6: EXPRESSION OF VARIANT GLOBINS

The mutant globin genes were cloned into yeast expression vector YEp51NT1. This vector contains GAL10 promoter and ADH terminator sequences. The following mutant genes were cloned into this yeast expression vector: i. β-Motown (127 Gln->Glu)

ii. α-Titusville (94 Asp->Asn)

iii. γ-Porto Alegre (9 Ala->Cys)

iv. β-Mississippi (44 Ser->Cys)

v. ζ-104 Ser (104 Cys->Ser)

vi. α-Titusville/104S (94 Asp->Asn)

(104 Cys->Ser)

vii. γ-Motown (127 Gln->Glu)

viii. β-BovII (Met Leu Thr Ala Glu Glu, .) ix. β-Deer Lodge (2 His->Arg)

x. β-Abruzzo (143 His->Arg)

xi. β-McKees Rock (145 Term)

xii. γ-Chico (66 Lys->Thr).

xiii. ζ-Titusville (94 Asp->Asn)

11.1. MATERIALS AND METHODS

specifications.

Oligonucleotides used in the Polymerase Chain Reaction (PCR) were obtained by chemical synthesis on

Applied Biosystems 380B DNA synthesizer.

The E. coli strain used for all bacterial transformations was DHSα. 11.1.1. DNA FRAGMENT ISOLATION

All DNA fragments were separated on a 0.6% or 1.0% agarose gels (IX TBE) and isolated by electroelution using Pharmacia Electroeluter.

11.1.2. DNA LIGATION AND E. COLI TRANSFORMATION All DNA ligations were carried out using standard ligation procedures ("Laboratory Cloning: A

Laboratory Manual"; Sambrook, J., Fritsch, E. F. and

Maniatis, T. eds. Cold Spring Harbor Laboratory Press, 1989, Second Edition pp: 1.631.71) and E. coli

transformation was carried out using standard

transformation procedures ("Laboratory Cloning: A

Laboratory Manual"; Sambrook, J., Fritsch, E. F. and

Maniatis, T. eds.; Cold Spring Harbor Laboratory Press 1989, Second Edition pp: 1.74 - 1.84). Transformed cells were plated on LB-media with 100 mg/L ampicillin. Plates were incubated at 37°C overnight. Colonies appearing on these plates were used to inoculate 5.0 ml LB media with 100 mg/L ampicillin and cultures were grown at 37°C overnight.

11.1.3. PLASMID DNA ANALYSIS

DNA was isolated from 1.5 ml of the overnight culture using alkaline lysis procedure. Plasmid DNA was analyzed by appropriate restriction enzyme digestion.

11.1.4. YEAST TRANSFORMATION

Yeast transformation was done using published procedures (Hinnen, A., Hicks, J. B. and Fink, G. R. (1978) Transformation in Yeast, Proc. Natl. Acad. Sci. U.S.A. 75, 1929-1933).

11.2. SYNTHESIS OF OLIGONUCLEOTIDES

Various oligonucleotides were synthesized as a preliminary step in the construction of several globin gene variants. The oligonucleotides to be used in the in vitro mutagenesis procedure with M13 were synthesized and purified. Polyacrylamide gel electrophoresis following kinasing demonstrated that the synthesis was efficient and that the oligonucleotides were ready for use in the M13 system.

The following oligonucleotides, Mu-145Cy, Mu- 66Th, and Mu-9Cy were synthesized on the Applied Biosystems DNA synthesizer and are described respectively in the

Sequence Description as SEQ ID NO:21, .SEQ ID NO:22, and SEQ ID NO:23. The bold print within the sequence indicates a change from the wild type beta-globin gene sequence . (See Figure 21). Following synthesis and incubation at 65°C, the oligonucleotides were purified using oligonucleotide purification columns (ABI). The purified oligonucleotides were lyophilized and suspended in 100 μl water and the concentration was determined by 0D260.

Approximately 100 ng of the synthetic DNA was used in a kinasing reaction to determine the efficiency of the synthesis. The [γ-³²P]ATP kinased oligonucleotides were analyzed on a 6% acrylamide sequencing gel containing 7M urea. The dye used in this electrophoresis was a mixture of bromphenol blue and xyno-cynol which separate during the procedure, with each dye migrating at different rates .

Autoradiography was performed following drying of the gel.

Following autoradiography of the sequencing gel, the results indicated that the synthesis was

efficient, as the majority of the radioactivity was

incorporated into the larger bands that moved between the two dye fronts. Under the electrophoresis conditions described above, fragments that are approximately 25 bases should migrate with the bromphenol blue dye front, while those of about 90 bases should migrate with the xyno-cynol dye front. The synthetic oligonucleotides ranged in size from 30 to 45 bases which should run between the two dye fronts as was observed.

11.3. IN VITRO MUTAGENESIS

The in vitro mutagenesis kit from Bio Rad provides the necessary components for mutagenesis with the M13 system. Included in this kit are two strains of E. coli to be used in the process. E. coli strain CJ236 contains mutations which result in the incorporation of uracil instead of thymine in DNA. E. coli strain MV1190 is a wild type strain that is used to produce the single stranded DNA following mutagenesis.

11.3.1. STRAINS

The E. coli strains that were received in the mutagenesis kit were subcultured on appropriate media according to the genetic markers for selection. The constituents of each type of media as well as a suggested protocol for mutagenesis may be found in the brochure that was received with the kit (New England BioLabs, "M13

Cloning and Sequencing System - A Laboratory Manual").

11.3.2. TRANSFECTION OF CJ236

CJ236 competent cells for use in transfection were prepared by inoculating 100 ml LB broth containing chloramphenicol with 5 ml of an overnight culture of CJ236. The culture was incubated at 37°C in an air shaker until the OD₆₀₀ reached 0.8. The cells were centrifuged at 3K rpm for 5 minutes, resuspended in 20 ml 50 mM cold CaCl₂, and held on ice for 30 minutes. The cells were centrifuged again and resuspended in 4 ml 50 mM CaCl₂.

The CJ236 competent cells were transfected with M13mp19BHS by adding 1 μl or 5 μl of DNA to 0.3 ml competent cells. The tubes were held on ice for 40

minutes, heat shocked at 42°C for 3 minutes and the contents were added to 4 ml of top agar (50°C) containing chloramphenicol and 300 μl of the overnight culture of CJ236. This top agar was poured onto H-medium plates containing chloramphenicol and incubated overnight at

37°C. The phage was isolated (from those cells which were infected) by touching a toothpick to plaques and

suspending in 0.5 ml TE.

11.3.3. ISOLATION OF URACIL CONTAINING DNA

Uracil containing DNA was isolated from CJ236 by inoculating 50 ml LB medium containing chloramphenicol with 1.0 ml of an overnight culture of CJ236. The culture was incubated at 37°C with shaking until it reached an OD₆₀₀ of 0.3. At this point, the culture was infected with 50 μl of a -70°C stock culture that was previously infected with phage in order to amplify the production of single stranded DNA. The infected culture was allowed to grow overnight at these conditions. The following day, 30 ml of the culture was centrifuged at 16K rpm for 15 minutes. The supernatant containing the phage particles was transferred to a new tube and centrifuged a second time. The supernatant from this second centrifugation was treated with 150 μg RNase A at room temperature for 30 minutes. Single stranded DNA was precipitated by adding 7.5 ml of PEG solution (3.5 M ammonium acetate, 20% PEG 8000) and held on ice for 30 minutes. The tube was centrifuged and the supernatant was discarded. The pellet was suspended in 200 μl of high salt buffer (300 mM NaCl, 100 mM Tris, pH 8.0, 1 mM EDTA), held on ice for 30 minutes, and centrifuged in a

microcentrifuge for 2 minutes. The supernatant was

transferred to a new tube.

The phage was titered on CJ236 and MV1190 to determine whether infection was productive. Following confirmation of productive infection, the DNA was

extracted with an equal volume of phenol, an equal volume of phenol-chloroform, and an equal volume of ether. The extracted DNA was precipitated with 1/10 volume 7.8 M ammonium acetate and 2.5 volumes ethanol at -20°C

overnight. The tube was centrifuged for 15 minutes and the pellet was resuspended in 20 μl TE . This DNA is the single stranded uracil-containing DNA which was used as a template for the synthesis of the mutagenic strand.

11.3.4. KINASING OF OLIGONUCLEOTIDES

The purified oligonucleotides were kinased by treating 5 μg of each of the six oligonucleotides with T4 polynucleotide kinase and ATP to ensure efficient ligation of the two ends of the newly synthesized DNA strand.

11.3.5. SYNTHESIS OF THE MUTAGENIC STRAND

The synthesis of the mutagenic strand was carried out by adding 0.25 μg (0.1 pM) of the

uracil-containing single stranded DNA template and 0.03 μg

(3 pM) of each of the synthetic oligonucleotide primers. The primer was annealed to the single stranded template

(final reaction volume 10 ml) in 1X annealing buffer (2 mM Tris-HCl, pH 7.4, 0.2 mM MgCl₂, 5 mM NaCl) in a water bath with an initial temperature of 70°C which was allowed to cool to 30°C. The reactions were then placed in an ice water bath and the following components were added to each: 1 μl 10X synthesis buffer (Final concentration = 0.4 mM each dNTP, 0.75 mM ATP, 17.5 mM Tris-HCl, pH 7.4, 3.75 mM MgCl₂, 21.5 mM DTT), 1 μl T4 DNA Ligase (2-5 units), and

1 μl T4 DNA Polymerase (1 unit). The reactions were incubated on ice for 5 minutes in order to stabilize the primer by initiation of DNA synthesis under conditions that favor the binding of the primer to the template. The reactions were then incubated at 25°C for 5 minutes and finally at 37°C for 90 minutes. Following the final incubation, 90 μl of stop buffer (10 mM Tris, pH 8.0, 10 mM EDTA) was added to each reaction and were placed at -20°C until use in the transfection of MV1190.

11.3.6. TRANSFECTION OF MV1190 CELLS

MV1190 cells were transfected with the

products of the synthesis reactions by adding 3 μl and 9 μl of each reaction to 0.3 ml of competent cells. The tubes were incubated on ice for 90 minutes, heat shocked at 42°C for 3 minutes, and then placed on ice. 50 and 100 μl of the transfected cells were added to tubes containing 0.3 ml of an overnight culture of MV1190, 50 μl 2% X-gal, 20 μl 100 mM IPTG, and 2.5 ml top agar (55°C). The mixture was vortexed and poured onto H-agar plates. The plates were incubated overnight at 37°C and observed for the formation of plaques the following morning. Those plaques that appeared blue did not contain the insert, while those that appeared clear were the plaques of interest.

11.3,7. ANALYSIS OF TRANSFORMANTS BY SEQUENCING

The clear plaques were picked by inserting a sterile Pasteur pipet into the agar and suspending the plug in 3 μl LB broth (24 plaques were chosen from each of the plates containing plaques). 100 μl of an overnight culture of MV1190 was added and the tubes were incubated with shaking overnight at 37°C. Following the incubation period, single-stranded DNA was isolated from the cultures and this DNA was used in sequencing reactions.

Dideoxy sequencing was performed to confirm the presence of mutations. The sequencing kit used in this case was obtained from New England Biolabs. Each sequencing reaction was set up using 8 μl of the single stranded DNA to be sequenced, 1 μl of the appropriate primer, and 1 μl 10X sequencing buffer. The primer was annealed to the single stranded template by placing the tubes at 90°C and allowing them to cool to 30°C. 2 μl of the DNA-primer mixture was used in each individual sequencing reaction along with 2 μl of the termination mix (50 μl of the appropriate dNTP's and ddNTP plus 5 μl [α-³²P] dATP) and 2 μl of Klenow enzyme diluted to 0.1 units/μl. The reaction was incubated at room temperature for 15 minutes and 2 μl of a chase mixture was added that consisted of a dNTP mixture containing cold dATP and Klenow enzyme. This reaction was incubated again at room temperature for 15 minutes and 4 μl of dye mix was added to stop the reaction. The samples were denatured by boiling for 2.5 minutes and, placed in an ice water bath, and loaded onto a 6%

polyacrylamide sequencing gel containing 7M urea. The gel was run at 55 watts for approximately 4 hours before it was dried under vacuum and placed in an X-ray film cassette for autoradiography.

Other sequencing kits were used to achieve the best results in conjunction with [α³⁵S] dATP. A

sequencing kit specifically for use with single-stranded DNA was obtained from IBI and a Pharmacia kit was used with T7 DNA polymerase rather than Klenow Enzyme in order to sequence mutants further from the point of primer

annealing.

Transfection of MV1190 with the synthesis reaction products resulted in clear plaques on the plates containing Mu-66Th, Mu-145Cy, and Mu9-Cy DNA._. The control which was included consisted of a transfection with

template DNA without a primer for synthesis of the second strand. This control revealed some plaques, but fewer than those in which a primer for mutagenesis was used.

11.4. CONSTRUCTION OF PLASMID YEp51NT1

Yeast shuttle vector YEp51 was modified to have ADH terminator sequences. The ADH terminator was inserted between the GAL10 promoter and the 2μ present on this vector. Specifically, plasmid YEp51 was digested with restriction enzyme Bcl1. The linearized DNA molecule was treated with Klenow enzyme and dNTPs to make it blunt ended. A double-stranded oligonucleotide was ligated to the blunt ended plasmid. This oligonucleotide was obtained from BRL and contained sequences for restriction enzyme

NotI. Ligation was carried out overnight at room

temperature. DNA from the ligation reaction was

precipitated using polyethylene glycol (PEG). This procedure removes all unligated oligonucleotides because only large DNA molecules are precipitated with PEG. After the PEG precipitation, DNA was cleaned by phenol extraction and ethanol precipitation. Plasmid DNA was digested with Notl and HindIII.

The ADH terminator was obtained from plasmid AAH5 (see Figure 6). Plasmid AAH5 was digested with restriction enzyme BamHI. DNA was blunt-ended with Klenow and dNTPs. Blunt-ended DNA was subjected to phenol

extraction and ethanol precipitation. The above-mentioned double-straiided oligonucleotide was ligated to the blunt- ended plasmid. Ligation was carried out overnight at room termperature. DNA from the ligation was precipitated using polyethylene glycol (PEG). After the PEG precipitation, DNA was cleaned by phenol extraction and ethanol

precipitation. DNA was then digested with restriction enzyme NotI and HindIII. A 400 bp NotI-HindIII fragment was isolated from a 1.0% agarose gel (IX TBE). DNA was electroeluted from the agarose slice and precipitated with ethanol. This purified DNA fragment was ligated to the above-mentioned plasmid. The ligation mixture was used to transform DH5α-cells. Transformed cells were spread on plates containing LB-media with 100 mg/L ampicillin.

Plates were incubated overnight at 37°C. Colonies

appearing on these plates were used to inoculate 5.0 ml LB-media containing 100 mg/L ampicillin . Cultures were grown at 37°C overnight. DNA was isolated from 1.5 ml of the overnight culture using the alkaline lysis procedure.

Plasmid DNA was digested with restriction enzyme NotI and HindIII. The resulting plasmid was called YEp51NTl and is shown in Figure 22 .

11.5. CLONING OF VARIANT GLOBINS

The vector for cloning the mutated β-globin gene(s) was prepared by digesting plasmids YEP51T/G (supra, Section 8.4) or YEp51NTl/γ-P0RT (infra, 11.5.1.) with SalI and HindIII. The vector for cloning the γ-globin gene was

YEp51NT1. This, digestion results in two fragments (7300 and 500 bp); the 7300 bp fragment was isolated.

11.5.1. CLONING OF PORTO ALEGRE (9 Ala->Cys. γ-GLOBIN GENE

The Porto Alegre γ-globin was created by substituting two bases in the natural γ-globin sequence using PCR. The γ-globin gene was obtained as a 450 bp fragment. The 5' and 3' primers used for synthesizing the sequence, respectively, G-5-9CY and GAM-3-H are shown in Figure 23 and are described in the Sequence Description as SEQ ID NO: 22 and SEQ ID NO: 12 respectively.

The mutated γ-globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector YEp51NTl cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (see Section 11.1., supra) . DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed clones that had expected fragments (three fragments when digested with PstI; two fragments from vector without insert). This plasmid was called YEp51NT1/γ-PORT. 11.5.2. CLONING OF THE MOTOWN (127 Gln->Glu) β-GLOBIN GENE

The Motown β-globin was created by a base substitution using PCR. The globin gene was isolated as two fragments. The 3'-end of the gene (EcoRI-HindIII) was obtained by PCR. The 5' and 3' primers used for

synthesizing the sequence, B-G127-5 and Beta-3-H are shown in Figure 24 and are shown in the Sequence Description as SEQ ID NO:25 and SEQ ID NO:26.

The mutated fragment of the β-globin gene obtained by PCR was digested with EcoRI and HindIII. This digested DNA fragment (80 bp) was purified by phenol extraction and ethanol precipitation. The 5'-end of the β-globin gene was isolated from plasmid YEp51T/NAT (supra.

6.4.). Plasmid YEp51T/NAT was digested with restriction enzymes BamHI and HindIII. A 360 bp fragment was isolated.

This purified 360 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NT1/γ-Port cut with SalI and HindIII. DNA ligation, E. coli

transformation and DNA isolation was performed as described (supra, 11.1). DNA isolated from the transformed cells was digested with restriction enzyme PstI. The results

obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when

digested with PstI; three fragments from vector without insert). This plasmid was called pNT1/β-Mot.

11 .5 .3 . CLONING OF THE TITUSVILLE (94 Asp->Asn)

α-GLOBIN GENE The Titusville α-globin was created by

substituting one base in the natural α-globin gene using

PCR. The α-globin gene was isolated as two fragments. The

3'-end of the gene (HindIII-HindIII) was obtained by PCR using plasmid pl9AlGT as template. The 5' primer and 3' primers used for PCR, A-Tit-5 and G10T3H, are shown in

Figure 25 and are described in the Sequence Description as

SEQ ID NO: 27 and SEQ ID NO: 28.

The 5'-end of the gene (SalI-HindIII) was obtained as a 450 bp fragment. Primers used for PCR were

51-A3-S.L (5'-end primer) and A-Hin3-3 (3'-end primer) and are described in the Sequence Description as SEQ ID NO: 29 and SEQ ID NO: 30 respectively and are shown in Figure 26.

The template for the PCR was plasmid pJW101. The PCR product was digested with restriction enzymes Sail and

HindIII. A 300 bp fragment was isolated. This purified 300 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NTl/γ-PORT (See Section 11.5.1., supra, cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (supra, 11.1). DNA isolated from the transformed cells was digested with restriction enzyme HindII. The results obtained from this analysis showed that one clone had expected fragments (six fragments when digested with

HindII). This plasmid was called pNT1/2ATit.

11.5.4. CLONING OF THE β-MISSISSIPPI (44 Ser->Cys)

β-GLQBIN GENE

The Mississippi β-globin was created by substituting two bases in the natural β-globin gene using

PCR. The globin gene was isolated as two fragments. The 3 '-end of the gene (AccI-HindIII) was obtained by PCR. Primers used for PCR were B-44C-5 (5'-end primer) and Beta- 3-H (3'-end primer) and are shown in Figure 27 with restriction sites and are described respectively in the Sequence Description as SEQ ID NO:31 and SEQ ID NO:26.

The mutated fragment of the β-globin gene was digested with Accl and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol

precipitation. The 5'-end of the β-globin gene was

isolated from plasmid YEp51T/NAT (see Section 6.4., supra). Plasmid YEp5IT/NAT was digested with restriction enzymes Accl and SalI. A 117 bp fragment was isolated. This purified 117 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NT1/γ-PORT. DNA ligation, E. coli transformation and DNA isolation was performed as described (see Section 11.1., supra). DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with Pstl; three fragments from vector without insert). This plasmid was called pNT1/β-Miss.

11.5.5.CLONING OF 104-Ser (104 Cys->Ser) ALPHA-GLOBIN GENE

The 104-Ser alpha-globin was created by

substituting one base in the natural alpha-globin gene using PCR. The alpha-globin gene was isolated as two fragments. The 3'-end of the gene (HindIII-HindIII) was obtained by PCR using plasmid pAlGT (Wilson et al., 1978, Nucl. Acids Res. 5:563-580) as template. The primers used for PCR, A-104Ser (5'-end primer) and G10T3H (3'-end primer), are described in the Sequence Description as SEQ ID NO:32 and SEQ ID NO:28, respectively, and are shown in Figure 28 with restriction sites. The mutated fragment of the alpha-globin gene was digested HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation.

The 5'-end of the gene (SalI-HindIII) was obtained by PCR using plasmid pJW101 as template. Primers used for PCR were 51-A3-SL (5'-end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as

SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are shown in Figure 26 with restriction sites. PCR product was digested with SalI and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol

precipitation. This purified 300 bp fragment along with the fragment obtained by PCR for the 5'-end were ligated to the vector pNTl/γ-PORT cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was digested with restriction enzyme HindII. The results obtained from this analysis showed that one clone had expected fragments (five fragments when digested with HindII). This plasmid was called pNTl/2A104S.

The 104 Ser alpha-globin gene was also cloned in yeast expression vector YEp51UT/NAT. Plasmid

Yep51UT/NAT was digested with SalI and BamHI. A 7000 bp fragment was gel purified. The 5'-end of the gene (SalI- HindIII) was obtained by PCR using plasmid pJW101 as template. Primers used for PCR 51-A3-SL (5'-end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are shown in Figure 26 with restriction sites. The PCR product was digested with SalI and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation. The 3'-end of the gene (Hindlll-HindllI) was obtained by PCR using plasmid pA1GT as template. Primers used for PCR were A-104Ser (5'-end primer) and 519-A-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO:32 and SEQ ID NO: 8, respectively. The mutated fragment of the alpha-globin gene was digested HindIII. This digested DNA fragment was purified by phenol

extraction and ethanol precipitation.

Purified fragments obtained by PCR for the 5' and 3'-ends were ligated to the vector YEp51UT/NAT cut with SalI and BamHI. DNA ligation, E. coli transformation and DNA isolation was performed as described in Section 11.1. DNA isolated from the transformed cells was digested with restriction enzyme HindII. The results obtained from this analysis showed that one clone had expected fragments (five fragments when digested with HindII). This plasmid was called pUT/2A104S.

11.5.6. CLONING OF 104-Ser (104 Cys->Ser) ZETA-GLOBIN GENE

The 104-Ser ζ-globin was created by substituting one base in the natural ζ-globin gene using

PCR. The ζ-globin gene was isolated as two fragments.

Both fragments were obtained by PCR. The 5'-end of the gene (SalI-BstEII) was obtained by PCR using plasmid 4p-7-7 as template. Primers used for PCR, Z-5-SAL (5'-end primer) and Z-104S-B (3'-end primer) are shown in Figure 29 and are described in the Sequence Description as SEQ ID NO:33 and SEQ ID NO:34 respectively.

The 3'-end of the gene (BstEII-HindIII) was obtained by PCR using plasmid 4p-7-7 as template. Primers used for PCR are Z-BST-5 (5'-end primer) and Z2-3-H (3'-end primer) are shown in Figure 30 and are described in the Sequence Description as SEQ ID NO: 35 and SEQ ID NO: 36 respectively.

The mutated fragment of the ζ-globin gene was digested with SalI and BstEII. This digested DNA fragment (330 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the ζ-globin gene obtained by

PCR was digested with restriction enzymes BstEII and

HindIII. A 100 bp fragment was isolated. Purified

fragments (330 and 100 bp) were ligated to the vector

YEp51NTl cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (see Section 11.1., supra). DNA isolated from the

transformed cells was digested with restriction enzyme

HindII. The results obtained from this analysis showed that one clone had expected fragments (five fragments when digested with HindII). This plasmid was called pNT1/ζ104S.

11.5.7. CLONING OF TITUSVILLE ζ-GLOBIN GENE (94 Asp->Asn).

The 94-Asn ζ-globin was created by substituting one base in the natural ζ-globin gene using PCR. The ζ-globin gene was isolated as two fragments. Both fragments were obtained by PCR. The 5'-end of the gene (SalI-BstEII) was obtained by PCR using plasmid 4p-7-7 as template. The 5' primer used for synthesizing the sequence, Z-5-SAL, is described in the Sequence Description as SEQ ID NO: 33 and the 3' primer used for synthesizing the sequence, Z-A95-3, is described in the Sequence Description as SEQ ID NO: 37. Restriction sites on these two primers are shown in Figure 31.

The 3'-end of the gene (BstEII-HindIII) was obtained by PCR using plasmid 4p-7-7 as template. The 5' primer used for synthesizing the sequence, Z-BST-5, is described in the Sequence Description as SEQ ID NO: 35 and the 3' primer used for synthesizing the sequence, Z2-3-H, is described in the Sequence Description as SEQ ID NO: 36. Restriction sites on these two primers are shown in Figure 30.

A mutated fragment of the ζ-globin gene obtained by PCR was digested SalI and BstEII. This digested DNA fragment (330 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the ζ-globin gene obtained by PCR was digested with restriction enzymes Bst EII and Hind III. A 100 bp fragment was isolated. Purified fragments (330 and 100 bp) were ligated to the vector

YEp51NTl cut with Sal I and HindIII. DNA ligation, E. coli transformation and DNA isolation were performed as

described (11.1., supra). DNA isolated from the

transformed cells was digested with restriction enzyme

HindII. The results obtained from this analysis showed that one clone had expected fragments (five fragments when digested with HindII). This plasmid was called pNT1/Z95An. 11.5.8. CLONING OF ALPHA-GLOBIN GENE CONTAINING ALPHA

TITUSVILLE AND ALPHA-104 Ser MUTATIONS:

94 Asp->Asn; 104 Cys->Ser)

The double mutant (Titusville + 104 Ser) alpha- globin was created by substituting one base in the 104 Ser alpha-globin gene using PCR. The alpha-globin gene was isolated as two fragments. The 3'-end of the gene (HindIII -HindIII) was obtained by PCR using plasmid pNT1/α104S as template. Primers used for PCR were A-Tit-5 (5'-end primer) and G10T3H (3'-end primer), are described in the Sequence Description as SEQ ID NO:27 and SEQ ID NO: 28, respectively, and are shown in Figure 25 with restriction sites.

The alpha-globin gene fragment containing double mutation was digested HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation. The 5'-end of the alpha-globin gene was obtained from plasmid pJW101 using PCR. The 5'-end of the gene (SalI-HindIII) was obtained by PCR using plasmid pJW101 as template. Primers used for PCR were 51-A3-SL (5'- end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are shown in Figure 26 with restriction sites. The PCR product was digested with SalI and HindIII.

This digested DNA fragment was purified by phenol

extraction and ethanol precipitation. This purified 300 bp fragment along with the fragment obtained by PCR for the 5' end were ligated to the vector (pNTl/γ-PORT cut with SalI and HindIII). DNA ligation, E. coli transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was

digested with restriction enzyme HindII. The results obtained from this analysis showed that one clone had expected fragments (five fragments when digested with

HindII). This plasmid was called pNTl/2ATiS.

11.5.9. CLONING OF THE MOTOWN GAMMA-GLOBIN GENE:

(127 Gln->Glu)

The Motown gamma-globin was created by base substitution in the natural gamma-globin sequence using

PCR. The gamma-globin gene was obtained as two fragments. The 5 'end of the gene was isolated as SalI-EcoRI fragment (320 bp) from plasmid YEp51T/G. The 3'end of the gene

(containing mutation) was obtained by PCR. Template used for the PCR was pJW151. Primers used for PCR, G2-Mot-5 (5' end primer) and GAM-3-H (3'-end primer), are described in the Sequence Description as SEQ ID NO: 38 and SEQ ID NO: 12, respectively, and are shown in Figure 32 with restriction sites. PCR product was digested with restriction enzymes EcoRI and HindIII. Digested fragment was purified by phenol extraction and ethanol precipitation.

The purified fragments obtained by PCR and isolated from plasmid was YEp51T/G were ligated to the vector (YEp51NTl cut with SalI and HindIII). DNA ligation, E. coli transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was digested with restriction enzyme

PstI. The results obtained from this analysis showed that the clones had expected fragments (three fragments when digested with Pstl; two fragments from vector without insert). This plasmid was called YEp51NT1/γ-Mot2.

11.5.10. CLONING OF THE BOVII β-GLOBIN GENE

(Met Leu Thr Ala Glu Glu ....)

The BovII (human globin gene 5'-end with four amino acids of the bovine globin gene) β-globin was created by replacing six amino acids of the human β-globin at the

5'-end with four amino acids from the bovine β-globin gene's 5'-end. The mutated β-globin gene was obtained as a

450 bp fragment. The 5' primer, B-Bov2-5, used for synthesizing the sequence is described in the Sequence Description as SEQ ID NO:39 and the 3' primer, Beta-3-H, used for synthesizing the sequence is described in the

Sequence Description as SEQ ID NO:26. Restriction sites on these two primers are shown in Figure 33.

The mutated β-globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by

PCR was ligated to the vector YEp51NT1/γ-P0RT cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (supra, 11.1.). DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with Pstl; three fragments from vector without insert). This plasmid was called pNT1/β-Bov2.

11.5.11. CLONING OF β-2 Arg GLOBIN GENE: (2 His->Arg)

The β-2 Arg beta-globin was created by

replacing amino acid His (amino acid #2) of the human beta-globin with amino acid Arg. The mutated beta-globin gene was obtained as a 450 bp fragment. Primers used for PCR, B-2ARG-5 (5'-end primer) and Beta-3-H (3'-end primer) are described in the Sequence Description as SEQ ID NO: 40 and SEQ ID NO: 26, respectively, and are shown in Figure 34 with restriction sites.

The mutated beta globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NTl/γ-PORT cut with Sal! cut with Hindlll). DNA ligation, E. coli transformation and DNA isolation was performed as described in Materials and Methods section. DNA isolated from the transformed cells was digested with restriction enzyme

PstI. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with PstI; three fragments from vector without insert). This plasmid was called

pNTl/β2Arg.

11.5.12. CLONING OF THE 143 Arg BETA-GLOBIN GENE :

(143 His->Arg)

The 143 Arg beta-globin was created by

replacing amino acid His (amino acid # 143) of the human beta-globin with amino acid Arg. The mutated beta-globin gene was obtained as a 450 bp fragment. Primers used for PCR, BN-5-SAL (5'-end primer) and B-143A-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 33 and SEQ ID NO: 41, respectively, and are shown in Figure 35 with restriction sites.

The mutated gamma-globin gene obtained by PCR was digested with Sail and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NTl/γ-PORT cut with SalI and HindIII). DNA ligation, E. coli

transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was digested with restriction enzyme PstI. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with PstI; three fragments from vector without insert). This plasmid was called pNTl/βl43Arg.

11.5.13. CLONING OF THE 145 Term BETA-GLOBIN GENE:

(145 Tyr->TAA)

The 145 Term beta-globin was created by replacing amino acid Tyr (amino acid #145) of the human beta-globin with protein termination codon (TAA). The mutated beta-globin gene was obtained as a 450 bp fragment. Primers used for PCR, BN-5-SAL (5'-end primer) and B-145T-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO:33 and SEQ ID NO: 42, respectively, and are shown in Figure 36 with restriction sites.

The mutated beta-globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NT1/γ-PORT cut with SalI and HindIII). DNA ligation, E. coli

transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was digested with restriction enzyme PstI. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with PstI; three fragments from vector without insert). This plasmid was called pNT1/β145T.

11.5.14. CLONING OF THE CHICO ( 66 Lys->Thr) γ-GLOBIN GENE

The Chico γ-globin was created by substituting one base in the natural γ-globin gene using PCR . The γ-globin gene was isolated as two fragments . The 5 ' -end of the gene (SalI-XcmI) was obtained by PCR using plasmid pJW151 as template. The 5' primer used for synthesizing the sequence, GAM-5-S is described in the Sequence

Description as SEQ ID NO: 11 and the 3' primer used for synthesizing the sequence, G66T-3' is described in the Sequence Description as SEQ ID NO: 43. Restriction sites on these two primers are shown in Figure 37.

The mutated fragment of the γ-globin gene was digested with SalI and XcmI. This digested DNA fragment (230 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the γ-globin gene was

isolated from plasmid YEp51NT1/γ-PORT. Plasmid YEp51NT1/γ- PORT was digested with restriction enzymes XcmI and

HindIII. A 220 bp fragment was isolated. This purified 220 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NTl cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation were performed as described (supra. 11.1). DNA isolated from the transformed cells was digested with restriction enzyme

PstI. The results obtained from this analysis showed that one clone had expected fragments (three fragments when digested with PstI; two fragments from vector without insert). This plasmid was called pNT1/γ-Chico.

11.6. EXPRESSION OF VARIANT GLOBINS 11.6.1. EXPRESSION OF GAMMA-GLOBIN MOTOWN IN A YEAST

11.6.1.1. TRANSFORMATION OF YEAST STRAIN Sc1114

WITH pNT1/γ-Mot2

Yeast strain Sc1114 was transformed with plasmid pNTl/γ-Mot2 (see Section 11.5.9., supra) using electroporation (see Section 9.4., supra..

concentration of 2%. At induction, the pH was adjusted to 7.27 with KH(2)PO(4) and hemin was added to a final

concentration of 40 μg/ml. Samples were collected between two and 30 hours after induction.

11.6.1.2. WESTERN BLOT ANALYSIS QF EXPRESSED GLOBIN

The expressed globins was quantitated by

Western Blot analysis using procedures described in Section 6.6. Samples taken after induction had detectable levels of globin (0.01%).

11.6.2. EXPRESSION OF BETA-GLOBIN BovII IN YEAST 11.6.2.1. TRANSFORMATION OF YEAST STRAIN Sc1111

WITH pNT1/β-Bov2

Yeast strain Sc1115 was transformed with plasmid pNT1/β-Bov2 (see Section 11.5.10., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tweenSO, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final

concentration of 2%. At induction, the pH was adjusted to 6.94 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between two and 24 hours after induction.

11.6.2.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins was quantitated by

Western Blot analysis using procedures described in section 6.6. Samples taken 5 hours after induction had detectable levels of globin (0.5%).

11.6.3. EXPRESSION OF ZETA-GLOBIN 104 SER IN YEAST

11.6.3.1. TRANSFORMATION OF YEAST STRAIN Sc340 WITH

pNT1/ζ104S

Yeast strain Sc340 was transformed with plasmid pNTl/ζ104S (see Section 11.5.6., supra) using electroporation (see Section 9.4., supra).

concentration of 2%. At induction, the pH was adjusted to 7.01 with KH(2)PO(4) and hemin was added to a final

concentration of 40 μg/ml. Samples were collected between two and 48 hours after induction.

11.6.3.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins was quantitated by

Western Blot analysis using procedures described in Section 6.6. Samples taken after induction had detectable levels of globin (0.09%).

12. EXAMPLE 7: COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN BOVII IN YEAST

12.1. TRANSFORMATION OF YEAST STRAINS Sc389 WITH pUT/2A

AND pNT1/β-Bov2

Yeast strain Sc389 was transformed with

plasmids pUT/2A (see Section 7, supra) and pNT1/β-Bov2 (see

Section 11.5.10., supra) using electroporation (see Section

9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween 80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.12 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between two and 30 hours after induction.

12.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.6% of soluble protein.

12.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc Natl Acad Sci USA, 1987, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the

suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same instrument.

If hemoglobin is present the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.

Functional hemoglobin was detected in this strain by this method.

13. EXAMPLE 8: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN

143 ARG IN YEAST

13.1. TRANSFORMATION OF YEAST STRAINS Sc340 WITH pUT/2A

AND pNT1/β143Arg

Yeast strain Sc340 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/β143Arg

(see Section 11.5.12., supra, using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2% . At induction, the pH was adjusted to 7.18 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between four and 74 hours after induction.

13.2. WESTERN BLOT ANALYSTS OF EXPRESSED GLOBIN

The expressed globins were quantitated by

Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.16% of soluble protein. 13.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:6961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly, the O.D. at 600 nm is measured on the same instrument.

If hemoglobin is present the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin .

Functional hemoglobin was detected in this strain by this method.

14. EXAMPLE 9: COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN 145 TERM IN YEAST

14.1. TRANSFORMATION OF YEAST STRAINS Sc1090 WITH pUT/2A

AND pNT1/β145T

Yeast strain Sc1090 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/β145T (see Section 11.5.13, supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.04 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between two and six hours after induction.

14.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.11% of soluble protein.

14.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same

instrument.

Functional hemoglobin was detected in this strain by this method.

14.4. WESTERN BLOT ANALYSIS OF EXPRESSED

ALPHA AND BETA GLOBINS

The expressed alpha and beta globins were separated using an 18% SDS polyacrylamide gel.

Phosphate-buffered saline (PBS, 0.9 M NaCl, 0.01 M phosphate, pH 7.6) solution (2 ml) was added to thawed yeast samples (0.2 g wet weight). The samples were centrifuged at 4°C for 10 minutes at 2700 rpm in a Sorvall RT6000B and the supernatant decanted. Cold disruption buffer (50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0) prepared immediately before use (0.2 ml) was added to the pellet, followed by enough ice-cold glass beads to just reach the top surface of the liquid. After vortexing for 30 seconds at maximum speed the samples were placed on ice for 5 minutes. This step was repeated twice. Ice-cold

disruption buffer (1 ml) was added to each sample and the homogenate was transferred to an Eppendorf tube. In another Eppendorf tube, 200 μl of homogenate was combined with 200 μl of freshly prepared standard discontinuous 2X sample buffer (Laemli, 1970, Nature 227:680-685) and the sample was boiled for 10 min. After centrifuging the samples for 10 min., the samples were loaded onto a 20 × 18 cm discontinuous denaturing gel in which the stacking gel was 3% acrylamide and the separating gel was 18% (Laemli, 1970, Nature 227: 680-685). Gels were run at a constant current of 15 mA per gel.

After the electrophoresis was complete and the dye band had reached the bottom of the separating gel, the gels were removed from the electrophoresis unit and the plates were pried apart under running deionized water. The stacking gel was discarded and the lower gel was separated from the plate. The transfer unit was filled with transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine in a final volume of 10L, pH 8.3) and 2L of the transfer buffer was put into a shallow pan. The transfer sandwich

consisting of large pore gauze, 3M blotting paper, the gel, a piece of nitrocellulose paper precut to just cover the gel, 3M blotting paper, and another piece of large pore gauze was assembled under the buffer in the shallow pan.

Protein was transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose was removed and placed in a shallow pan with 50 ml of blocking solution [5% (w/v) BSA in PBS]. The nitrocellulose membrane was incubated for 1 hour with agitation, after which the blocking solution was replaced with washing solution [0.1% Tween 20 (v/v) in PBSI . Three washings of 15, 5 and 5 minutes were carried out. The final wash solution was discarded and 25 μl of primary antibody in 25 ml of PBS was added to the pan. After incubation for 2 hours, with agitation, the nitrocellulose was washed three times (1 × 15 and 2 × 5 minutes). The final wash was discarded and 2.5 μl of secondary antibody in 25 ml of PBS added for a 1 hour incubation with agitation. After three washes (1 × 15 and 2 × 5 minutes), 5 μl of streptavidin-HRP (horseradish peroxidase) was added in 25 ml of PBS containing 0.1% Tween 20 (v/v). After a 20 minute incubation with agitation, the membrane was washed three times (1 × 15 and 2 × 5 minutes). The nitrocellulose was then placed in ECL (enhanced

chemiluminescent) developing solution that had been

prepared immediately prior to use by mixing equal volumes of detection reagent 1 and detection reagent 2 (Amersham). The membrane was incubated for 1 minute with agitation, removed from the developing solution, the excess reagent drained off and the membrane then wrapped in SaranWrap. The wrapped nitrocellulose was then exposed to X-ray film for an appropriate length of time. After development, the X-ray film was scanned using a laser densitometer and the quantity of globin in each sample estimated by comparison with globin standards run on the same gel.

Alpha and beta globins are separated on this gel by molecular weight. Alpha and beta globin were

detected in the protein extracts of the cotransformed yeast.

15. EXAMPLE 10: COEXPRESSION OF ALPHA-GLOBIN AND GAMMA- GLOBIN MOTOWN IN YEAST

15.1. TRANSFORMATION OF YEAST STRAINS Sc1114 WITH pUT/2A

AND pNT1/γ-Mot2

Yeast strain Se1114 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/γ-Mot2 (see

Section 11.5.9., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween 80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2% . At induction, the pH was adjusted to 7.09 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between four and 51 hours after induction.

15.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.3% of soluble protein.

15.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:8961). Approximately 1.2 ml of

suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same instrument.

If hemoglobin is present, the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.

Functional hemoglobin was detected in this strain by this method.

16. EXAMPLE 11: COEXPRESSION OF ZETA-GLOBIN 104 SERINE

AND BETA-GLOBIN IN YEAST

16.1. TRANSFORMATION OF YEAST STRAINS Sc1114 WITH

pNT1/ζ104S AND YEp51T/NAT

Yeast strain Sc1114 was transformed with plasmids pNT1/ζ104S (see Section 11.5.6., supra) and

YEp51T/NAT (see Section 6, supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 6.96 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between two and 30 hours after induction.

16.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBTN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.09% of soluble protein. 16.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:6961). Approximately 1.2 ml of

suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same

instrument.

If hemoglobin is present the difference spectrum will produce a peak around 420 nm and a valley around 435nm. A single peak at 420 nm does not indicate the presence of hemoglobin.

Functional hemoglobin was detected in this strain by this method.

17. EXAMPLE 12: COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN 2 ARG IN YEAST

17.1. TRANSFORMATION OF YEAST STRAINS Scl090 WITH pUT/2A

AND pNT1/β2Arg

Yeast strain Sc1090 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/β2Arg (see

Section 11.5.11., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 6.93 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between four and eight hours after induction.

17.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6.

Globin was detected at a level of 0.17% of soluble protein.

17.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc Natl Acad Sci USA, 1987, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette.

This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the

suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D, at 600 nm is measured on the same instrument.

Functional hemoglobin was detected in this strain by this method.

18. EXAMPLE 13: EXPRESSION OF HEMOGLOBIN

PORTLAND I IN YEAST

18.1.TRANSFORMATION OF YEAST STRAINS Sc1012 WITH pYES2-ζ2

AND YEP51T/G

Yeast strain Scl012 was cotransformed with plasmids pYES2-ζ2 (see Section 10, supra) and YEp51T/G (see

Section 8, supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, 4ppm aminolevulinic acid (ALV) , and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 4ppm ALV, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was

adjusted to 6.93 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between two and 30 hours after induction.

18.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by

Western .Blot analysis using procedures described in Section

6.6. Globm was detected at a level of 0.06% of soluble protein.

18.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

instrument.

Functional hemoglobin was detected in this strain by this method. 19. EXAMPLE 14: EXPRESSION OF BETA-GLOBIN IN A YEAST

EXPRESSION VECTOR CONTAINING A HYBRID PROMOTER AND

ADH1 TRANSCRIPTION TERMINATION SEQUENCE

A hybrid promoter was constructed by the fusion of the upstream activating sequence of GAL1-10 promoter with the downstream promoter elements of the TDH3 promoter (referred to hereafter as the 3' end of the TDH3 promoter or TDH3-3'). The cassette containing the hybrid promoter + beta-globin gene + ADH1 terminator were excised and cloned into the yeast shuttle vector, YEp13. Yeast strain Sc340 was transformed with the resulting plasmid, pNML-V-G-1 and the proteins expressed were analyzed by Western Blot Analysis.

19.1. MATERIALS

(Biolabs), Bethesda Research Laboratories (BRL) or

Boehringer Mannheim (BM). All enzymes were used according to the suppliers specifications. Plasmid DNA was isolated from a one liter culture of the transformed cells and purified by CsCl gradient centrifugation.

19.2. CONSTRUCTION OF PLASMID L19βt CONTAINING BETA-GLOBIN

GENE AND THE ADH1 TERMINATOR

The beta-globin gene was obtained by digestion of plasmid mp18βHS with SalI and HindIII (see Figure 3δ for map of mp18βHS). The 600 bp fragment was isolated by electroelution.

Plasmid AAH5 was digested with HindIII and BamHl (Ammerer, G., Methods in Enzymology, 101, pp. 192-201, 1963). The resulting 450 bp fragment was isolated by gel electrophoresis. Subsequently, the band containing the 450 bp fragment was precipitated with ethanol and digested with SphI. The 320 bp fragment (HindIII-SphI) containing ADH1 transcription termination sequences was isolated by electroelution.

Plasmid pUC19 was cut with SalI and SphI. A three way ligation reaction mixture was set up between the pUC19 fragment, the SalI-HindIII beta-globin fragment, and the HindIII-SphI ADH1 terminator fragment. The ligation was used for transforming competent E.. coli cells (DH5α).

The transformants were selected on ampicillin plates (100 mg/L). Plasmid DNA was isolated from twenty transformants (clones) and analyzed by restriction digestion with SalI-HindIII. The resulting plasmid containing the above two inserts in pUC19 was called L19βAt, and is shown in Figure

39. The DNA was digested with Sphl and ApaLI and a 920 bp fragment containing the beta-globin gene and the ADH1 terminator was isolated by electroelution and used for the construction of the plasmid containing the TDH3 promoter, the beta-globin gene and the ADH1 terminator. 19.3. CONSTRUCTION OF PLASMID pUC19-HβAt

The TDH3-3' promoter fragment was synthesized by PCR using appropriate primers and template DNA from plasmid gp491. The primers, TDH3-5' (5'-primer) and TDH3-3' (3'-primer) are shown in Figure 40 and are described in the Sequence Description as SEQ ID NO: 42 and SEQ ID NO: 43. The 180 bp promoter fragment (TDH3-3') synthesized by PCR was digested with ApaLI and SmaI. The plasmid pUC19 was cut with Smal and Sphl. The DNA from plasmid L19βAt was cut with ApaLI and Sphl and 920 bp fragment was isolated. Three way ligation was set between these three fragments. The transformation of E. coli DH5α cells was carried out as described earlier. The DNA isolated from the transformants were screened by restriction enzyme analysis with PvuII, ApaLI, and PvuII-HindII to check for the correct insert . The map of the resulting plasmid, pUC19-HβAt, is shown in Figure 41. 19.4 . CLONING OF GAL1-1Q UAS INTO pUC19-HβAt

GAL1-10 upstream activator sequence (UAS) , which is shown in Figure 42 and described in the Sequence Description as SEQ ID NO : 44, was synthesized by polymerase chain reaction using GAL1-10-5 ' and GAL1-10-3 ' primers and DNA from YEp51 as a template . The sequences of these primers, GAL1-10-5 ' and GAL1-10-3 ' are shown in Figure 43 and are described in the Sequence Description as SEQ ID NO: 45 and SEQ ID NO : 46. The restriction sites Sacl and

SmaI were added to facilitate cloning .

The GAL1-10 UAS PCR product was digested with

SacI, blunt ended and cut with SmaI . It was cloned by blunt end ligation into SmaI digested ρUC19-HβAt which contains the 3 ' end of the T DH3 promoter with the betaglobin gene and ADH1 terminator . The structure of the resulting plasmid, pUC19-GHβAt is shown in Figure 44 .

Transformation was carried out using E . coli DH5α cells .

The DNA isolated from the transformants were screened by restriction enzyme analysis with PvuII, EcoRI, and HindIII to check for the correct insert .

19 .5. CLONING OF THE HYBRID PROMOTER-BETA-GLOB IN GENE

CASSETTE IN SHUTTLE VECTOR, YEp13

pUC19-GHβAt was digested with SacI-SphI to excise the GAL10-UAS + TDH3-3 ' + beta-globin gene + ADH1-terminator cassette from pUC19 which was subsequently blunt-ended. The resulting 1. 43 kb fragment was isolated by electroelution .

Plasmid YEp13 (obtained from Fred Winston, Harvard Medical School) which contains LEU2 (yeast) and Amp^R (E. coli) markers, was digested with BamHI and blunt-ended; the resulting linear DNA was isolated by

electroelution.

Ligation was set between the insert and the vector and the ligation mixture was used for transforming competent E. coli cells (DH5α). The transformants were selected on ampicillin plates (100 mg/L). The plasmid DNA was isolated from 24 transformants and analyzed by

restriction digestion with HindIII, EcoRI, EcoRI/SalI. A map of the resulting plasmid, pNML-V-G-1 is shown in Figure 45.

19.6. TRANSFORMATION OF YEAST STRAIN Sc340 CELLS

WITH pNML-V-G-1

Strain Sc340 has the following genotype:

MATa, ura3-52, leu2, ade1, MEL+, [his3 : :GAL10 (UAS+P) +

GAL4 + URA31. Yeast strain Sc340 was transformed with plasmid pNML-V-G-1 using the spheroplast procedure (Rose, M. et al., 1989, Methods in Yeast Genetics, Cold Spring

Harbor Laboratory, Cold Spring Harbor, N.Y., pp 112-115). To minimize background in the control plates and increase efficiency of transformation, the regeneration media contained 1 M sorbitol, 10 mM CaCl₂, 0.1% yeast nitrogen base, and 2% glucose. The medium was filter sterilized.

The plating media was prepared by mixing 182 g sorbitol, 20 g agar, 6.7 g Difco YNB without amino acids, glucose, required amino acids except leucine in 1 L distilled water. The top agar was made by mixing 18.2 g sorbitol, 2 g agar, 0.67 g Difco YNB without amino acids, 2 g glucose and required amino acids in 100 ml distilled water.

For the starter culture, cells were grown overnight in minimal media containing 0.67% yeast nitrogen base, 0.5% glucose, and supplemented with uracil, adenine, and histidine. 500 ml of SD media supplemented with 200 μM ferric citrate and 20 mg/L each of adenine, uracil, and histidine was inoculated with the starter culture to an OD₆₀₀ of 0.02. The culture was incubated with shaking (300 rpm) at 30°C, and was induced with 2% galactose for a period of 4 hours before sampling for analysis.

19.7. WESTERN BLOT ANALYSTS OF EXPRESSED BETA-GLOBIN

The expressed beta-globin was quantitated by Western Blot analysis using procedures described in Section 6.6., supra. The results indicated that up to 5.4% of the total yeast protein expressed in transformed Sc340 cells was beta-globin.

20. EXAMPLE 15: EXPRESSION OF HEMOGLOBIN

PORTLAND IT IN YEAST

20.1. TRANSFORMATION OF YEAST STRAINS Sc1041 WITH pYES2-ζ2

AND pNM-V-Gβ1

Yeast strain Sc1041 was cotransformed with plasmids pYES2-ζ2 (see Section 10, supra) and pNM-V-Gβ1

(see Section 19, supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.16 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between two and 30 hours after induction. 20.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.04% of soluble protein.

20.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM

instrument.

Functional hemoglobin was detected in this strain by this method. 21 . EXAMPLE 16 : COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN MISSISSIPPI IN YEAST

21.1. TRANSFORMATION OF YEAST STRAINS Sc389 WITH pUT/2A

AND pNT1/β-Miss

Yeast strain Sc389 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/β-Miss (see

Section 11.5.4., supra) using electroporation (see Section 9.4., supra),

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.10 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between two and 30 hours after induction.

21 . 2 . WESTERN BLOT ANALYSTS OF EXPRESSED GLOBIN

The expressed globins were quantitated by

Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.16% of soluble protein.

21.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1967, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

instrument.

Functional hemoglobin was detected in this strain by this method.

22. EXAMPLE 16: COEXPRESSION OF ALPHA-GLOBIN

TITUSVILLE AND BETA-GLOBIN IN YEAST

22.1. TRANSFORMATION OF YEAST STRAINS Sc1114 WITH

pNT1/2ATit and YEp51T/NAT

Yeast strain Sc1114 was transformed with plasmids pNT1/α-Tit (see Section 11.5.3., supra) and

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2% . At induction, the pH was adjusted to 6.9δ with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between four and 51 hours after induction.

22.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.01% of soluble protein.

22.3. DETECTION OF HEMOGLOBIN IN YEAST BY

CARBON MONOXIDE DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1967, 84:6961). Approximately 1.2 ml of

spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly, the O.D. at 600 nm is measured on the same

instrument. If hemoglobin is present the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.

Functional hemoglobin was detected in this strain by this method.

23. EXAMPLE 18: COEXPRESSION OF ALPHA-GLOBIN

TITUSVILLE/104 SERINE AND BETA-GLOBIN IN YEAST

23.1. TRANSFORMATION OF YEAST STRAINS Sc1114 WITH

pNT1/2ATiS and YEp51T/NAT

Yeast strain Sc1114 was transformed with plasmids pNT1/2ATiS (see Section 11.5.8., supra) and

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.10 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between four and 51 hours after induction.

23.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by

Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.2% of soluble protein. 23.3. DETECTION OF HEMOGLOBIN IN YEAST BY

CARBON MONOXIDE DIFFERENCE SPECTRUM

instrument.

Functional hemoglobin was detected in this strain by this method.

24. EXAMPLE 19: COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN MOTOWN IN YEAST

24.1. TRANSFORMATION OF YEAST STRAINS Sc389 WITH pUT/2A

AND pNT1/ß-Mot

Yeast strain Sc389 was transformed with

plasmids pUT/2A (see Section 7, supra) and pNT1/ß-Mot (see Section 11.5.2., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.17 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were

collected between four and 51 hours after induction.

24.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.4% of soluble protein.

24.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 64:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same

instrument.

Functional hemoglobin was detected in this strain by this method.

24.4. WESTERN BLOT ANALYSIS OF EXPRESSED

ALPHA AND BETA GLOBINS

Phosphate-buffered saline (PBS, 0.9 M NaCl, 0.01 M phosphate, pH 7.6) solution (2 ml) was added to thawed yeast samples (0.2 g wet weight) . The samples were centrifuged at 4°C for 10 minutes at 2700 rpm in a Sorvall RT6000B and the supernatant decanted. Cold disruption buffer (50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0) prepared immediately before use (0.2 ml) was added to the pellet, followed by enough ice-cold glass beads to just reach the top surface of the liquid. After vortexing for 30 seconds at maximum speed the samples were placed on ice for 5 minutes. This step was repeated twice . Ice-cold

disruption buffer (1 ml) was added to each sample and the homogenate was transferred to an Eppendorf tube. In another Eppendorf tube, 200 μl of homogenate was combined with 200 μl of freshly prepared standard discontinuous 2X sample buffer (Laemli, 1970, Nature 227:680-685) and the sample was boiled for 10 min. After centrifuging the samples for 10 min., the samples were loaded onto a 20 × 18 cm discontinuous denaturing gel in which the stacking gel was 3% acrylamide and the separating gel was 18% (Laemeli, 1970, Nature 227: 680-665). Gels were run at a constant current of 15 mA per gel.

Protein was transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose was removed and placed in a shallow pan with 50 ml of blocking solution [5% (w/v) BSA in PBS]. The nitrocellulose membrane was incubated for 1 hour with agitation, after which the blocking solution was replaced with washing solution [0.1% Tween 20 (v/v) in PBS]. Three washings of 15, 5 and 5 minutes were carried out. The final wash solution was discarded and 25 μl of primary antibody in 25 ml of PBS was added to the pan. After incubation for 2 hours, with agitation, the nitrocellulose was washed three times (1 × 15 and 2 × 5 minutes). The final wash was discarded and 2.5 μl of secondary antibody in 25 ml of PBS added for a 1 hour incubation with agitation. After three washes (1 × 15 and 2 × 5 minutes), 5 μl of streptavidin-HRP (horseradish peroxidase) was added in 25 ml of PBS containing 0.1% Tween 20 (v/v). After a 20 minute incubation with agitation, the membrane was washed three times (1 × 15 and 2 × 5 minutes). The nitrocellulose was then placed in ECL (enhanced

chemiluminescent) developing solution that had been

prepared immediately prior to use by mixing equal volumes of detection reagent 1 and detection reagent 2 (Amersham). The membrane was incubated for 1 minute with agitation, removed from the developing solution, the excess reagent drained off and the membrane then wrapped in SaranWrap.

The wrapped nitrocellulose was then exposed to X-ray film for an appropriate length of time. After development, the X-ray film was scanned using a laser densitometer and the quantity of globin in each sample estimated by comparison with globin standards run on the same gel.

detected in the protein extracts of the cotransformed yeast.

25. EXAMPLE 20: EXPRESSION OF THE PORTO ALEGRE BETA- GLOBIN IN A YEAST EXPRESSION VECTOR CONTAINING THE

GAL10 PROMOTER

As detailed herein, the natural beta-globin was modified to obtain a Porto Alegre beta-globin gene by replacing a 104 bp AccI-NcoI fragment from the natural beta-globin gene with a synthetic oligonucleotide containing a cysteine as amino acid 9 (instead of a

serine). The Porto Alegre beta-globin gene was

subsequently cloned into the yeast expression vector YEp51 to obtain plasmid YEpWB51/P0RT. YEpWB51/PORT was

transformed into yeast strain Sc340, a hem1 strain.

Quantitation of RNA by scanning the autoradiograph showed that mRNA for the Porto Alegre beta-globin was around 6.0% of total yeast RNA. Western blot analysis indicated that Porto Alegre beta-globin was expressed.

25.1. MATERIALS

The restriction enzymes, Klenow enzyme and

T4-DNA ligase were obtained from New England Biolabs

(Biolabs), Bethesda Research Laboratories (BRL) or

Boehringer Mannheim (BM) . All enzymes were used according to the suppliers specifications. Plasmid DNA was isolated from a one liter culture of the transformed E. coli cells and purified by CsCl gradient centrifugation.

25.2. CLONING OF THE PORTO ALEGRE BETA-GLOBIN GENE

INTO THE YEAST EXPRESSION VECTOR YEp51

The general procedure used to clone the Porto

Alegre beta-globin gene into the yeast expression vector

YEp51 is shown in Figure 3B. The plasmid pSPβC (see Figure

2 for the partial restriction map of pSBβC) was digested with AccI and HindIII. Digestion with this combination of enzymes generated two fragments. A 500 base pair (bp) DNA containing the beta-globin gene and a 2800 bp fragment from the plasmid. The 500 bp fragment was isolated from a 0.6% agarose gel. After the band was excised from the gel, the DNA was electroeluted, and ethanol precipitated. The precipitated DNA was spun in an Eppendorf Centrifuge, the supernatant was removed and the DNA pellet was dried under vacuum.

The 500 bp DNA fragment carrying the natural beta-globin gene fragment isolated from pSPβC was Accl compatible at the 5'-end while the 3'-end was HindIII compatible. To modify the 5'-end of the isolated fragment, a synthetic oligonucleotide was used. This double stranded oligonucleotide (104 bp) contained a codon for cysteine as amino acid 9 instead of a codon for serine and had a AccI compatible end at its 3'-end and a SalI compatible end at it 5 '-end (see Figure 3B). The 3'-end of the isolated fragment did not receive any adapter as the HindIII site was compatible with the HindIII site introduced into the YEp51.

The recipient plasmid YEp51 was cleaved with SalI and HindIII restriction enzymes. To insert the isolated fragment containing the beta-globin gene, a three- way ligation was set up (see Figure 3B). The ligation reaction was carried out using the standard ligation procedures (Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The ligation mixture was transformed into the E. coli HB101 cells using standard transformation procedure. Cells were spread on plates containing LB-media with 100 mg/L ampicillin. Plates were incubated overnight at 37°C. Forty eight colonies from the ampicillin plates were picked and a 5 ml culture was inoculated with individual transformant. Cultures were grown overnight at 37°C with vigorous shaking. The plasmid DNA was isolated from 1.5 ml of the overnight culture using the quick alkaline plasmid isolation procedure (Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The plasmid from each transformant was digested with EcoRI to confirm the presence of a DNA fragment containing the Porto Alegre beta-globin gene. The plasmid carrying the Porto Alegre beta-globin gene was called YEpWB51/PORT. The map of plasmid YEpWB51/Port is shown in Figure 46.

25.3. TRANSFORMATION OF Sc340 CELLS WITH YEPWB51/PORT

The yeast strain Sc340 was obtained from Dr. J.E. Hopper of Hershey Medical Center. The genotype of this strain is:

MATa ura3-52, leu2, ade1, his3: :GAL10^uas-GAL4-URA3⁺,MEL⁺. Sc340 cells were transformed with the plasmids

YEpWB51/Port and YEp51 (control). The spheroplast method of transformation was performed according to the published procedure (Hinnen et al., 1978, Proc. Natl. Acad. Sci.

The plates were incubated at 28°C for three days and were examined for colony formation.

Colonies were picked from these plates

following incubation and were precultured in yeast minimal media (0.67% yeast nitrogen base without amino acids) containing 0.5% glucose plus 20 mg/L each of adenine, uracil, and histidine. The overnight culture was then used to inoculate 1000 ml of the yeast minimal media containing

2% lactic acid, 3% glycerol and appropriate amino acids.

The cultures were inoculated to OD₆₀₀ of 0.02. Cultures were grown at 30°C until they reached OD₆₀₀ of 0.20 (usually after 48 hours). Induction was initiated by the addition of galactose to a final concentration of 2% in the media. After four hours, cultures were harvested by centrifugation and the pellet was washed with 150 mM NaCl . The pellet was divided into two parts. One part was used for RNA

isolation and the other was kept at -70°C for Western blot analysis.

25.4.QUANTITATION OF RNA FROM SC340 CELLS TRANSFORMED

WITH PLASMIDS YEp51, YEp51WB/NAT, AND YEpWB51/PORT

RNA was isolated using published procedures

(Meyhack et al., 1982, The EMBO Journal 1:675-680 or

Carlson and Botstein, 1982, Cell 28:145). Yeast cells were washed with 150 mM NaCl and the pellet was resuspended in RNA buffer (0.5 M NaCl, 0.2 M Tris-HCl, pH 7.6, 0.1 M EDTA and 1% SDS). Approximately 0.5 g of glass beads (0.45-0.5 mm) were added to the tubes. An equal volume of phenol mixture (phenol: chloroform:isoamyl alcohol 25:24:1, equilibrated with RNA buffer without SDS) was added. Yeast cells were broken by vortexing at maximum speed for 2.5 minutes and the sample was placed on ice for 3 minutes.

The above step was repeated twice more. Equal volumes of

Colowhich, S. P. and Kaplan, N. O. eds. Vol.

Amersham Hybond-N paper (Thomas, P., 1983, in "Methods in Enzymology" Colowhich, S. P. and Kaplan, N. O. eds. Vol. 100: pp 255-266, Academic Press, New York).

Total yeast RNA bound to the filter paper was hybridized to the radioactive labelled beta-globin DNA.

Hybridizations were carried out at 42°C overnight in 50% (v/v) formamide with 5X SSC (SSC: 3.0 M NaCl, 0.3 M Na citrate, pH 7.5); 50 mM NaPO₄, pH 6.5; 250 μg/ml Salmon sperm DNA; and IX Denhardt's solution; (Denhardt's

solution: 0.02% Ficoll, 0.02% polyvinylcarbonate, and 0.02% BSA, fraction V). The CYH2 mRNA which codes for yeast ribosomal protein L19 was used as control. The probe was plasmid mpl0CYH22 which carries the yeast CYH2 gene. After the hybridizations, filters were washed three times at room temperature in 2X SSC and 0.1% SDS and four times at 50°C in 0.1X SSC and 0 .1% SDS . Filters were exposed to X-ray films for 1 hour to overnight depending on the

radioactivity. X-ray films were developed in a Konica automated film developer.

The results from these RNA blot hybridizations are shown in Figure 8, which show results from RNA isolated from yeast transformed with a control plasmid containing no globin sequences (lane 1), a plasmid containing sequences encoding beta-globin (lane 2), and a plasmid containing sequences encoding Porto Alegre beta-globin (lane 3). The results indicate that the mRNA samples from all sources were intact and no degradation was detected. It was also observed that no beta-globin mRNA could be detected in lane 1, which contains the parent plasmid only. These results indicate that nonspecific hybridization of the beta-globin probe is minimal.

Autoradiographs containing bands corresponding to both beta-globin and CYH2 mRNA were scanned using the LKB gel scanner. Results obtained from the scanner are shown in Figure 9. It can be clearly seen that the abundance of CYH2 mRNA in all three lanes is approximately the same while the abundance of the Porto Alegre beta-globin mRNA in 340g2P was high.

25.5. WESTERN BLOT ANALYSIS OF EXPRESSED PORTO

ALEGRE BETA-GLOBIN

The expressed Porto Alegre Beta-Globin was analyzed by Western Blot analysis (see Section 6.6.,

supra). Globin was detected at a level of 0.09% of soluble protein.

26. EXAMPLE 21: COEXPRESSION OF ZETA-GLOBIN TITUSVILLE

AND GAMMA-GLOBIN MOTOWN IN YEAST

26.1. TRANSFORMATION OF YEAST STRAINS Sc1115 WITH

pNTl/Z95An AND YEp51T/G Yeast strain Sc1115 was transformed with plasmids pNTl/Z95An (see Section 11.5.7., supra) and

YEp51T/G (see Section 8, supra) using electroporation (see Section 9.4., supra).

collected between four and 51 hours after induction.

26.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

26.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:6961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

instrument.

Functional hemoglobin was detected in this strain by this method.

27. EXAMPLE 22: COEXPRESSION OF ALPHA-GLOBIN AND GAMMA- GLOBIN CHICO IN YEAST

27.1. TRANSFORMATION OF YEAST STRAINS Sc340 WITH pUT/2A

AND pNT1/γ-Chi

Yeast strain Sc340 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/γ-Chi (see

Section 11.5.14., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2% . At induction, the pH was adjusted to 7.17 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml. Samples were collected between four and 51 hours after induction. 27.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by

Western Blot analysis using procedures described in Section

6.6. Globin was detected at a level of 0.1% of soluble protein.

27.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

instrument.

If hemoglobin is present the difference

spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin. Functional hemoglobin was detected in this strain by this method.

28. EXAMPLE 23: COEXPRESSION OF ALPHA-GLOBIN AND GAMMA- GLOBIN PORTO ALEGRE IN YEAST

28.1. TRANSFORMATION OF YEAST STRAINS Sc1115 WITH pUT/2A

AND pNT1/γ-PORT

Yeast strain Sclll5 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/γ-PORT (see

Section 11.5.1., supra) using electroporation (see Section 9.4., supra).

For the starter culture, cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase. The starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking. The culture was induced by adding galactose to a final concentration of 2%. At induction, the pH was adjusted to 7.17 with KH(2)PO(4) and hemin was added to a final concentration of 40 μg/ml . Samples were

collected between four and 51 hours after induction.

28.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

28.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DTFFERENCE SPECTRUM The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:8961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

instrument.

Functional hemoglobin was detected in this strain by this method.

29. EXAMPLE 19: COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN PORTO ALEGRE IN YEAST

29.1. TRANSFORMATION OF YEAST STRAINS Sc1090 WITH pUT/2A

AND YEpWB51T/PORT

Yeast strain Scl090 was transformed with plasmids pUT/2A (see Section 7, supra) and YEpWB51T/PORT

(see Section 25, supra) using electroporation (see Section

9.4., supra).

collected between four and 51 hours after induction.

29.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN

The expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.02% of soluble protein.

29.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE

DIFFERENCE SPECTRUM

The whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1967, 84:6961). Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO₄, pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording

instrument.

Functional hemoglobin was detected in this strain by this method.

30. DEPOSIT OF MICROORGANISMS

The following yeast strains of the species

Saccharomyces cerevisiae carrying the listed plasmids were deposited with the Agricultural Research Culture Collection (NRRL) , Peoria, IL.

Accession

Yeast strain Plasmid Number Date of Deposit

340g2P YEpWB51WB/PORT Y-18640 April 2, 1990 340VGTB pNML-V-G-1 Y-18641 April 2, 1990 340g2G YEp51T/G Y-18695 August 7, 1990 389gBv22A pUT/2A October 24, 1991 pNT1/β-Bov2

340g22AB143Ar pUT/2A October 24, 1991 pNT1/βl43Arg

1090g2B145T2A PUT/2A October 24, 1991 pNT1/βl45T

1114g2GM22A pUT/2A October 24, 1991 pNT1/γ-Mot2

1114g2BZ104S PNT1/Z104S October 24, 1991

YEp51T/NAT

1041-2-GZ2 pYES2-ζ2 October 24, 1991 1012-G-Z2G-Cot19 pYES2-ζ2 October 25, 1991

YEp51T/G

1041-G-Z2B1-Cot18 YES2-ζ2 October 24, 1991 pNML-V-G-1

1041-5-GE3 YEp51T/ε3 October 24, 1991 1090g2B2Arg2A YEpNTl/B2Arg October 24, 1991 pUT/2A

389g2BMs2A pUT/2A October 24, 1991 pNTl/β-Miss

1114g2B2ATiS pNTl/2ATiS October 24, 1991

YEp5IT/NAT

1114g2B2ATit pNT1/2ATit October 24, 1991

YEp51T/NAT

389g2BMot2A pUT/2A October 24, 1991 pNT1/β-Mot

1115g2GZTi pNT1/Z95An October 24, 1991

YEp51T/G

340g22AGC pUT/2A October 24, 1991 pNT1/γ-Chi

1115g22AGP pUT/2A October 24, 1991 pNT1/γ-PORT

1090g2BP2A pUT/2A October 24, 1991

YEpWB51T/PORT

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also

intended to fall within the scope of the appended claims. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Agricultural Research Culture Collection

1815 North University Street

Peoria, IL 61604

US

Date of Deposit: April 2, 1990 Accession Number Y-18641

Date of Deposit: August 7, 1990 Accession Number Y-18695

Date of Deposit: October 24, 1991 Accession Number N.A.

Claims

WHAT IS CLAIMED IS:

1. A substantially pure hemoglobin variant which comprises (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian adult beta-globin chain, and (ii) comprises an N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme, and which variant has the ability to bind to oxygen at a low oxygen affinity and is free of erythrocyte membrane components and E. coli

endotoxins.

2. A substantially pure hemoglobin variant which (a) is alkali stable; (b) has the ability to bind to oxygen at a low oxygen affinity; and (c) is free of erythrocyte membrane components and E. coli endotoxins.

3. The substantially pure hemoglobin variant of claim 2 which comprises (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially

homologous to a mammalian embryonic alpha-globin chain; (ii) comprises an asparagine at the alpha-94 position; and (iii) comprises a serine at the alpha-104 position; (b) a mammalian beta-like globin chain or heme binding fragment thereof; and (c) heme.

4. A substantially pure hemoglobin variant which comprises (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian fetal gamma-globin chain, and (ii) comprises a glutamic acid at the gamma-127 position; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme, and which variant is stable in alkali and is free of erythrocyte membrane components and E. coli endotoxins.

5. A substantially pure hemoglobin variant which comprises (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian embryonic zeta-globin chain, and (ii) comprises a serine at the zeta-104 position; (b) a mammalian beta-like globin chain or heme binding fragment thereof; and (c) heme, and which variant is stable in alkali and is free of erythrocyte membrane components and E. coli endotoxins.

6. A substantially pure hemoglobin variant of claim 2 which comprises (a) a variant globin chain or heme- binding fragment thereof which (i) is substantially

homologous to a mammalian embryonic zeta-globin chain and

(ii) comprises an asparagine at the zeta-94 position; and (b) a mammalian gamma globin chain or heme binding fragment thereof; and (c) heme, and which variant is stable in alkali and is free of erythrocyte membrane components and E. coli endotoxins.

7. A substantially pure hemoglobin variant which has the ability to bind to oxygen at a high oxygen affinity and is free of erythrocyte membrane components and E. coli endotoxins.

8. The substantially pure hemoglobin variant of claim 7 in which the hemoglobin variant comprises (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian adult beta-globin chain, and (ii) comprises an arginine at the beta-2 position; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme.

9. The substantially pure hemoglobin variant of claim 7 comprising (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially

homologous to a mammalian adult beta-globin chain, and (ii) comprises an arginine at the beta-143 position; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme.

10. The substantially pure hemoglobin variant of claim 7 comprising (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially

homologous to a mammalian adult beta-globin chain, and (ii) which terminates at the beta-145 position; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme.

11. A substantially pure hemoglobin variant which comprises a (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian fetal gamma-globin chain, and (ii) comprises a threonine at the gamma-66 position; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme, and which variant has the ability to bind to oxygen at a low oxygen affinity and is free of erythrocyte

membrane components and E. coli endotoxins.

12. A recombinant DNA vector capable of

expressing a human embryonic epsilon-globin chain in a yeast cell comprising:

(a) a yeast inducible transcriptional

promoter regulated by galactose;

(b) a DNA sequence located downstream from the GAL10 promoter, encoding a human embryonic epsilon-globin chain;

(c) a LEU2 selectable marker or functionally active portion thereof; (d) a 2μ plasmid replication system or functionally active portion thereof; and

(e) a transcription termination sequence located downstream from the DNA sequence encoding the human fetal epsilon-globin chain, which comprises the

transcription termination region of the ADH2 gene

13. The recombinant DNA vector of claim 12 which is plasmid YEp51T/ε3.

14. A yeast cell containing the recombinant DNA vector of claim 13.

15. The yeast cell of claim 14 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof.

16. A recombinant DNA vector capable of

expressing a human embryonic zeta-globin chain in a yeast cell comprising:

(a) a yeast inducible transcriptional

promoter regulated by galactose;

(b) a DNA sequence located downstream from the promoter, encoding a human embryonic zeta-globin chain;

(c) a URA3 selectable marker or functionally active portion thereof;

(d) a 2μ plasmid replication system or functionally active portion thereof; and

(e) a transcription termination sequence located downstream from the DNA sequence encoding the fetal zeta-globin chain, which comprises the transcription termination region of the CYC1 gene.

17. The recombinant DNA vector of claim 16 which is plasmid pYES2-ζ2.

18. A yeast cell containing the recombinant DNA vector of claim 17.

19. The yeast cell of claim 18 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

20. A yeast cell containing

(a) a recombinant DNA vector capable of expressing a human embryonic zeta-globin chain in a yeast 'cell comprising:

(i) a yeast inducible transcriptional promoter regulated by galactose;

(ii) a DNA sequence located downstream from the hybrid promoter, encoding a human zeta-globin chain;

(iii) a URA3 selectable marker or functionally active portion thereof;

(iv) a 2μ plasmid replication system or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the human embryonic zeta-globin chain, which comprises the

transcription termination region of the CYC1 gene; and

(b) a recombinant DNA vector capable of

expressing a human fetal gamma-globin chain in a yeast cell comprising:

(i) a GAL10 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human fetal gamma- globin chain;

(iii) a LEU2 selectable marker or functionally active portion thereof;

(v) a transcription termination sequence located downstream from the DNA sequence encoding the fetal gamma-globin chain, which comprises the transcription termination region of the alcohol dehydrogenase I gene.

21. The yeast cell of claim 20 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

22. A yeast cell containing

(a) a recombinant DNA vector capable of expressing a human embryonic zeta-globin chain in a yeast cell comprising:

(i) a yeast inducible transcriptional promoter regulated by galactose;

(iii) a URA3 selectable marker or functionally active portion thereof;

transcription termination region of the CYC1 gene; and

(b) a recombinant DNA vector capable of

expressing a human adult beta-globin chain in a yeast cell comprising:

(i) a hybrid promoter comprising a GAL1-10 promoter and a TDH3 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult beta-globin chain;

(iii) a LEU2 selectable marker or functionally active portion thereof;

(v) a transcription termination sequence located downstream from the DNA sequence encoding the adult beta-globin chain, which comprises the transcription termination region of the alcohol dehydrogenase I gene.

23. The yeast cell of claim 22 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

24. A recombinant DNA vector capable of

expressing a human variant adult beta-globin chain

comprising an N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu, and comprising

(a) a first DNA sequence encoding a human variant globin chain which (i) is substantially homologous to an adult beta-globin chain; and (ii) comprises an N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu;

(b) a yeast transcriptional promoter which promotes the transcription of the first DNA sequence;

(c) a second DNA sequence encoding a yeast selectable marker or functionally active portion thereof;

(d) a yeast replication origin; and

(e) a transcription termination sequence located downstream from the DNA sequence encoding the va riant globin chain .

25 . The recombinant DNA vector of claim 24 which is plasmid pNT1/β-Bov2 .

26. A yeast cell containing the recombinant DNA vector of claim 25.

27. The yeast cell of claim 26 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

28. A recombinant DNA vector capable of expressing in a yeast cell a variant globin chain

substantially homologous to a human embryonic zeta-globin chain and comprising a serine at the zeta-104 position, comprising:

(a) a first DNA sequence encoding a human variant globin chain which (i) is substantially homologous to an embryonic zeta-globin chain; and (ii) comprises a serine at the zeta-104 position;

(d) a yeast replication origin; and

(e) a transcription termination sequence located downstream from the DNA sequence encoding the variant globin chain.

29. The recombinant DNA vector of claim 28 which is plasmid pNT1/Z104S.

30. A yeast cell containing the recombinant DNA vector of claim 29.

31. The yeast cell of claim 30 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof,

32. A recombinant DNA vector capable of expressing in a yeast cell a variant globin chain

substantially homologous to a human fetal gamma-globin chain and comprising a glutamic acid at the gamma-127 position, comprising:

(a) a first DNA sequence encoding a human variant globin chain which (i) is substantially homologous to a fetal gamma-globin chain; and (ii) comprises a glutamic acid at the gamma-127 position;

(d) a yeast replication origin; and

33. The recombinant DNA vector of claim 32 which is plasmid YEpNT1/γ-Mot2.

34. A yeast cell containing the recombinant DNA vector of claim 33.

35. The yeast cell of claim 34 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof,

36. A yeast cell containing

(a) a recombinant DNA vector capable of expressing a human adult alpha-globin chain in a yeast cell comprising:

(i) a first DNA sequence located

encoding a human alpha-globin chain;

(ii) a yeast transcriptional promoter which promotes the transcription of the first DNA sequence;

(iii) a second DNA sequence encoding a selectable marker or functionally active portion thereof;

(iv) a yeast replication origin or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the human adult alpha-globin chain; and

(b) A recombinant DNA vector capable of expressing in a yeast cell a variant globin chain

substantially homologous to a human adult beta-globin chain and comprising an arginine at the beta-143 position, comprising:

(i) a first DNA sequence encoding a variant globin chain substantially homologous to a human variant adult beta-globin chain and comprising an arginine at the beta-143 position;

(iv) a yeast replication origin or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the human variant adult beta-globin chain.

37. The yeast cell of claim 36 which is a

Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

38. A yeast cell containing

(i) a first DNA sequence encoding a human alpha-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

substantially homologous to a human adult beta-globin chain, and terminating at the beta-145 position:

(i) a first DNA sequence encoding a variant globin chain substantially homologous to a human variant adult beta-globin chain, and terminating at the beta-145 position;

(iv) a yeast replication origin or functionally active portion thereof; and

39. The yeast cell of claim 38 which is a

40. A yeast cell containing

(i) a first DNA sequence encoding a human adult alpha-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

substantially homologous to a human adult beta-globin chain and which comprises an arginine at the beta-2 position:

(i) a first DNA sequence encoding a variant globin chain substantially homologous to a human variant adult beta-globin chain, and comprising an arginine at the beta-2 position;

(ii) a yeast transcriptional promoter which, promotes the transcription of the first DNA sequence;

(iii) a second DNA sequence encoding a selectable marker or functionally active portion thereof; (iv) a yeast replication origin or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the human variant adult beta-globin chain .

41. The yeast cell of claim 40 which is a Saccharomyces ςereyjsjae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

42. A yeast cell containing

(i) a first DNA sequence encoding a human adult alpha-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

substantially homologous to a human adult beta-globin chain, and comprising an N-terminal sequence of Met Leu Thr Ala Glu Glu:

(i) a first DNA sequence encoding a variant globin chain substantially homologous to a human variant adult beta-globin chain, and comprising an N-terminal sequence of Met Leu Thr Ala Glu Glu; (ii) a yeast transcriptional promoter which promotes the transcription of the first DNA sequence;

(iv) a yeast replication origin or functionally active portion thereof; and

43. The yeast cell of claim 42 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof.

44. A yeast cell containing

(a) a recombinant DNA vector capable of expressing in a yeast cella variant globin chain which is substantially homologous to a human embryonic zeta-globin chain and comprises a serine at the zeta-104 position, comprising:

(i) a first DNA sequence encoding a variant globin chain which is substantially homologous to a human embryonic zeta-globin chain, and comprises a serine at the zeta-104 position;

(iv) a yeast replication origin or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human embryonic zeta-globin chain; and

(b) a recombinant DNA vector capable of expressing in a yeast cell an adult beta-globin chain:

(i) a first DNA sequence encoding a human adult beta-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the human adult beta-globin chain.

45. The yeast cell of claim 44 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

46. A yeast cell containing

(a) a recombinant DNA vector capable of expressing in a yeast cell a variant globin chain which is substantially homologous to a human adult alpha-globin chain and comprises a serine at the alpha-104 position and an asparagine at the alpha-94 position, comprising:

(i) a first DNA sequence encoding a variant globin chain which is substantially homologous to a human adult alpha-globin chain and comprises a serine at the alpha-104 position and an asparagine at the alpha-94 position;

(iv) a yeast replication origin or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human adult alpha-globin chain; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a human adult beta-globin chain:

(i) a first DNA sequence encoding a human adult beta-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

47. The yeast cell of claim 46 which is a

Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof.

48. A yeast cell containing

(i) a GAL10 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult alpha-globin chain;

(iii) a DNA sequence encoding an LEU2 selectable marker;

(iv) a 2μplasmid replication system or functionally active portion thereof; and

(v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human adult alpha-globin chain, which comprises the transcription termination region of an alcohol

dehydrogenase gene; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human adult beta-globin chain, and comprises a cysteine at the beta-44 position, comprising:

(i) a GAL10 transcriptional promoter;

(ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human adult beta-globin chain, and comprises a cysteine at the beta-44 position;

(iii) a DNA sequence encoding an LEU2 selectable marker or functionally active portion thereof;

(v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human adult beta-globin chain, which comprises the transcription termination region of an alcohol

dehydrogenase gene.

49. The yeast cell of claim 48 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

50. A yeast cell containing

(i) a GAL10 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult alpha- globin chain;

(iii) a DNA sequence encoding an LEU2 selectable marker;

dehydrogenase gene; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human adult beta- globin chain, and comprises a glutamic acid at the beta-127 position, comprising:

(i) a GAL10 transcriptional promoter; (ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human adult beta-globin chain, and comprises a glutamic acid at the beta-127 position;

dehydrogenase gene.

51. The yeast cell of claim 50 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof.

52. A yeast cell containing

(a) a recombinant DNA vector capable of expressing a variant globin chain substantially homologous to a human adult alpha-globin chain and comprising an asparagine at the alpha-94 position in a yeast cell comprising:

(i) a GAL10 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a variant globin chain substantially homologous to a human adult alpha-globin chain and comprising an asparagine at the alpha-94

position;

(iii) a DNA sequence encoding an LEU2 selectable marker;

dehydrogenase gene; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a human adult beta-globin chain, comprising:

(i) a hybrid promoter comprising a GAL1-10 promoter and a TDH3 promoter;

(ii) a DNA sequence located downstream from the hybrid promoter, encoding a human adult beta-globin chain;

dehydrogenase I gene.

53. The yeast cell of claim 52 which is a

54. A yeast cell containing

(a) a recombinant DNA vector capable of expressing a variant globin chain substantially homologous to a human embryonic zeta-globin chain and comprising an asparagine at the zeta-94 position in a yeast cell

comprising:

(i) a GAL10 promoter;

(ii) a DNA sequence located downstream from the GAL10 promoter, encoding a variant globin chain substantially homologous to a human embryonic zeta-globin chain and comprising an asparagine at the zeta-94 position;

(iii) a DNA sequence encoding an LEU2 selectable marker;

dehydrogenase gene; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a human fetal gamma-globin chain, comprising:

(i) a hybrid promoter comprising a GAL1-10 promoter and a TDH3 promoter; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human fetal gamma-globin chain;

dehydrogenase I gene.

55. The yeast cell of claim 54 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

56. A yeast cell containing

(i) a first DNA sequence encoding a human alpha-globin chain;

(iii). a second DNA sequence encoding a selectable marker or functionally active portion thereof;

(iv) a yeast replication origin or functionally active portion thereof; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human fetal gamma- globin chain, and comprises a glutamic acid at the gamma- 127 position, comprising:

(i) a GAL10 transcriptional promoter;

(ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human fetal gamma-globin chain, and comprises a glutamic acid at the gamma-127 position;

(v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human fetal gamma-globin chain, which comprises the transcription termination region of an alcohol

dehydrogenase gene.

57. The yeast cell of claim 56 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

58. A yeast cell containing

(i) a first DNA sequence encoding a human adult alpha-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the human adult alpha-globin chain; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human fetal gamma-globin chain, and comprises a threonine at the gamma-66 position, comprising:

(i) a GAL10 transcriptional promoter;

(ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human fetal gamma-globin chain, and comprises a threonine at the gamma-66 position;

dehydrogenase gene.

59. The yeast cell of claim 58 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

60. A yeast cell containing

(i) a first DNA sequence encoding a human adult alpha-globiri chain;

(iv) a yeast replication origin or functionally active portion thereof; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human fetal gammaglobin chain, and comprises a cysteine at the gamma-9 position, comprising:

(i) a GAL10 transcriptional promoter; (ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human fetal gamma-globin chain, and comprises a cysteine at the gamma-9 position;

dehydrogenase gene.

61. The yeast cell of claim 60 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant,

recombinant, or genetically engineered derivative thereof.

62. A yeast cell containing

(i) a first DNA sequence encoding a human adult alpha-globin chain;

(iv) a yeast replication origin or functionally active portion thereof; and

(b) a recombinant DNA vector capable of expressing in a yeast cell a variant human globin chain which is substantially homologous to a human adult betaglobin chain, and comprises a cysteine at the beta-9 position, comprising:

(i) a GAL10 transcriptional promoter;

(ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human adult beta-globin chain, and comprises a cysteine at the beta-9 position;

dehydrogenase gene.

63. The yeast cell of claim 62 which is a Saccharomyces cerevisiae as deposited with the NRRL and assigned accession number or a mutant, recombinant, or genetically engineered derivative thereof.

64. A method for producing hemoglobin comprising a human embryonic zeta-globin or heme-binding fragment thereof, and a human adult beta-globin chain or hemebinding fragment thereof in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a yeast inducible transcriptional promoter regulated by galactose; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human embryonic zeta-globin chain or heme-binding fragment thereof; (iii) a URA3 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the human embryonic zeta-globin chain or heme-binding fragment thereof, which comprises the transcription termination region of a CYC1 gene; and in which the second recombinant vector comprises: (i) a GAL10 promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult beta-globin chain or heme-binding fragment thereof; (iii) a LEU2 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the adult beta-globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase I gene; and

(b) growing the yeast cell in an appropriate medium such that the zeta-globin chain or heme-binding fragment and the beta-globin or heme-binding fragment thereof are expressed and assembled together with heme in the yeast cell to form hemoglobin.

65. A method for producing hemoglobin comprising a human embryonic zeta-globin or heme-binding fragment thereof, and a human fetal gamma-globin chain or heme-binding fragment thereof in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a yeast inducible transcriptional promoter regulated by galactose; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human embryonic zeta-globin chain or heme-binding fragment thereof; (iii) a URA3 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the human embryonic zeta-globin chain or heme-binding fragment thereof, which comprises the transcription termination region of a CYC1 gene; and in which the second recombinant vector comprises: (i) a GAL10 promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human fetal gamma-globin chain or heme-binding fragment thereof; (iii) a LEU2 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the human fetal gamma-globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase I gene; and

(b) growing the yeast cell in an appropriate medium such that the zeta-globin chain or heme-binding fragment and the gamma-globin or heme-binding fragment thereof are expressed and assembled together with heme in the yeast cell to form hemoglobin.

66. A method for producing hemoglobin comprising a human adult alpha-globin or heme-binding fragment thereof, and a variant beta-globin chain which is substantially homologous to a human adult beta-globin chain, and

comprises a cysteine at the beta-44 position or hemebinding fragment thereof, in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a GAL10 promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult alpha-globin chain or heme-binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker; (iv) a 2μplasmid replication system or functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the variant human adult alphaglobin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase gene; and which second recombinant DNA vector comprises: (i) a GAL10 transcriptional promoter; (ii) a DNA sequence encoding a variant human globin chain which is substantially homologous to a human adult beta-globin chain, and comprises a cysteine at the beta-44 position or heme-binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker or functionally active portion thereof; (iv) a 2μplasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the variant human adult beta- globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase gene; and

(b) growing the yeast cell in an appropriate medium such that the alpha-globin chain or heme-binding fragment and the variant beta-globin or heme-binding fragment thereof are expressed and assembled together with heme in the yeast cell to form hemoglobin.

67. A method for producing hemoglobin comprising a human adult alpha-globin or heme-binding fragment thereof, and a variant beta-globin chain which is substantially homologous to a human adult beta-globin chain, and

comprises a glutamic acid at the beta-127 position or hemebinding fragment thereof, in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two

recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a GAL10 promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human adult alpha-globin chain or heme-binding fragment thereof; (iii) a DNA sequence encoding a LEU2 selectable marker; (iv) a 2μplasmid replication system or functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the variant human adult alpha-globin chain, which comprises the transcription termination region of an alcohol dehydrogenase gene; and which second recombinant DNA vector comprises: (i) a GAL10

transcriptional promoter; (ii) a DNA sequence encoding a variant human globin chain which is substantially

homologous to a human adult beta-globin chain, and

comprises a glutamic acid at the beta-127 position or heme-binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker or functionally active portion thereof; (iv) a 2μplasmid replication system or

functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the variant human adult beta- globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase gene; and

68. A method for producing hemoglobin comprising a variant globin which is substantially homologous to a human adult alpha-globin chain and comprises an asparagine at the alpha-94 position or heme-binding fragment thereof and a human adult beta-globin chain or heme-binding fragment thereof in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a GAL10 promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a variant globin chain substantially homologous to a human adult alpha-globin chain and comprising an

asparagine at the alpha-94 position, or heme-binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker; (iv) a 2μplasmid replication system or functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the variant human adult alpha-globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase gene; and which second recombinant DNA vector comprises: (i) a hybrid promoter comprising a GAL1-10 promoter and a TDH3 promoter; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human adult beta-globin chain or heme-binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker or functionally active portion thereof; (iv) a 2μ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the human adult beta-globin chain or heme-binding fragment † ereof, which comprises the transcription termination region of an alcohol dehydrogenase I gene

(b) growing the yeast cell in an appropriate medium such that the variant alpha-globin chain or hemebinding fragment and the adult beta-globin or heme-binding fragment thereof are expressed and assembled together with heme in the yeast cell to form hemoglobin.

69. A method for producing hemoglobin comprising a human adult alpha-globin or heme-binding fragment thereof, and a variant beta-globin chain which is substantially homologous to a human fetal gamma-globin chain, and comprises a cysteine at the gamma-9 position, or hemebinding fragment thereof, in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a first DNA sequence encoding a human alpha-globin chain or heme-binding fragment thereof; (ii) a yeast transcriptional promoter which promotes the

transcription of the first DNA sequence; (iii) a second DNA sequence encoding a selectable marker or functionally active portion thereof; (iv) a yeast replication origin or functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the human adult alpha-globin chain or heme-binding fragment thereof; and which second recombinant recombinant DNA vector comprises: (i) a GAL10 transcriptional promoter; (ii) a DNA sequence encoding a variant human globin chain which is substantially

homologous to a human fetal gamma-globin chain, and comprises a cysteine at the gamma-9 position, or heme- binding fragment thereof; (iii) a DNA sequence encoding an LEU2 selectable marker or functionally active portion thereof; (iv) a 2μplasmid replication system or

functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the variant human fetal gamma- globin chain or heme-binding fragment thereof, which comprises the transcription termination region of an alcohol dehydrogenase gene.

70. A method for producing hemoglobin comprising a human adult alpha-globin or heme-binding fragment thereof, and a variant beta-globin chain which is substantially homologous to a human adult beta-globin chain, and

comprises a cysteine at the beta-9 position, or heme-binding fragment thereof in a yeast cell comprising the steps of:

(a) introducing into a yeast cell two recombinant DNA vectors in which the first recombinant DNA vector comprises: (i) a first DNA sequence encoding a human adult alpha-globin chain or heme-binding fragment thereof; (ii) a yeast transcriptional promoter which promotes the transcription of the first DNA sequence; (iii) a second DNA sequence encoding a selectable marker or functionally active portion thereof; (iv) a yeast replication origin or functionally active portion thereof; and (v) a

transcription termination sequence located downstream from the DNA sequence encoding the human adult alpha-globin chain or heme-binding fragment thereof; and which second