Tissue specific hypoxia regulated therapeutic constructs

FIELD OF THE INVENTION

This invention relates to chimeric genes (e.g., carried on expression vectors) containing therapeutic genes whose expression is under the control of tissue specific and hypoxia response enhancer elements.

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BACKGROUND OF THE INVENTION

Each year, over a half-million Americans die from heart attacks. Even more--close to 700,000--have non-fatal heart attacks. For these surviving victims, a portion of the heart is usually damaged irreparably. Such cell death of cardiac tissue, called myocardial infarction, is due in large part to tissue damage caused by ischemia and/or ischemia followed by reperfusion.

Similar ischemic damage may occur in many other tissues when the blood supply to the tissue is reduced or cut off. Stroke, deep vein thrombosis, pulmonary embolus, and renal failure are examples.

Surviving victims of ischemic episodes, such as heart attacks, are at substantially greater risk for subsequent episodes of ischemia, which in many cases prove debilitating or fatal. Thus, it would be desirable to have therapeutic methods and compositions by which survivors of heart attacks and other types of ischemic insults could lower the risk of tissue damage due to recurrent ischemic/reperfusion episodes.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method for reducing ischemic injury to a cell exposed to hypoxic conditions. The method includes introducing into the cell a chimeric gene containing a hypoxia response element, a therapeutic gene, and a tissue-specific promoter operably linked to the therapeutic gene to control transcription of the therapeutic gene in the cell, where the element is effective to modulate expression of the therapeutic gene. Exposing the cell to hypoxic conditions enhances expression of the gene and expression of the gene is effective in reducing ischemic injury to the cell. The method may be applied to, for example, cardiac cells using a cardiac-specific promoter, kidney cells using a kidney-specific promoter, brain cells using a brain-specific promoter, and vascular endothelium cells using a vascular endothelium-specific promoter. The hypoxia response element may be selected from, for example, the erythropoietin HRE element (HREE1), muscle pyruvate kinase (PKM) HRE element, .beta.-enolase (enolase 3; ENO3) HRE element and endothelin-1 (ET-1) HRE element. The therapeutic gene may be selected from, for example, nitric oxide synthase (NOS), B-cell leukemia/lymphoma 2 (bcl-2), superoxide dismutase (SOD) and catalase.

In a related aspect, the invention includes a chimeric gene, containing a hypoxia response element, a tissue-specific promoter heterologous to the element, and a therapeutic gene. The promoter is operably linked to the therapeutic gene and the element is effective to modulate expression of the therapeutic gene. The method may be used with a variety of cell types and corresponding promoters, for example, as identified above. Suitable cardiac-specific promoters include the .alpha.-MHC.sub.5.5 promoter, .alpha.-MHC.sub.86 promoter, and human cardiac actin promoter. Suitable kidney-specific promoters include the renin promoter. Suitable brain-specific promoters include the aldolase C promoter and the tyrosine hydroxylase promoter. Suitable vascular endothelium-specific promoters include the Et-1 promoter and vonWillebrand factor promoter. Hypoxia response enhancer element useful with the method include HREE1, PKM HRE element, ENO3 HRE element and ET-1 HRE element. Therapeutic genes useful with the method include NOS, Bcl-2, SOD and catalase. The chimeric gene may also be contained in an expression vector.

Another aspect of the present invention includes the above-described chimeric gene carried in an expression vector. The expression vector may be a plasmid, adenovirus vector, retrovirus vector, or the like.

In still another aspect, the invention includes a chimeric gene which contains a hypoxia response element, a tissue-specific promoter heterologous to the element, and a deleterious gene. The promoter is operably linked to the deleterious gene, and the element is effective to modulate expression of the deleterious gene. Suitable promoters include tumor-specific promoters, such as alpha fetoprotein (AFP) promoter. Suitable hypoxia response elements are as articulated above. Deleterious genes useful in this aspect include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk.

In a related aspect, the invention includes a method of causing injury to a cell exposed to hypoxic conditions. The method includes introducing into the cell a vector containing a hypoxia response element, a deleterious gene, and a tissue-specific promoter operably linked to the gene and capable of controlling transcription of the gene in the cell. Exposing the cell to hypoxic conditions enhances expression of the gene, and expression of the gene is effective to cause injury to the cell. Promoters useful with this method include tumor-specific promoters such as the AFP promoter. Specific hypoxia response elements and deleterious genes useful with the method are also as identified above.

The invention further includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response enhancer element(s) (HREE(s)) present in a control region of the muscle pyruvate kinase gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from a muscle pyruvate kinase gene.

Also included in the invention is a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the endothelin-1 gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from an endothelin-1 gene.

Another aspect of the invention includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the enolase 3 (ENO3) gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from an ENO3 gene.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show a schematic diagram of the construction of plasmid pGLHRE (FIG. 1B) from plasmid pGL2PV (FIG. 1A).

FIGS. 2A, 2B, 2C and 2D show a schematic diagram of the construction of plasmids pGLHSA-150HRE (FIG. 2B), pGL.alpha.MHC.sub.86 -HRE (FIG. 2C), and pGLHCA.sub.118 HRE (FIG. 2D), from plasmid pGLHRE (FIG. 2A).

FIGS. 3A and 3B show a schematic diagram of the construction of plasmid pGL.alpha.MHC.sub.1.2 HRE (FIG. 3B) from plasmid pGLHRE (FIG. 3A).

FIGS. 4A and 4B show a schematic diagram of the construction of plasmid pGL.alpha.MHC.sub.1.2 HRE-NOS (FIG. 4B) from plasmid pGL.alpha.MHC.sub.1.2 HRE (FIG. 4A).

FIGS. 5A and 5B show a schematic diagram of the construction of plasmid p.alpha.MHC.sub.1.2 -HRE-Bcl-2 (FIG. 5B) from plasmid pSFFV-Bcl-2 (FIG. 5A).

FIGS. 6A, 6B, 6C, 6D and 6E show a schematic diagram of the construction of plasmids pGLPKM.sub.460 (FIG. 6C), pGLPKMD (FIG. 6D), and pGLPKM.sub.285 (FIG. 6E) from plasmid pGL2BV (FIG. 6B) and a fragment of the PKM promoter (FIG. 6A; SEQ ID NO:7).

FIGS. 7A, 7B and 7C show a schematic diagram of the construction of plasmid pGLET-1.sub.700 (FIG. 7C) from plasmid pGL2BV (FIG. 7B) and a fragment of the ET-1 promoter (FIG. 7A; SEQ ID NO:8).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the sense strand nucleotide sequence of a GATA4 enhancer element (Molkentin, et al., 1984).

SEQ ID NO:2 is the nucleotide sequence of muscle pyruvate kinase (PKM) sense strand primer F.

SEQ ID NO:3 is the nucleotide sequence of PKM reverse strand primer R.

SEQ ID NO:4 is the nucleotide sequence of endothelin-1 (Et-1) sense strand primer F.

SEQ ID NO:5 is the nucleotide sequence of Et-1 reverse strand primer R.

SEQ ID NO:6 is the nucleotide sequence of hypoxia response enhancer element 1 (HREE1), derived from the erythropoietin (EPO) gene (Semenza and Wang), and containing 4 tandem copies of a hypoxia response enhancer (HRE) sequence and cloning linkers.

SEQ ID NO:7 is the nucleotide sequence of a rat muscle pyruvate kinase (PKM) promoter region (Takenaka, et al.).

SEQ ID NO:8 is the nucleotide sequence of a human Et-1 promoter region (Inoue, et al.).

SEQ ID NO:9 is the nucleotide sequence of a human cardiac actin promoter region (Minty and Kedes).

SEQ ID NO:10 is a nucleotide sequence containing a portion of the rat cardiac .alpha.-myosin heavy chain promoter region (Mahdavi, et al.; GenBank Accession # K01464).

SEQ ID NO:11 is a nucleotide sequence containing a portion of the mouse cardiac .alpha.-myosin heavy chain promoter region (Gulick, J., et al.; GenBank Accession # M62404).

SEQ ID NO:12 is the nucleotide sequence of a human B-cell leukemia/lymphoma 2 (bcl-2) gene (Tsujimoto, et al.; GenBank Accession # M13994).

SEQ ID NO:13 is the predicted amino acid sequence from SEQ ID NO:12.

SEQ ID NO:14 is the nucleotide sequence of a rat nitric oxide synthase (bNOS) gene (Bredtet al.; EMBL Accession # X59949).

SEQ ID NO:15 is the predicted amino acid sequence from SEQ ID NO:14.

SEQ ID NO:16 is the nucleotide sequence of a human bcl-2 fusion gene (Seto, et al.; EMBL Accession # X06487).

SEQ ID NO:17 is the predicted amino acid sequence from SEQ ID NO:16.

SEQ ID NO:18 is the nucleotide sequence of a human NOS-1 gene (Fujisawa, et al.); DDBJ Accession # D16408; NCBI Seq ID 506339)

SEQ ID NO:19 is the predicted amino acid sequence from SEQ ID NO:18.

SEQ ID NO:20 is the nucleotide sequence of a human NOS-SN gene (Nakane, et al.; GenBank Accession # L02881)

SEQ ID NO:21 is the predicted amino acid sequence from SEQ ID NO:20.

SEQ ID NO:22 is the nucleotide sequence of a 256 base pair (bp) 3' EPO-1 hypoxia response enhancer element (Semenza and Wang).

SEQ ID NO:23 is the nucleotide sequence of a 42 bp 3' EPO-1 hypoxia response enhancer element (Madan, et al.).

SEQ ID NO:24 is the nucleotide sequence of an 86 bp rat .alpha.MHC promoter region.

SEQ ID NO:25 is the nucleotide sequence of a mouse catalase gene (Reimer, et al.; GenBank # L25069).

SEQ ID NO:26 is the predicted amino acid sequence from SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence of a human manganese superoxide dismutase (SOD) gene (Clair, et al.; EMBL # X59445).

SEQ ID NO:28 is the predicted amino acid sequence from SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence of a human .beta.-enolase (ENO3) gene (Giallongo, et al.; EMBL # X56832) between nucleotides -628 to +63.

SEQ ID NO:30 is the predicted amino acid sequence from SEQ ID NO:29.

SEQ ID NO:31 is a consensus sequence of a region present in both the PKM and ENO3 promoters.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Ischemia" is defined as an insufficient supply of blood to a specific organ or tissue. A consequence of decreased blood supply is an inadequate supply of oxygen to the organ or tissue (hypoxia). Prolonged hypoxia may result in injury to the affected organ or tissue. "Anoxia" refers to a virtually complete absence of oxygen in the organ or tissue, which, if prolonged, may result in death of the organ or tissue.

"Hypoxic condition" is defined as a condition under which a particular organ or tissue receives an inadequate supply of oxygen.

"Anoxic condition" refers to a condition under which the supply of oxygen to a particular organ or tissue is cut off.

"Reperfusion" refers to the resumption of blood flow in a tissue following a period of ischemia.

"Ischemic injury" refers to cellular and/or molecular damage to an organ or tissue as a result of a period of ischemia and/or ischemia followed by reperfusion.

An "element", when used in the context of nucleic acid constructs, refers to a region of the construct or a nucleic acid fragment having a defined function. For example, a hypoxia response enhancer element is a region of DNA that, when associated with a gene operably linked to a promoter, enhances the transcription of that gene under hypoxic conditions.

The term "operably linked", as used herein, denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the coding region of a gene, whereby the transcription of the coding region is under the control of the regulatory region.

Two nucleic acid elements are said to be "heterologous" if the elements are derived from two different genes, or alternatively, two different species. For example, a hypoxia response enhancer element from a human erythropoietin gene is heterologous to a promoter from a human myosin gene. Similarly, a hypoxia response enhancer element from a human erythropoietin gene, for example, is heterologous to a promoter from a mouse erythropoietin gene.

"Control region" refers to specific sequences at the 5' and 3' ends of eukaryotic genes which may be involved in the control of either transcription or translation. For example, most eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription initiation site. Similarly, most eukaryotic genes have a CXCAAT region (X may be any nucleotide) 70 to 80 bases upstream from the start of transcription. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may be the signal for addition of the polyadenylation tail to the 3' end of the transcribed mRNA.

"Chimeric gene" also termed "chimeric DNA construct" refers to a polynucleotide containing heterologous DNA sequences, such as promoter and enhancer elements operably linked to a therapeutic gene. For example, a construct containing a human .alpha.-myosin heavy chain (.alpha.-MHC) promoter fragment operably linked to a human bcl-2 gene and containing a human erythropoietin gene hypoxia response element comprises an exemplary chimeric gene.

I. Overview of the Invention

The present invention relates to chimeric genes having at least three functional elements: (i) a therapeutic gene, (ii) a tissue-specific promoter, and (iii) a hypoxia response enhancer (HRE) element. The tissue-specific promoter in combination with the HRE element directs expression of the therapeutic gene in a selected tissue under hypoxic conditions.

The gene is preferably introduced into a target tissue as part of a complete expression vector in a pharmaceutically-acceptable vehicle, either by direct administration to the target tissue (e.g., injection into the target tissue), or by systemic administration (e.g., intravenous injection). In the latter case, the gene may be targeted to a selected tissue, for example, by incorporating it in a virion expressing a modified envelope protein designed to bind to receptors preferentially expressed on cells from the selected, or targeted, tissue. Regardless of the delivery means, expression of the gene in tissues other than the target tissue, and under conditions other than hypoxic or anoxic is preferably minimal.

As described below, a variety of therapeutic genes, promoters, HRE elements and gene delivery means may be employed in the practice of the present invention.

II. Tissue Specific Promoters

A promoter, in the context of the present specification, refers to a polynucleotide element capable of regulating the transcription of a gene adjacent and downstream (3') of the promoter. The promoter may contain all of, or only a portion of, the complete 5' regulatory sequences of the gene from which it is derived. A sequence in the promoter region is typically recognized by RNA polymerase molecules that start RNA synthesis.

A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the chimeric gene containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult).

Promoters useful in the practice of the present invention are preferably tissue-specific--that is, they are capable of driving transcription of a gene in one tissue while remaining largely "silent" in other tissue types. It will be understood, however, that tissue-specific promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 5. Preferably, the selectivity ratio is greater than about 15.

It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the present invention. For example, promoters that are active in a variety of central nervous system (CNS) neurons may be therapeutically useful in protecting against damage due to stroke, which may effect any of a number of different regions of the brain.

Tissue-specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different tissues. For example, a variety of promoters have been identified which are suitable for upregulating expression in cardiac tissue. Included are the cardiac .alpha.-myosin heavy chain (AMHC) promoter and the cardiac .alpha.-actin promoter.

A further desirable characteristic of promoters useful in the present invention is that they possess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissues. One means of achieving this is to select promoters of genes encoding proteins that have a relatively low turnover rate in adult tissue, such as the actin and .alpha.-MHC promoters described herein. Another means is to use "silencer" elements, which suppress the activity of a selected promoter in the absence of hypoxia.

The level of expression of a gene under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis). Vectors used for such experiments typically contains a reporter gene, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities.

This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring hypoxia sensitivity.

A number of tissue specific promoters, described below, may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector.

Alternatively, promoter fragments may be isolated using the polymerase chain reaction (PCR; Mullis, Mullis, et al.). Cloning of amplified fragments may be facilitated by incorporating restriction sites at the 5' ends of the primers.

Promoters suitable for cardiac-specific expression include the promoter from the murine cardiac .alpha.-myosin heavy chain gene. The gene contains a 5.5 kbp promoter region which may be obtained as a 5.5 kbp SacI/Sall fragment from the murine .alpha.MHC gene (Subramaniam, et al., 1991). Reporter gene constructs utilizing this 5.5 kbp .alpha.MHC promoter are expressed at relatively high levels selectively in cardiac tissue (whether or not an HREE is present) and, when present in transgenic animals, are regulated in a similar fashion to the endogenous gene (Subramaniam, et al., 1991).

A smaller fragment of the rat .alpha.-MHC promoter may be obtained as a 1.2 kbp EcoRI/HindIII fragment (Gustafson, et al.). As shown in Example 1 and Table 1, below, constructs utilizing the 1.2 kbp rat .alpha.MHC promoter are expressed at a low level in the absence of an HREE, and at an intermediate level in the presence of an HREE. These results indicate that the .alpha.MHC.sub.1.2 promoter is an exemplary promoter to target expression of chimeric genes of the present invention to cardiac tissue. Expression of genes under the control of this promoter fragment is very low in cardiac cells under normal oxygenation conditions, but is increased by about a factor of four under hypoxic conditions when the construct contains HREE1. Expression in cells other than cardiac cells is at background levels.

An 86 bp fragment of the rat .alpha.MHC promoter, presented herein as SEQ ID NO:24, restricts expression of reporter genes to cardiac and skeletal muscle (i.e., it has lost some tissue selectivity). Additional cardiac specificity may be conferred to the fragment by ligating (e.g., blunt end ligating) a 36-mer oligonucleotide (SEQ ID NO:1) containing cardiac-specific GATA4 enhancer elements just upstream of base pair -86 (Molkentin, et al., 1984). This promoter fragment also results in low levels of expression in the absence of additional enhancers such as HRE elements. The low level of basal expression induced by the 86 bp fragment, and the ability to upregulate this basal level of expression with a hypoxia response enhancer element are useful properties for a promoter for use with the present invention.

The sequences of exemplary cardiac-specific promoter regions from the rat and mouse AMHC genes are presented herein as SEQ ID NO:10 and SEQ ID NO:11, respectively. Both sequences end just upstream of the ATG initiation codons of their respective genes. Other cardiac-specific promoters include the cardiac .alpha.-actin promoter and the myosin light chain-2 (MLC-2) promoter. Constructs described herein utilizing a 118 bp fragment (SEQ ID NO:9) from the human cardiac .alpha.-actin (HCA) promoter result in a relatively low level of cardiac-specific expression, which may be increased by the inclusion of an HREE in the expression construct (Example 1, Table 1). The cardiac-specific myosin light chain-2 promoter may be obtained as a 2.1 kbp KpnI/EcoRI fragment from the rat cardiac myosin light chain-2 (MLC-2) gene (Franz, et al.).

Prostate-specific promoters include the 5'-flanking regions of the human glandular kallikrein-1 (hKLK2) gene and the prostate-specific antigen (hKLK3; PSA) gene (Murtha, et al.; Luke, et al.). The renin promoter is suitable for directing kidney-specific expression (Fukamizu, et al.), while the aldolase-C promoter (Vibert, et al.) or the tyrosine hydroxylase promoter (Sasaoka, et al.) may be used to direct expression in the brain. Promoters specific for vascular endothelium cells include the Et-1 promoter (Inoue, et al.) and vonWillebrand factor (Jahrondi and Lynch) promoter.

Tumor-specific promoters include the .alpha.-fetoprotein (AFP) promoter, contained in a 7.6 kbp fragment of 5'-flanking DNA from the mouse AFP gene (Marci, et al.). This promoter normally directs expression of the AFP gene in fetal liver and is transcriptionally silent in adult tissues. However, it can be abnormally reactivated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissue (Marci, et al.).

The above promoters are exemplary promoters for use with the present invention. Other promoters suitable for use with the present invention may be selected by one of ordinary skill in the art following the guidance of the present specification.

III. Hypoxia Response Enhancer Elements

Therapeutic genes contained in constructs of the present invention are preferably expressed at low levels, if at all, under conditions of normal oxygenation (minimizing any side effects). Under conditions of hypoxia, however, expression of the genes is increased, affording protection to the target tissue. The elevated expression under hypoxic conditions is conferred by the presence of one or more hypoxia response enhancer (HRE) elements.

HRE elements contain polynucleotide sequences that may be located either upstream (5') or downstream (3') of the promoter and/or therapeutic gene. The HRE element (HREE) is typically a cis-acting element, usually about 10-300 bp in length, that acts on a promoter to increase the transcription of a gene under the control of the promoter. Preferably, the promoter and enhancer elements are selected such that expression of a gene regulated by those elements is minimal in the presence of a healthy supply of oxygen, and is upregulated under hypoxic or anoxic conditions.

Hypoxia response enhancer elements are found in association with a number of genes, including the erythropoietin (EPO) gene. Exemplary HRE elements from the EPO gene are presented herein as SEQ ID NO:6, SEQ ID NO:22 and SEQ ID NO:23. The element having the sequence represented as SEQ ID NO:22 results in approximately a five-fold induction of reporter gene expression under hypoxic conditions (Semenza and Wang), while, the element having the sequence represented as SEQ ID NO:23 results in approximately a 17-fold increase in activity under hypoxic conditions (Madan, et al.)

Experiments performed in support of the present invention (e.g., Example 1) demonstrate that expression of constructs containing HREE1 (SEQ ID NO:6) is increased by approximately 5- to 7-fold in response to hypoxic conditions. These results indicate that the HREE1 element is fully functional when fused to muscle and cardiac specific promoters and that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.

Expression of constructs containing a fragment (SEQ ID NO:29) from the control region of the enolase 3 (ENO3) gene was induced approximately 5 to 8 fold by hypoxia in C2C12 cells and cardiac myocytes respectively (see Table 1). These results suggest that the HREE in the ENO3 promoter fragment may be a particularly effective HREE for hypoxia induction in constructs containing a tissue-specific promoter, such as a cardiac or skeletal muscle promoter.

According to the present invention, exemplary hypoxia response enhancer elements may also be isolated from regulatory regions of both the muscle glycolytic enzyme pyruvate kinase (PKM) gene (Takenaka, et al.), the human muscle-specific .beta.-enolase gene (ENO3; Peshavaria and Day), and the endothelin-1 (ET-1) gene (Inoue, et al.). The HRE regions from the PKM gene and the ET-1 gene, identified in experiments performed in support of the present invention (see Materials and Methods, Examples 4 and 5), are presented herein as SEQ ID NO:7 and SEQ ID NO:8, respectively.

Example 4 demonstrates that the expression of pGLPKM, a plasmid containing the HRE element from the PKM gene, in transfected C2C12 myotubes and neonatal cardiac myocytes was increased by 6.+-.2 (n=4) fold in both cell types by incubation of the cells in a hypoxic atmosphere. A portion of this HRE element, obtained by digesting with SmaI to cut at an internal SmaI site, localized the hypoxia response sequence to a 200 bp fragment. This fragment, termed HREPKM.sub.285, confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes that is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).

Both PKM and ENO3 promoters contain a common sequence element (SEQ ID NO:31) located at 5' -88 and -70 bp respectively from the transcription start sites. An oligonucleotide containing this sequence may be sufficient to confer hypoxia response characteristics to constructs of the present invention.

Data presented in Example 5 show that expression of pGLET-1.sub.700, containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), in transfected human arterial endothelial cells was increased approximately 5 -fold by incubation of the cells in a hypoxic atmosphere. No hypoxia-induced increase in pGLET-1.sub.700 expression was seen in other cell types, including HeLa cells, C2C12 cells, and cardiac myocytes. Accordingly, the 700 bp fragment may be used to target hypoxia regulated genes specifically to cells of the vascular endothelium, since the fragment contains element(s) conferring tissue specificity (i.e., elements effective to target expression exclusively to the vascular endothelium), as well as HRE element(s) effective to upregulate transcription of a gene under control of the fragment during hypoxic conditions.

It will be appreciated that the present invention includes the use of HRE elements not explicitly identified above. Additional HRE elements may be identified, for example, as detailed in Examples 4 and 5. Further, promoter deletion and mutation analyses (e.g., as described above and in Webster and Kedes) may be used to identify such elements in other hypoxia responsive genes. A number of such responsive target genes have been shown to be induced when cells are exposed to hypoxia in vitro (e.g., Heakock and Sutherland).

It will also be appreciated that, in certain circumstances, the tissue-specific promoter and the hypoxia response enhancer element(s) of the present invention may be derived from a contiguous polynucleotide sequence from a single gene (e.g., as shown above for the ET-1 promoter region, which contains HRE element(s) and also imparts endothelial cell-specific expression).

IV. Therapeutic Genes

The present invention may be used to alleviate a number of disease conditions resulting from hypoxic and/or anoxic conditions due to ischemia where cell and tissue damage results from ischemia and ischemia followed by reperfusion. The invention is particularly suitable in cases where the subject is diagnosed to be at risk for an ischemic episode in a particular tissue.

For example, it is recognized that virtually all surviving heart attack victims are at significantly increased risk for recurrent episodes of myocardial ischemia. Such subjects would benefit from the introduction of constructs capable of expressing therapeutic genes into their cardiac tissue in order to decrease the risk of injury to the tissue during any subsequent ischemic episodes. Such constructs may serve to protect, for example, cardiac and vascular endothelial tissues from ischemic damage and thereby prevent the progression of the heart disease.

Recurrent ischemia and reperfusion typically results in oxidative damage to cells from reactive oxygen species (free radicals), such as peroxides, that are generated during redox switching (Frei). Contact of fresh blood with damaged or dead cells induces the influx of neutrophils, or pus cells, which kill heart cells which would otherwise have recovered. Much of the damage caused by neutrophils has been attributed to superoxide ions. The superoxide anion can damage tissue in several ways. The interaction of the superoxide anion with hydrogen peroxide leads to the production of hydroxyl radicals which are potentially toxic and react rapidly with most organic molecules. Lipids, proteins, and nucleic acids may all be primary targets for such oxidative damage. The extent and type of damage depend on the severity and nature of the hypoxic stress. For example, the stress may cause cellular damage, initiating an inflammatory response with neutrophil attack and subsequent tissue necrosis. Alternatively, the stress may initiate apoptosis (programmed cell death) to eliminate the damaged cells.

Regardless of the mechanism by which tissue death occurs (necrosis or apoptosis), the damage caused by ischemia-reperfusion episodes is typically the result of redox reactions and is quantitatively related to the severity and duration of the ischemia. For example, in the case of the myocardium, a severe heart attack may result in extensive damage (e.g., infarction of 30% to 40% of the left ventricle), whereas moderate angina and silent repetitive ischemia may result in relatively minor damage during each episode.

While the pathology of ischemia in tissues is complex, resulting in multiple potential targets for therapeutic intervention, several classes of targets are particularly suitable for therapeutic intervention in accordance with the teachings of the present invention. These include anti-oxidant systems, that may intervene immediately at the sites of intracellular redox reactions to minimize damage, and vasodilator systems, that may minimize the severity of the ischemia by increasing blood flow to vulnerable tissues. Antioxidant proteins amenable for use with the present invention include gene products of Bcl-2, catalase and superoxide dismutase (SOD) genes, while proteins with vasodilative properties include nitric oxide synthase (NOS), which produces the vasodilator nitric oxide (NO).

Bcl-2, an integral inner mitochondrial membrane protein of relative molecular mass .about.25 kDa, has been shown to protect certain cells against apoptosis (Hockenbery, et al., 1990) by acting as an antioxidant (Hockenbery, et al., 1993). Bcl-2 may be an effective therapeutic gene for reducing damage to tissues during ischemic episodes because apoptosis may be a common response of many tissues, including the heart, to oxidative stress (Williams and Smith; Gottlieb, et al.

The enzyme superoxide dismutase (SOD) catalyzes the decomposition of the superoxide anion to peroxide. Enzymes such as superoxide dismutase, free radical scavengers or agents which prevent the influx on neutrophils are able to increase the salvage of heart muscle cells. The enzyme catalase in turn catalyzes the conversion of peroxides to water. Exemplary sequences of a SOD gene and a catalase gene are presented herein as SEQ ID NO:27 and SEQ ID NO:25, respectively. The sequence presented herein as SEQ ID NO:27 encodes a manganese SOD, which has a relatively long half-life. A related sequence, of a human Cu/Zn SOD, may be found in Gorechi, et al. The Cu/Zn SOD has a shorter half-life than the manganese SOD.

Endothelial-derived nitric oxide (NO) regulates the expression of vasoconstrictors and growth factors by the vascular endothelium (Kourembanas, et al.). Under hypoxia, endothelial cells typically increase expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor. This increase in expression can be reduced or prevented by exposure to NO (Kourembanas, et al.). One of the effects of ET-1 induced vasoconstriction is decreased blood flow to the affected organ or tissue, which can exasperate hypoxic damage due to ischemia. According to the present invention, such damage may be reduced by providing NO to the affected tissue through the expression of a NOS gene under the control of a vascular epithelium or cardiac-specific promoter and hypoxia response enhancer element.

Therapeutic genes of the present invention may be preferably derived from the same or related species as the one to which the methods and compositions of the present invention are applied. For example, for therapeutic treatment of a dog, it may be desirable to utilize a construct containing a therapeutic gene cloned from a dog. Similarly, for treatment of human conditions, it may be desirable to utilize therapeutic genes cloned from human-derived nucleic acids.

The genes encoding the proteins discussed above represent exemplary therapeutic genes useful in the practice of the present invention. It will be appreciated, however, that following the teachings and guidance of the present specification, one of skill in the art may select other therapeutic genes effective to reduce cellular damage due to hypoxia or ischemia, and that the use of such genes is considered to be within the scope of the present invention.

V. Deleterious Genes

In another aspect, the present invention includes constructs containing deleterious genes, rather than therapeutic genes. Expression of the deleterious genes is targeted to tissues which are harmful (e.g., malignant tumors) or otherwise undesirable. Promoters and hypoxia response elements may be selected as described above. Promoters useful in this aspect of the invention preferably restrict expression only to the undesirable tissue. For example, as discussed above, the AFP promoter can be activated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissues (Marci, et al.).

Deleterious genes include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk. This gene is not deleterious by itself, but when expressed, viral TK can phosphorylate ganciclovir (GCV), turning GCV into a cytotoxic compound. Since tumor cells are typically hypoxic, constructs having a tumor-specific promoter operably linked to a viral tk and an HREE may be used in conjunction with GCV to selectively kill tumor cells.

VI. Expression Vectors

Chimeric genes of the present invention are preferably incorporated into expression vectors capable of expressing a therapeutic gene product in a selected eukaryotic host cell (i.e., a target tissue). Such expression vectors may contain, in addition to the chimeric gene, various other sequences useful for effective expression of the therapeutic gene in selected tissues. Such sequences may include, for example, sequences necessary for the termination of transcription. These sequences are transcribed as polyadenylated segments in the untranslated portion of the MRNA encoding the desired therapeutic protein. The 3' untranslated regions may also include transcription termination sites.

Molecular techniques and methods useful in the construction of expression vectors are well known in the art (e.g., Ausubel, et al., Sambrook, et al.). Vector constructs made in support of the present invention are designed to express either a reporter gene (e.g., luciferase), or therapeutic genes (e.g., Bcl-2 or NOS). Therapeutic gene expression is under the control of either a ubiquitous promoter (e.g., SV40), or a tissue-specific promoter (e.g., striated muscle or cardiac-specific promoter). Further regulation of expression by hypoxia or anoxia is provided by inclusion of hypoxia response enhancer (HRE) elements (e.g., from the erythropoietin (EPO) gene, muscle specific pyruvate kinase (PKM) gene, enolase 3 (ENO3) gene or the endothelial cell endothelin-1 (Et-1) gene).

The generation of exemplary constructs is described in the Materials and Methods section, below. The results of in vitro experiments to assess the performance of constructs having HREE1 and tissue specific promoters are presented in Example 1 and Table 1. The relative amount of gene expression was measured using a reporter gene (luciferase) in place of a therapeutic gene.

The data shown in Table 1 demonstrate that cells containing constructs having a hypoxia response enhancer element, such as HREE1, in combination with a compatible promoter, express the reporter at levels that are 5 to 7 times greater under hypoxic conditions than under aerobic conditions, and that HREE1 is equally active in different cells and independent of the promoter. The data also demonstrate that expression of constructs containing .alpha.-MHC promoters is cardiac specific, and that the basal (aerobic) expression from .alpha.-MHC.sub.1.2 and HCA promoters is relatively low. Further, the data indicate that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.

In vivo experiments conducted with plasmids pGLHRE and pGLHCA.sub.118 HRE (Example 2, Table 2) demonstrate that gene expression in hearts of rats injected with the plasmids and subjected to ischemia was approximately 2-fold higher than expression in hearts from control animals (not subjected to ischemia). These results indicate that the direct injection of therapeutic constructs of the present invention into cardiac tissue in vivo is effective to result in the expression of genes carried on those plasmids. Further, these results indicate that expression vectors carrying chimeric genes of the present invention are effective to result in significantly increased levels of expression in response to hypoxia caused by ischemia in vivo.

Since expression was measured at 20 hours after a brief (20 minute) episode of ischemia, it will be appreciated that (i) hypoxia-induced expression may peak significantly earlier than 20 hours, and (ii) repeat ischemic episodes may upregulate expression more the single experimental episode used herein. Accordingly, the 2-fold induction may be an underestimate of the level of enhancement of transcription/expression caused by ischemia.

While the experiments described above were performed with cardiac tissue, it will be appreciated that one of ordinary skill in the art having the benefit of the present specification may perform similar manipulations with other tissues subject to ischemic and or ischemic/reperfusion injury, and that such procedures are within the scope of the present invention.

In vitro experiments (Example 3) demonstrate that cells transfected with reporter (pGLHRE, pGLHCA.sub.118 HRE, pGL.alpha.MHC.sub.1.2 HRE) and therapeutic (pSFFV-Bcl-2 and pNOS-HRE) constructs appear normal and respond to stimuli as expected. Reporter-transfected cells differentiate normally and respond to hypoxia with the predicted induction of reporter, while NOS and bcl-2-transfected cells appear normal both during the hypoxia and during subsequent reoxygenation. These results suggest that inclusion of HRE elements, Bcl-2 over-expression, and hypoxia-induced over-expression of NOS is not toxic or deleterious to muscle cells in vitro.

These results also suggest that expression vectors carrying therapeutic genes of the present invention may be effective to protect tissues from ischemic damage. Such protective effects may be assayed in an animal model by, for example, infecting myocardial tissue with an expression vector containing a chimeric gene of the present invention, such as an adenoviral vector expressing a therapeutic gene (e.g., Bcl-2 or SOD), a cardiac-specific promoter, and an HRE element, as described, for instance, in Example 2.

Following infection, the animals may be subjected to repeat ischemic episodes (e.g., 30 minutes to 1 hour) followed by reperfusion (e.g., 1 to 8 hours). Following the last reperfusion, the animals may be sacrificed and the ischemic regions of the myocardium may be tested for the presence and extent of infarction as described, for example, by Thornton, et al., and for the presence of apoptosis as described, for example, in Gottlieb, et al. Sample biopsies may also be assayed for expression of the therapeutic gene by Northern blots.

Similar experiments may be performed using constructs direct (e.g., via an appropriate promoter) to other tissues, such as brain, kidney and vascular endothelium.

VII. Delivery of Constructs to Cells and Tissues

Any of a variety of methods known to those skilled in the art may be used to introduce chimeric genes of the present invention into selected target tissue cells. For example, gene therapy of cardiac tissue has included lipofection, retrovirus and adenovirus-mediated gene transfer, and injection of naked DNA directly into the vascular endothelium or cardiac tissue (Nabel, et al.; Lin, et al.; Leclere, et al.; Flugelman, et al.). These and other methods are discussed more fully in the sections below.

Viral-Mediated Gene Transfer.

Host cells may be transfected with chimeric genes of the present invention by infection with mature virions containing hybrid vectors (the chimeric genes along with selected viral sequences). The virions used to transfect host cells are preferably replication-defective, such that the virus is not able to replicate in the host cells.

The virions may be produced by co-infection of cultured host cells with a helper virus. Following coinfection, the virions are isolated (e.g., by cesium chloride centrifugation) and any remaining helper virus is inactivated (e.g., by heating). The resulting mature virions contain a chimeric gene of the present invention and may be used to infect host cells in the absence of helper virus. Alternatively, high titers of replication-defective recombinant virus, free of helper virus, may be produced in packaging cell lines containing those components for which the virus is defective (Miller).

Several types of viruses, including retroviruses, adeno-associated virus (AAV), herpes virus, vaccinia virus, and several RNA viruses may be amenable for use as vectors with chimeric gene constructs of the present invention. Each type of virus has specific advantages and disadvantages, which are appreciated by those of skill in the art. Methods for manipulating viral vectors are also known in the art (e.g., Grunhaus and Horowitz; Hertz and Gerard; and Rosenfeld, et al.)

Retroviruses, like adeno-associated viruses, stably integrate their DNA into the chromosomal DNA of the target cell. Unlike adeno-associated viruses, however, retroviruses typically require replication of the target cells in order for proviral integration to occur. Accordingly, successful gene transfer with retroviral vectors depends on the ability to at least transiently induce proliferation of the target cells.

Retroviral vectors are attractive in part due to the efficiency of transfection--some vectors can stably transduce close to 100% of target cells. The use of retroviral vectors for in vivo gene therapy has been limited, in part, by the requirement of appropriate viral receptors on the target cell. Because the identities of most retroviral receptors are unknown, it has not been possible to determine the distribution of receptors in different cell types. Accordingly, the targeting of specific cell types by retroviral vectors has in many cases proven problematic.

This difficulty may be circumvented by modifying the envelope protein of the retrovirus to contain a ligand for a known endogenous (not necessarily viral) receptor expressed on the target cells. An application of this technique is described in detail by Kasahara. Preferably, the virus also contains an unmodified envelope protein to facilitate cell entry. A number of receptors, such as desmin, E-selectin, and A-CAM, are expressed preferentially on cardiac cells and may be amenable to this approach (e.g., Hansen and Stawaski; Lefer, et al.; Youker, et al.).

Adeno-associated viruses are capable of efficiently infecting nondividing cells and expressing large amounts of gene product. Furthermore, the virus particle is relatively stable and amenable to purification and concentration. Replication-defective adenoviruses lacking portions of the E1 region of the viral genome may be propagated by growth in cells engineered to express the E1 genes (Jones and Shenk; Berkner; Graham and Prevea). Most of the currently-used adenovirus vectors carry deletions in the E1A-E1B and E3 regions of the viral genome. A number of preclinical studies using adenoviral vectors have demonstrated that the vectors are efficient at transforming significant fractions of cells in vivo, and that vector-mediated gene expression can persist for significant periods of time (Rosenfeld, et al.; Quantin, et al.; Stratford-Perricaudet, et al., 1992a; Rosenfeld, et al.; L. D. Stratford-Perricaudet, et al., 1992b; Jaffe, et al.). Several studies describe the effectiveness of adenovirus-mediated gene transfer to cardiac myocytes (Kass-Eisler, et al.; Kirshenbaum, et al.).

Herpes virus vectors (Breakefield and DeLuca; Freese, et al.) are particularly well suited for the delivery and expression of foreign DNA in cells of the central nervous system (CNS), since they can efficiently infect mature, postmitotic neurons. Methods for manipulating the vectors and transfecting CNS cells are well known (see, e.g., Kennedy and Steiner; Yung). A number of studies describe methods for transplanting genetically modified cells into different regions of the brain (Malim, et al.; Rossi and Sarver; Sullenger, et al.; Morgan, et al.; Chatterjee, et al.; Malin, et al.; Hope, et al.). Studies utilizing direct injection of vectors into CNS tissue have also been performed (e.g., Zhang, et al.).

Naked DNA Iniection

Plasmids bearing chimeric genes of the present invention may be purified and injected directly into a target tissue, as exemplified in Example 2 for rat cardiac tissue. The data discussed in Example 2 demonstrate that cardiac injection of plasmid suspended in saline buffer is effective to result in expression of the plasmid in the cardiac cells. Similar approaches have been used successfully by others to express, for example, exogenous genes in rodent cardiac and skeletal muscle (Wolf, et al.; Ascadi, et al., 1991a; Ascadi, et al., 1991b; Lin, et al.; Kitsis, et al..

Liposome-Mediated Gene Transfer

Liposomes may be employed to deliver genes to target tissues using methods known in the art. The liposomes may be constructed to contain a targeting moiety or ligand, such as an antigen, an antibody, or a virus on their surface to facilitate delivery to the appropriate tissue. For example, liposomes prepared with ultraviolet (UV) inactivated Hemagglutinating Virus of Japan (HVJ) may be used to deliver DNA to selected tissues (Morishita, et al.).

The liposomes may also be surface-coated, e.g., by incorporation of phospholipid--polyethyleneglycol conjugates, to extend blood circulation time and allow for greater targeting via the bloodstream. Liposomes of this type are well known.

Receptor-Mediated Gene Transfer

Receptor-mediated endocytic pathways for the uptake of DNA may permit the targeted delivery of genes to specific cell types in vivo. Receptor-mediated methods of gene transfer involve the generation of complexes between plasmid DNA and specific polypeptide ligands (Wu) that can be recognized by receptors on the cell surface. One of the problems with receptor-mediated uptake for gene delivery is that the endocytic vesicles formed during this process may be transported to the lysosome, where the contents of the endosome are degraded. Methods have been developed to facilitate escape of the DNA from the endosome during the course of its transport. For example, either whole adenovirus (Wagner, et al., 1992a; Christiano, et al.) or fusogenic peptides of the influenza HA gene product (Wagner, et al., 1992b) may be used to induce efficient disruption of DNA-containing endosomes.

Administration of Constructs

In cases such as those outlined above, where a vector may be targeted to selectively transfect a specific population of cells, it will be understood that in addition to local administration (such as may be achieved by injection into the target tissue), the vector may be administered systemically (e.g., intravenously) in a biologically-compatible solution or pharmaceutically acceptable delivery vehicle. Vector constructs administered in this way may selectively infect the target tissue. According to the present invention, the presence of a target tissue-specific promoter on the construct provides an independent means of restricting expression of the therapeutic gene.

VIII. Applications

A. Therapeutic Applications

Compositions and methods of the present invention may be useful to prevent tissue damage and/or death, due to ischemia and/or subsequent reperfusion, in a variety of tissues. As stated above, an exemplary application is in the reduction of damage due to recurrent myocardial ischemia following a heart attack. The expression of therapeutic genes in the cardiac tissue of heart attack victims may decrease the risk of injury to the tissue during any subsequent ischemic episodes.

Similarly, subjects who have been diagnosed with transient cerebral ischemia, blood clots or other risk factors for stroke may benefit from the use of hypoxia-inducible brain-specific constructs. Subjects diagnosed with acute or chronic renal failure are at greater risk for further ischemic damage to the kidneys (e.g., Rosenberg and Paller). Such subjects may benefit from a therapeutic gene under the control of a kidney-specific promoter, expression of which is enhanced by hypoxic conditions. A variety of other tissues diagnosed as "at risk" for ischemia may be similarly protected, as will be appreciated by one of skill in the art having the benefit of the present specification.

In addition to the utilities discussed above, compositions (e.g., expression vectors containing chimeric genes of the present invention) and methods of the present invention also have a number of applications in animal medicine. Although animals do not usually develop classical atherosclerosis, cardiomyopathies are very common. A number of species develop ischemia-related syndromes, including arteritis, vasculitis, and related vasculopathies, that result in direct redox damage to cells and tissues, particularly to vascular walls and myocardial tissues. Such conditions may be alleviated by administration of chimeric genes of the present invention.

A common and serious condition in horses and ponies involves ascending colonic ischemia, usually caused by strangulation obstruction (Dabareiner, et al.; Sullivan, et al.; Wilson and Stick). A related disease in dogs is called gastric dilation-volvulus (Lantz, et al.). Treatment of these disorders typically involves surgical removal of the obstruction. Reperfusion following such surgery can result in significant injury to reperfused tissues, and typically triggers an inflammatory response with progressive tissue necrosis. The reperfusion may also results in death of the animal due to cardiogenic shock. Compositions and methods of the present invention may be used therapeutically to treat such conditions, and to provide protection to vulnerable tissues, including heart and vascular endothelium, during the treatment of the above syndromes.

Another utility of the present invention is the treatment of cardiac disease in cats and dogs (Miller, et al.). A variety of forms of cardiovascular disease have been described in both cats and dogs, including dilated cardiomyopathy, left ventricular hypertrophy, and hyperthyroidism (Fox, et al.; Atkins, et al.). Systemic necrotizing vasculitis, a condition that may be analogous to atherosclerosis in humans (with regard to plaque formation and intimal proliferation), has been described in Beagles (Scott-Moncrieff, et al.). Each of these conditions may involve ischemia and reperfusion redox injuries to cardiac and vascular tissue that may be treated using the methods and compositions of the present invention.

B. Reporter Constructs for Diagnostic Applications

The present invention may also be employed in diagnostic applications, where it is desirable to localize the site of hypoxia or anoxia. According to this aspect of the invention, therapeutic genes are replaced by reporter genes, such as those used in experiments performed in support of the present invention (e.g., luciferase). The chimeric genes containing the reporter genes under the control of a selected promoter and a hypoxia response element are introduced into a tissue where it is desirable to localize the site of hypoxia. Hypoxia is localized by increased expression of the reporter gene.

The following examples illustrate but in no way are intended to limit the present invention.

MATERIALS AND METHODS

Unless indicated otherwise, chemicals and reagents were obtained from Sigma Chemical Company (St. Louis, Mo.) or Mallinckrodt Specialty Chemicals (Chesterfield, Mo.), restriction endonucleases were obtained from New England Biolabs (Beverly, Mass.), and other modifying enzymes and biochemicals were obtained from Pharmacia Biotech (Piscataway, N.J.), Boehringer Mannheim (Indianapolis, Ind.) or Promega Corporation (Madison, Wis.). Materials for media for cell culture were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO (Detroit, Mich.). Unless otherwise indicated, manipulations of cells, bacteria and nucleic acids were performed using standard methods and protocols (e.g., Titus; Sambrook, et al.; Ausubel, et al.).

A. Definitions

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Several transformation methods are commonly used in the art, and may be found, for example, in Ausubel, et al., and Sambrook, et al.

"Transfection" refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO.sub.4 and electroporation. Successful transfection is generally recognized when any indication of the operation of the expression vector occurs within the host cell.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences (restriction sites) in the DNA. The various restriction enzymes used herein are commercially available (e.g., New England Biolabs, Beverly, Mass.) and their reaction conditions are known to the ordinarily skilled artisan. For analytical purposes, typically 1 .mu.g of a plasmid or of a DNA fragment is used with about 2 units of enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 10 .mu.g of DNA are digested with about 20 to 40 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about one hour at 37.degree. C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion, the reaction products are run on a gel (e.g., agarose) to isolate desired fragments.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (e.g., Sambrook, et al.). Unless otherwise noted, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0.5 .mu.g of approximately equimolar amounts of the DNA fragments to be ligated.

"Filling" or "blunting" refer to the procedures by which the single stranded end in the cohesive terminus of a restriction enzyme-cleaved nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus. Typically, blunting is accomplished by incubating 2-15 .mu.g of the target DNA in a buffer containing 10 mM MgCl.sub.2, 1 Mm dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) at about 37.degree. C. in the presence of 8 units of the Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, Ind.) and 250 .mu.M of each of the four deoxynucleoside triphosphates (Boehringer Mannheim). The incubation is generally terminated after about 30 min. The reaction products may be purified using standard phenol and chloroform extraction methods followed by ethanol precipitation.

"Northern" blotting is a method by which the presence of a cellular MRNA is confirmed by hybridization to a known, labelled oligonucleotide, DNA or RNA fragment. For the purposes herein, unless otherwise provided, Northern analysis shall mean electrophoretic separation of RNA, typically MRNA, on agarose (e.g., 1%) in the presence of a denaturant (e.g., 7% formaldehyde), transfer to nitrocellulose or nylon membrane, hybridization to the labelled fragment, washing, and detection of the labeled fragment, as described by Sambrook, et al.

B. Cells and Media

HeLa cells, Hep G2 cells and C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC; Rockville, Md.). Human arterial endothelial cells were obtained from Clonetics Corp. (San Diego, Calif.). Unless otherwise indicated, the cells were grown at 37.degree. C. under 5 or 10% CO.sub.2 in MEM or DMEM medium (Gibco/BRL) containing 10% fetal bovine serum (Gibco/BRL).

Cardiac myocytes were isolated and cultured as described previously (Bishopric, et al., Webster and Bisphopric, 1992). Briefly, hearts from about 30 (three litters) were minced and subjected to serial trypsin digestion to release single cells. After the final digestion, the cells were washed and preplated for 0.5 h in minimal essential medium (MEM; Gibco/BRL, Gaithersburg, Md.) with 5% fetal calf serum (FCS; Gibco/BRL). Nonattached cells were re-plated in 60-mm Falcon dishes (Becton Dickinson Labware, Lincoln Park, N.J.) at a density of about 2.5.times.10.sup.6 cells per dish in MEM containing 5% fetal calf serum, 2.0 g/l glucose and 10 mM HEPES, and grown at 37.degree. C. under 5 or 10% CO.sub.2.

C. DNA

1. Therapeutic Genes

Bcl-2 cDNA was obtained in the expression vector pSFFV-Bcl-2 from Dr. Stanley Korsemeyer (Washington University, St. Louis, Mo.; Hockenbery, et al., 1990). Nitric oxide synthase (bNOS) cDNA was obtained from Dr. Solomon Snyder in the vector pNOS (Johns Hopkins University, Baltimore, Md.; Bredt, et al., 1991).

2. Promoters

(i) Cardiac-specific

p.alpha.MHC.sub.5.5 CAT, containing 5.5 kilobases (Kb) 5' of the mouse .alpha.-myosin heavy chain (.alpha.MHC) promoter ligated to the chloramphenicol acetyl transferase (CAT) gene, was obtained from Dr. Jeffrey Robbins (University of Cincinnati, College of Medicine, Cincinnati, Ohio; Subramaniam, et al.).

p.alpha.MHC.sub.2.0 CAT, containing 2.0 Kb of the rat .alpha.MHC promoter ligated to the CAT gene, was obtained from Dr. Thomas Gustafson (University of Maryland, Baltimore, Md.; Gustafson, et al.).

p.alpha.MHC.sub.86 CAT, containing 86 base pairs (bp) of the rat .alpha.MHC promoter ligated to the CAT gene, was obtained from Dr. Bruce Markham (Medical College of Wisconsin, Milwaukee, Wis.). The construct was made by 5' truncation of p.alpha.MHC2.0CAT and blunt end ligation to the CAT gene. The sequence of the 86 bp promoter fragment is provided herein as SEQ ID NO:24.

pHCA.sub.118 CAT, containing 118 bp of the region 5' of the human cardiac .alpha.-actin promoter ligated to the CAT gene, was also obtained from Dr. Larry Kedes (Minty and Kedes).

(i) Skeletal muscle-specific

pHSA-150CAT, containing 150 bp of the human skeletal muscle .alpha.-actin promoter ligated to the CAT gene, was obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Muscat and Kedes).

3. Hypoxia Response Elements

A construct containing four tandem copies of the erythropoietin gene 3' hypoxia inducible enhancer element cloned into the BamHI site of pGEM-4Z (Promega Corp., Madison, Wis.) was obtained from Dr. Greg Semenza (Johns Hopkins University School of Medicine, Baltimore, Md.; Semenza and Wang, 1992). The enhancer element fragment, termed herein as HREE1 (SEQ ID NO:6), was excised from the pGEM vector by cleavage with SmaI and HincII for blunt end subcloning into constructs of the present invention (below).

A construct containing 691 bp (-628 to +63) of the .beta.-enolase (ENO3) gene was obtained from Dr. Charlotte Peterson (Veterans Administration Medical Center, University of Arkansas, Little Rock, Ark.). A sequence containing this region is presented herein as SEQ ID NO:29.

4. Chimeric Genes and Expression Vectors of the Present Invention

The vector pGL2PV (plasmid-gene-1 lght-promoter-vector; Promega Corp., Madison, Wis.), was used as the base vector for the construction of most of the plasmids described below. pGL2PV is a eukaryotic expression vector containing the SV40 early promoter upstream of the luciferase gene. The vector multiple cloning (MCS) site is just upstream of the SV40 promoter, and is designed for the insertion of DNA fragments containing enhancer sequences. pGL2BV (Promega Corp.) is similar to pGL2PV, but it does not contain an SV40 early promoter.

(i) HREE1/luc Constructs with Different Tissue-Specific Promoters

Plasmid pGLHRE (FIGS. 1B, 2A, 3A) was made by blunt-1 lighting the 240 bp HREE1 fragment (SEQ ID NO:6) into the SmaI site of the MCS of pGL2PV (FIG. 1A).

Plasmid pGLHSA-150HRE (FIG. 2B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 150 bp HindIII-SmaI fragment from pHSA-150CAT containing a fragment of the human skeletal actin (HSA) promoter.

Plasmid pGL.alpha.MHC.sub.86 HRE (FIG. 2C) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 120 bp HindIII-EcoRI fragment from p.alpha.MHC.sub.86 CAT containing 86 bp (SEQ ID NO:24) of the human .alpha.-myosin heavy chain (.alpha.-MHC) promoter. The EcoRI end of the 120 bp fragment was filled in with DNA polymerase I using standard methods (Sambrook, et al.) before blunt end ligation to the vector SmaI site.

Plasmid DGL.alpha.MHC.sub.86 -GATA-HRE was made by cloning a 36 bp oligonucleotide (SEQ ID NO:1; described above), containing a duplicated GATA 4 box into the HindIII site (filled in with polymerase) of plasmid pGL.alpha.MHC.sub.86 HRE, upstream of the 86 bp promoter fragment.

Plasmid pGLHCA.sub.118 HRE (FIG. 2D) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 188 bp HindIII-EcoRI fragment from pHCA.sub.118 CAT, containing 118 bp of the human cardiac actin (HCA) promoter plus 70 bp of actin exon 1. The EcoRI end of the 188 bp fragment was filled in with DNA polymerase I as above before blunt end ligation to the vector SmaI site.

Plasmid pGL.alpha.MHC.sub.1.2 HRE (FIG. 3B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 1.2 kb HindIII-EcoRI fragment from p.alpha.MHC.sub.20 CAT containing 1.2 kb of the human .alpha.-MHC promoter. The EcoRI end of the 1.2 kb fragment was filled in as above in prior to cloning.

(ii) PKM Promoter/luc Constructs

Plasmid pGLPKM.sub.460, containing 460 bp of the rat muscle specific pyruvate kinase (PKM) gene promoter and 140 bp of the PKM coding sequence (SEQ ID NO:7), was created using polymerase chain reaction (PCR) as follows. PKM-specific primers containing endonuclease restriction sites near their 5' end were designed based on the nucleotide sequence of the PKM gene (Takenaka, et al., 1989). PKM primer F (SEQ ID NO:2) contained a KpnI site, while PKM primer R (SEQ ID NO:3) contained a XhoI site. PCR was carried out using the above primers and 1 .mu.g of rat heart genomic DNA as a template for 25 cycles using standard procedures and a Perkin-Elmer (Norwalk, Conn.) DNA thermal cycler. The PCR product (FIG. 6A) was purified by agarose gel electrophoresis, cut with KpnI and XhoI, and cloned into KpnI/XhoI cut pGL2BV (FIG. 6B; Promega Corp., Madison, Wis.), generating pGLPKM.sub.460 (FIG. 6C).

Plasmid pGLPKM.sub.285 (FIG. 6E) was generated by digesting pGLPKM.sub.460 with SmaI to drop out the -460 to -285 portion of the promoter, and religating the vector. pGLPKM.sub.D (FIG. 6D) was generated by digesting pGLPKM.sub.460 with SmaI to isolate the -460 to -285 portion of the promoter, and cloning that fragment into pGL2PV (Promega Corp.) that had been cut with SmaI.

(iii) Et-1 Promoter/luc Constructs

Plasmid pGLET-1.sub.700 (FIG. 7C), containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), was created using PCR to amplify HeLa cell genomic DNA as described above. ET-1 specific primers were designed based on the promoter sequence (Inoue, et al., 1989) of the ET-1 gene. The forward primer (SEQ ID NO:4) contained PstI and KpnI sites, while the reverse primer (SEQ ID NO:5) contained HindIII and XbaI sites. The PCR product (FIG. 7A) was purified by gel electrophoresis, cut with KpnI and HindIII, and cloned into KpnI/HindIII cut pGL2BV (FIG. 7B; Promega Corp.).

(iv) ENO3 Promoter/luc Constructs

Plasmid pGLENO.sub.628 was constructed by cloning a blunt ended genomic DNA containing an ENO3 promoter fragment (-628 to +63; SEQ ID NO:29), isolated from a lambda gt10 human genomic library, into the SmaI site of pGL2BV.

(v) Therapeutic Gene Constructs

Plasmid p.alpha.MHC.sub.1.2 HRE-NOS (FIG. 4B) was made by digesting plasmid pGL.alpha.MHC.sub.1.2 HRE (FIG. 4A) with HindIII and EcoRV to drop out the luciferase cDNA and replacing it with a HindIII/XbaI fragment from pNOS containing a full length NOS CDNA.

Plasmid p.alpha.MHC.sub.1.2 HRE-Bcl-2 (FIG. 5B) was made by digesting pSFFV-Bcl-2 with SalI, blunting the vector as described above, removing the SFFV promoter from the linearized vector with an EcoRI digest, and replacing the SFFV promoter with a SmaI/EcoRI fragment from pgL.alpha.MHC.sub.1.2 HREE containing the 1.2 kb .alpha.MHC promoter fragment and the 240 bp HREE1.

(vi) Other Plasmid Constructs

Plasmid p.alpha.MHC.sub.5.5 HRE-CAT was made by inserting the 240 bp HREE1 immediately 5' of the AMHC promoter of p.alpha.MHC.sub.5.5 CAT.

(vi) Adenoviral Constructs

Adenoviral constructs are made using standard methods (e.g., Friedman,, et al., 1986; Hertz and Gerard, 1993), as follows.

Construct Ad.alpha.MHC1.2Bcl2HREE is made by inserting a 3.34 Kb EcoRI/HindIII fragment from p.alpha.MHC1.2-Bcl-2 (containing 1.2 Kb of the .alpha.-MHC promoter, 1.9 Kb Bcl-2 CDNA, and 240 bp HREE1) into pAPLCMV digested with EcoRI and HindIII to drop out the CMV promoter and CAT gene. pAPLCMV, which may be obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Kass-Eisler, et al., 1993), is a base replication deficient adenoviral expression vector. The backbone adenoviral vector for recombination, p9M17, may also be obtained from Dr. Larry Kedes.

Recombinant pAPLCMV (pAd.alpha.MHC1.2bcl-2HRE) and p9M17 are used to co-transfect 293 cells (ATCC) to propagate the adenovirus.

EXAMPLE 1

Tissue Specific Hypoxia Induced Expression In Vitro

Constructs pGLHRE, pGLHSA-150HRE, p.alpha.MHC.sub.5.5 HRE-CAT, pGL.alpha.MHC.sub.1.2 HRE, pGLHCA.sub.118 HRE and pGL-Eno.sub.628 were tested for tissue-specific expression and hypoxia inducibility in HeLa cells, Hep G2 cells, differentiated C2C12 muscle myotubes, and cardiac myocytes.

______________________________________ A. Buffers and Solutions HEPES buffered saline (HeBS; 2X solution) 16.4 g NaCl 11.9 g HEPES acid 0.21 g Na.sub.2 HPO.sub.4 H.sub.2 O to 1 liter Titrate Ph to 7.05 with 5 M NaOH. PBS Buffer 137 mM NaCl 2.7 mM KCl 4.3 mM Na.sub.2 HPO.sub.4 1.4 mM KH.sub.2 PO.sub.4 Adjust pH to 7.1. Reconstituted Luciferase Assay Reagent (LAR) 20 mM Tricine 1.07 mM (MgCO.sub.3).sub.4 MG(OH).sub.2.5H.sub.2 O 2.67 mM MgSO.sub.4 0.1 mM EDTA 33.3 mM DTT 270 .mu.M coenzyme A 470 .mu.M luciferin 530 .mu.M ATP Cell Culture Lysis Reagent (CCLR; 1X Solution) 25 mM Tris-phosphate, pH 7.8 2 mM DTT 2 mM 1,2-diaminocyclohexane-N,N,N',N'- tetraacetic acid 10% glycerol 1% Triton X-100 ______________________________________

A. Cell Transfection

HeLa cells, C2C12 myocytes, and cardiac myocytes were transfected with the indicated plasmid DNA by the standard calcium phosphate procedure (Ausubel, et al.).

Briefly, 10.sup.5 cells were plated on a 10-cm tissue culture dish and grown for 3 days. The cells were split 1:10 into 10 ml of medium one day before application of plasmid DNA. DNA for transfection was prepared by resuspending an ethanol-precipitated pellet containing 20 .mu.g of the plasmid DNA in 450 .mu.l ddH.sub.2 O and adding 50 .mu.l of 2.5 mM CaCd.sub.2.

500 .mu.l of 2.times. HeBS were added to a 15 ml conical centrifuge tube, and the solution was aerated by bubbling air with a 10 ml pipette attached to an automatic pipettor (Drummond Instruments, Fisher Scientific, Pittsburgh, Pa.). The DNA/CaCl.sub.2 solution was added dropwise, and the resultant mixture was vortexed for 5 seconds and then allowed to sit for 20 minutes at room temperature to form precipitate.

The precipitate was added to the dishes containing the cells and the dishes were incubated overnight.

The cells were washed twice with 5 ml PBS and fed with 10 ml of complete medium. The cells were then allowed to recover for 24 hours before incubation under an atmosphere of 1.0% O.sub.2, 5% CO.sub.2, 94% N.sub.2 for an additional 20 hours.

B. Exposure to Hypoxic Conditions

Two to three days after transfection, the cells were exposed to atmospheric oxygen (approximately 21% O.sub.2, 5% CO.sub.2, balance N.sub.2 ; pO.sub.2 =.about.160 mmHg), or to hypoxic conditions (approximately 0.5-2.0% O.sub.2, 5% CO.sub.2, balance N.sub.2 ; pO.sub.2 =.about.4-8 mmHg) in an environmental chamber (Anaerobic Systems, San Jose, Calif., USA) which was equipped with a Nikon TMS microscope and a continuous readout oxygen electrode (Controls Katharobic, Philadelphia, Pa., USA). Unless otherwise indicated, the cells were kept in the chambers for one day prior to assaying for luciferase expression.

C. Luciferase Expression

Cells transfected and treated as above were assayed for expression of the luciferase enzyme using a standard reaction protocol (Titus). Briefly, 1 ml of CCLR and 1 ml of LAR were allowed to equilibrate at room temperature. The culture medium in the dish containing the cells to be assayed was removed and the cells were rinsed twice in PBS buffer.

Approximately 300 .mu.l of the room-temperature CCLR was added to the dish containing the cells, and the dish was incubated at room temperature for 10-15 minutes. The cells were then scraped off the bottom of the culture dish, and the solution containing the cells was transferred to a micro-centrifuge tube. The tube was centrifuged in a table-top microcentrifuge briefly (about 5 seconds) to pellet large debris.

20 .mu.l of the supernatant (cell extract) were mixed with 100 .mu.l of LAR at room temperature, and the light produced was measured for a period of 5 minutes, starting approximately 5 seconds after mixing, with a model # 1250 LKB luminometer (Bioorbit, Gaithersburg, Md.).

D. Results

Data from HeLa, C2C12, and cardiac cells are given in Table 1, below. Values, presented in arbitrary units, represent averages of three or more experiments for each condition.

TABLE 1 __________________________________________________________________________ REGULATED EXPRESSION OF UBIQUITOUS-MUSCLE- AND CARDIAC-SPECIFIC PROMOTERS BY HYPOXIA GL2PV GLHRE GLHSA.sub.150 HRE .alpha.MHC.sub.1.2 HRE GLHCA.sub.118 HRE GLENO.sub.628 A Hx A Hx A Hx A Hx A Hx A Hx __________________________________________________________________________ HeLa 18 27 56 387 BG BG BG -- C2Cl2 189 204 350 1680 46 278 BG 48 248 320 1560 Cardiac 24 27 22 165 18 94 21 85 38 263 210 1610 __________________________________________________________________________ BG Background

Data shown in the table demonstrate that (i) none of the tested constructs carrying tissue-specific promoters are expressed above background in fibroblast-derived HeLa cells under either normal or hypoxic conditions, (ii) cells containing constructs having HREE1 and a compatible promoter (including the SV40 and tissue-specific promoters) express the reporter at levels that are .about.5 to .about.7 times greater under hypoxic conditions than under aerobic conditions; (iii) the HREE1 element is equally active in different cells and independent of the promoter; (iv) the .alpha.-MHC.sub.1.2 promoter expresses in cardiac, but not in skeletal or fibroblast-derived cells, the HCA.sub.118 promoter expresses in both cardiac and skeletal muscle cells, but not in fibroblast-derived cells, and the HSA.sub.150 promoter expresses in both skeletal and cardiac muscle, with stronger expression in skeletal muscle; and (v) basal (aerobic) expression from .alpha.-MHC.sub.1.2 HCA.sub.118, and HSA.sub.150 promoters is weak.

These results indicate that the HREE1 element is fully functional when fused to muscle and cardiac specific promoters and that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters, and suggest that the .alpha.MHC.sub.1 2 promoter is an exemplary promoter for moderate levels of cardiac-specific expression.

The data also show that both the HREE present in the ENO3 promoter and HREE1, when present in constructs with the SV40 promoter, result in comparable levels of hypoxia induction in skeletal muscle cells. In cardiac cells, however, constructs containing the ENO3 HREE are expressed at significantly higher levels than those containing HREE1. Further, hypoxia increases the level of expression of the ENO3 HREE containing constructs in cardiac cells by over seven-fold, as compared with less than 5-fold in skeletal muscle cells. Plasmid pGLENO.sub.628 confers induced expression in C2C12 myotubes and cardiac myocytes that is at least equivalent to four copies of the erythropoietin HRE (HREE1) in these cells. These results suggest that the HREE in the ENO3 promoter fragment may be a particularly effective HREE for hypoxia induction in constructs targeted with a tissue-specific promoter to cardiac or skeletal muscle cells.

EXAMPLE 2

Tissue Specific Hypoxia Induced Expression In Vivo Following Injection of Constructs into Target Animal Tissue

Constructs of the present invention were injected directly into cardiac tissue using techniques described in Buttrick, et al., (1992) and Buttrick, et al., (1993). Briefly, adult female Wistar rats were anesthetized with an intraperitoneal injection of chloral hydrate (0.7 ml/100 g of a 4% solution). Cardiac injections were made directly into the apex of the heart through a lateral thoractomy, after which the heart was replaced in the chest, the rats were briefly hyperventilated, and the incision closed. Fifty microliters of a DNA solution containing 2 .mu.g/.mu.l of either pGLHRE or pGLHCA.sub.118 HRE in 20% sucrose and 2% Evans blue were injected through a 27-gauge needle. Following injection the rats were subjected to a 20 min ischemia by cannulation of the coronary artery as described by Smith, et al. (1988).

Hypoxia-inducibility of vector expression was assayed as follows. Hearts were excised approximately 20 hours after the induced ischemia and the ventricles were washed with ice-cold phosphate buffered saline (PBS). The tissue was suspended in 1 ml of ice-cold PBS containing 20% sucrose and homogenized with a Polytron (Kinematica, Switzerland) for 45 sec. After centrifugation at 10,000.times.g for 10 min supernatants were analyzed for luciferase expression by the assay method described above. Protein was measured using a BioRad assay kit (BioRad Laboratories, Hercules, Calif.).

The results of the experiments are shown in Table 2, below. Luciferase expression in hearts from rats injected with pGLHRE or pGLHCA118HRE and subjected to ischemia was approximately 2-fold higher than expression in hearts from control animals injected with saline (n=3).

TABLE 2 ______________________________________ ISCHEMIA INDUCIBLE EXPRESSION OF pGLHRE AND pGLHCA.sub.118 HRE IN RAT HEART Luciferase Activity Light Units/mg Protein Plasmid Aerobic 20 min. Ischemic ______________________________________ pGLHRE 1180 2440 pGLHCA.sub.118 HRE 88 127 Control 15 21 ______________________________________ Rat hearts were injected with plasmids as described above. A 20 min. ischemia was imposed on one group (3 rats) and the other (1 control) was sham operated. Tissue samples were harvested and assayed for luciferase expression 20 hr. later.

These results indicate that the direct injection of plasmid DNA, made in accordance with the teachings of the present specification, into hearts of living mammals is effective to result in the expression of genes carried on those plasmids. Further, these results indicate that expression vectors carrying chimeric genes of the present invention are effective to result in significantly increased levels of expression in response to hypoxia caused by ischemia in vivo.

EXAMPLE 3

Stable Expression of Hypoxia Regulated NOS and Bcl-2 Genes In Vitro

10.sup.6 C2C12 myoblasts were cotransfected with pSV2Neo (Minty and Kedes) and a test plasmid at a ratio of 1:19 (1 .mu.g pSV2Neo+19 .mu.g test plasmid) using standard methods (Minty and Kedes, 1986). Test plasmids were pGLHRE, pGLHCA.sub.118 HRE, pGL.alpha.MHC.sub.1.2 HRE, pSFFV-Bcl-2, and pNOS-HRE. Cultures were selected on day 2 following transfection with 400 .mu.g/ml of the neomycin drug G418 (Gibco/BRL). Colonies of cells resistant to G418 appeared after 10 to 14 days. The resistant cells were pooled. Mass cultures were assayed for the expression of luciferase as described above or by Northern blot assay (Webster, et al., 1993) for the expression of Bcl-2 or NOS RNA. Stable lines were positive for expression of the transfected genes.

Mass cultures were subjected to differentiation conditions by transferring them to low mitogen medium (DMEM with 2% horse serum) and were analyzed visually for differentiation into myotubes. There was no apparent difference between transfected and control cells. Approximately 40% of cells were fused into multinucleate myotubes after 24 h in low mitogen medium. All cultures contained approximately 74% myotubes after 48 h.

Reporter-transfected cells differentiated normally and respond to hypoxia with the predicted induction of reporter. NOS-transfected cells appeared normal both during the hypoxia and during subsequent reoxygenation. A stable line of C2C12 cells that constitutively over-expresses Bcl-2 (without HREE1) was also constructed as described above, and the cells showed normal growth and differentiation characteristics.

Taken together, the data presented above suggest that inclusion of HRE elements, Bcl-2 over-expression, and hypoxia-induced over-expression of NOS is not toxic to muscle cells in vitro. Further, the data indicate that the cells may be protected from the deleterious effects of hypoxia by the expression of therapeutic genes (e.g., NOS).

EXAMPLE 4

Expression of DGLPKM Plasmids under Hypoxic Conditions

Plasmid pGLPKM.sub.460 was transfected into C2C12 cells and cardiac myocytes and assayed for luciferase activity as described in Example 1. The expression of PGLPKM in both transfected C2C12 myotubes and neonatal cardiac myocytes was increased by 6.+-.2 fold (n=4) in both cell types by incubation of the cells in an atmosphere containing 0.5% O.sub.2, 5% CO.sub.2, balance N.sub.2 (hypoxic conditions) relative to normal conditions, as described in Example 1.

A portion of this HRE element, obtained by digesting with SmaI to cut at an internal SmaI site, is also effective as a hypoxia response enhancer element. This fragment, termed HREPKM.sub.285, confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes similar to that obtained with pGLPKM.sub.460. This level of hypoxia induction is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).

These results indicate that the PKM promoter fragment contained in the sequence represented as SEQ ID NO:7 contains an HRE element that is effective at enhancing the expression of chimeric genes containing the element under conditions of hypoxia.

The PKM promoter sequence has no significant homology with the erythropoietin HRE consensus, but does share a consensus sequence (SEQ ID NO:31) with the ENO3 promoter fragment (SEQ ID NO:29). This consensus, located approximately 88 bp upstream of the transcription start site of PKM and approximately 70 bp upstream of the transcription start site of ENO3, may represent an important element for conferring enhancement of expression in response to hypoxia.

EXAMPLE 5

Expression of pGLET-1.sub.700 Plasmids under Hypoxic Conditions

Plasmid pGLET-1.sub.700 was transfected into human arterial endothelial cells as described in Example 1. The expression of pGLET-1.sub.700 in these cells was increased 5 fold by incubation of the cells in a hypoxic atmosphere as described above. No significant induction of pGLET-1.sub.700 was observed in any other cell types tested, including HeLa, C2C12, and cardiac myocytes. Elements contained within the 700 bp sequence have no significant homology with the erythropoietin HRE consensus.

These results indicate that the 700 bp fragment of the human ET-1 gene promoter corresponding to the sequence represented herein as SEQ ID NO:8 is effective to (i) restrict expression of genes under its control to the vascular endothelium, and (ii) confer hypoxia-inducibility on the expression of those genes. Accordingly, this fragment, in conjunction with a therapeutic or reporter gene, may be used in the methods of the present invention to both target expression to a selected tissue (vascular endothelium), and confer enhancement of expression by hypoxia.

While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.

__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 31 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: GATA4 Enhancer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CAAAGGGCCGATGGGCAGATAGAGGAGAGACAGGA35 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: PKM primer F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AATTGGTACCCGGGCGAGCGCCGGGAGGGTGGA33 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: PKM primer R (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTAACTCGAGGCACTATGGCATTGGCTCTGGG32 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: ET-1 primer F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TATATCTGCAGGTACCGATAGGGAAAAGACTGGCATGTGCC41 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: ET-1 primer R (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TATATAAGCTTCTAGAGACCCGTTCGCCTGGCGCGCAGATGCA43 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 240 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: HREE1 (Hypoxia responsive enhancer element 1) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC60 CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC120 CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC180 CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCGATCCCG240 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 560 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: sequence containing PKM promoter frag. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GAGTCACCGGGCGGGGCTGGAGGAATGTCCGGGACCTATAAATCTGGGCAACGCCCTGGT60 AGGCCAGGGCAGATGGGGCACCTGGGCAGAATTCCAAAATGGGATTATGTAGCCTCTGAG120 GTCCTAAAGCAACAGGTGGCGGACCACCCGGGGATCTAGGGGTGGTGGCGGCGGTGGACC180 CGAGGGCGGGTCCTGCCTCCTCACCACTTCCCCATTGGCCATCAGAATGACCCATGCGCA240 ATTTTGGTTTGCAATGTCCTTCCGCCACGGAAGGTAGTCCCCCTCAAAAGGGCAACCTGC300 TTGTCCCGCCTACCCTGCGACTCTCTCAGAAGGTGCGGGTGCCTGTTGAGAGGCGGGGCT360 CTGCTAGCTCCTGCCCGGATTGGGCGAGGGGCGGGGCTGCGGAGGGATTGCGGCGGCCCG420 CAGCAGTGATAACCTTGAGGCCCAGTCTGCGCAGCCCCGCACAGCAGCGACCCGTCCTAA480 GTCGACAGACGTCCTCTTTAGGTATTGCAACAGGATCTGAAGTACGCCCGAGGTGAGCGG540 GGAGAACCTTTGCCATTCTC560 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 713 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Sequence containing ET-1 promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GATAGGGAAAAGACTGGCATGTGCCTAAACGAGCTCTGATGTTATTTTTAAGCTCCCTTT60 CTTGCCAATCCCTCACGGATCTTTCTCCGATAGATGCAAAGAACTTCAGCAAAAAAGACC120 CGCAGGAAGGGGCTTGAAGAGAAAAGTACGTTGATCTGCCAAAATAGTCTGACCCCCAGT180 AGTGGGCAGTGACGAGGGAGAGCATTCCCTTGTTTGACTGAGACTAGAATCGGAGAGACA240 TAAAAGGAAAATGAAGCGAGCAACAATTAAAAAAAATTCCCCGCACACAACAATACAATC300 TATTTAAACTGTGGCTCATACTTTTCATACCAATGGTATGACTTTTTTTCTGGAGTCCCC360 TCTTCTGATTCTTGAACTCCGGGGCTGGCAGCTTGCAAAGGGGAAGCGGACTCCAGCACT420 GCACGGGCAGGTTTAGCAAAGGTCTCTAATGGGTATTTTCTTTTTCTTAGCCCTGCCCCC480 GAATTGTCAGACGGCGGGCGTCTGCTTCTGAAGTTAGCAGTGATTTCCTTTCGGGCCTGG540 CTTATCTCCGGCTGCACGTTGCCTGTTGGTGACTAATAACACAATAACATTGTCTGGGGC600 TGGAATAAAGTCGGAGCTGTTTACCCCCACTCTAATAGGGGTTCAATATAAAAAGCCGGC660 AGAGAGCTGTCCAAGTCAGACGCGCCTCTGCATCTGCGCCAGGCGAACGGGTC713 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: HCA118 promoter fragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGAAGGGGACCAAATAAGGCAAGGTGGCAGACCGGGCCCCCCACCCCTGCCCCCGGCTGC60 TCCAACTGACCCTGTCCATCAGCGTTCTATAAAGCGGCCCTCCTGGAGCCAGCCACCC118 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Rat alpha MHC promoter fragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GAATTCTCTTACTATCAAAGGGAAACTGAGTCATGCACCTGCAAAATGAATGCCCTCCCT60 GGACATCATGACTTTGTCCCTGGGGAGCCAGCACTGTGGAACTCCAGGTCTGAGAGTAGG120 AGGCACCCCTCAGCCTGAAGCTGTGCAGATAGCTAGGGTGTAAAAGAGGGAAGGGGGGAG180 GCTGGAATGGGAGCTTGTGTGTTCGGAGACAGGGGACAAATATTAGGCCCGTAAGAGAAG240 GTGACCCTTACCCAGTGTGTTCAACTCAGCCTTTCAGATTAAAAATAACTAAGGTAAGGG300 CCATGTGGGTAGGGGAGGTGGTGTGAGACGGTCCTGTCTCTCCTCTATCTGCCCATCGGC360 CCTTTGGGGAGGAGGAAATGTGCCCAAGGACTAAAAAAGGCCTGGAGCCAGAGGGGCTAG420 GGCTAAGCAGACCTTTCATGGGCAAACCTCAGGGCTGCTGTCCTCCTGTCACCTCCAGAG480 CCAAGGGATCAAAGGAGGAGGAGCCAGACAGGAGGGATGGGAGGGAGGGTCCCAGCAGAT540 GACTCCAAATTTAGGCAGCAGGCACGCGGAATGAGCTATAAAGGGGCTGGAGCGCTGAGA600 GCTGTCAGACCGAGATTTCTCCATCCCAAGTAAGAAGGAGTTTAGCGTGGGGGCTCTCCA660 ACCGCACCAGACCTGTCCCACCTAGAGGGAAAGTGTCTTCCCTGGAAGTGGGCTCCTCCC720 ACCGGCCTGGGAAGATTCCTCGGTGGGCAGGATGTTCTACTGGATGCCCCTTCCCTTCCA780 CTGCCTCCTCCCTCCCTTGTCTTGATTAATCTTGGCTCTTAGTGTTCAGAAAGATTTGCC840 CGGTGCTGTCTACTCCATCTGTCTCTACTCTCTCTGCCTTGCCTTCTTGTGTGTTCTCCT900 TTTCCACGTGTTTCTCACTCCACTGCCTCCCCCCCCCCCTTCATTTTTATCCTTCCTTTC960 TTTCTGTGTCAGAATGCTGGGAATCAAACCCAGGGCTTCATACACGTCAAGTAAGCAATC1020 TCCCAGTGAGTCAAAGCTTTAATCCTCTGGGTGCTGTCTTACCGAGCCTCACTCCCTGTC1080 TTGTCCTGTTCCGTCCTAGTCAGGATCTCTGGTCCGTCTCTCAGCTTCTGCTACTCCTCT1140 CCCTGCCTGCTCTTCTCTCCGTCCAGCTGCACCTCTGTGGCGCTCATTCCAGCCGTGGTC1200 CAAATTCTCTGTGAAAAGATTAACCGGGTGAGAATGCCCCCAGTTTCCCCTGTAGACAGC1260 AGATCATGATTTTCCCCAGAAGCCAGACTTCCAGCGCCCGCCCTCTGCCCAGCAACTTGA1320 CACTCTTAGCAAACTTCAGCCACCCTTCCCCCACATAGACCAAGTCTTGCAGAGAGCCTT1380 CCTTCAGATGACTTCGAGTTCTTGCAAAGGAAGGAGAACTCTTTGTGGCGGGGAAGCAGG1440 CACTTTACACGGAGTCTGACGGGAGGTCATAGGCTATGGCATAGCAGAGGCAGGGAGGTG1500 GTGGAATTGGACTTCGCGCAGAAGCTAAGCACACACCAGGAATGACATATCCCTCCTATC1560 TCCCCCATAAGAGTTTAAGAGTGACAGG1588 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1679 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Mouse alpha MHC promoter fragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAATTCTCTTACTATCAAAGGGAAACTGAGTCGTGCACCTGCAAAGTGGATGCTCTCCCT60 AGACATCATGACTTTGTCTCTGGGGAGCCAGCACTGTGGAACTTCAGGTCTGAGAGAGTA120 GGAGGCTCCCCTCAGCCTGAAGCTATGCAGATAGCCAGGGTTGAAAGGGGGAAGGGAGAG180 CCTGGGATGGGAGCTTGTGTGTTGGAGGCAGGGGACAGATATTAAGCCTGGAAGAGAAGG240 TGACCCTTACCCAGTTGTTCAACTCACCCTTCAGATTAAAAATAACTGAGGTAAGGGCCT300 GGGTAGGGGAGGTGGTGTGAGACGCTCCTGTCTCTCCTCTATCTGCCCATCGGCCCTTTG360 GGGAGGAGGAATGTGCCCAAGGACTAAAAAAAGGCCATGGAGCCAGAGGGGCGAGGGCAA420 CAGACCTTTCATGGGCAAACCTTGGGGCCCTGCTGTCCTCCTGTCACCTCCAGAGCCAAG480 GGATCAAAGGAGGAGGAGCCAGGACAGGAGGGAAGTGGGAGGGAGGGTCCCAGCAGAGGA540 CTCCAAATTTAGGCAGCAGGCATATGGGATGGGATATAAAGGGGCTGGAGCACTGAGAGC600 TGTCAGAGATTTCTCCAACCCAGGTAAGAGGGAGTTTCGGGTGGGGGCTCTTCACCCACA660 CCAGACCTCTCCCCACCTAGAAGGAAACTGCCTTTCCTGGAAGTGGGGTTCAGGCCGGTC720 AGAGATCTGACAGGGTGGCCTTCCACCAGCCTGGGAAGTTCTCAGTGGCAGGAGGTTTCC780 ACAAGAAACACTGGATGCCCCTTCCCTTACGCTGTCTTCTCCATCTTCCTCCTGGGGATG840 CTCCTCCCCGTCTTGGTTTATCTTGGCTCTTCGTCTTCAGCAAGATTTGCCCTGTGCTGT900 CCACTCCATCTTTCTCTACTGTCTCCGTGCCTTGCCTTGCCTTCTTGCGTGTCCTTCCTT960 TCCACCCATTTCTCACTTCACCTTTTCTCCCCTTCTCATTTGTATTCATCCTTCCTTCCT1020 TCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTTCTCCCTTCCTTCCTTCCTTCCT1080 TCCTTCCTTCCTTCCTTCCTTCCTGTGTCAGAGTGCTGAGAATCACACCTGGGGTTCCCA1140 CCCTTATGTAAACAATCTTCCAGTGAGCCACAGCTTCAGTGCTGCTGGGTGCTCTCTTAC1200 CTTCCTCACCCCCTGGCTTGTCCTGTTCCATCCTGGTCAGGATCTCTAGATTGGTCTCCC1260 AGCCTCTGCTACTCCTCTTCCTGCCTGTTCCTCTCTCTGTCCAGCTGCGCCACTGTGGTG1320 CCTCGTTCCAGCTGTGGTCCACATTCTTCAGGATTCTCTGAAAAGTTAACCAGGTGAGAA1380 TGTTTCCCCTGTAGACAGCAGATCACGATTCTCCCGGAAGTCAGGCTTCCAGCCCTCTCT1440 TTCTCTGCCCAGCTGCCCGGCACTCTTAGCAAACCTCAGGCACCCTTACCCCACATAGAC1500 CTCTGACAGAGAAGCAGGCACTTTACATGGAGTCCTGGTGGGAGAGCCATAGGCTACGGT1560 GTAAAAGAGGCAGGGAAGTGGTGGTGTAGGAAAGTCAGGACTTCACATAGAAGCCTAGCC1620 CACACCAGAAATGACAGACAGATCCCTCCTATCTCCCCCATAAGAGTTTGAGTGACAGA1679 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5057 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: rat bNOS cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 349..4638 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ACGTCTGACAAGCTGGTGACCAAGATGCCCAGAGACTAGACCCTATGCTTGTGAGTCACA60 GTCATCAGACACGGCAAACCTCCAGTCTTCCTGACCTGTTGCTTAGGGACACATCCCGTT120 GCTGCCCCTGACGTCTGCCTGGTCAACCTTGACTTCCTTTGAGAGTAAGGAAGGGGGCGG180 GGACACGTTGAAATCATGCCACCCAAGGCCGAATCGGAATGAGCAGATGACGCCAAGTTG240 ACGTCAAAGACAGAGGCGACAGAAACTCTGCAGCCAGCTCTTGCCCCCGAGGAGCTCAGG300 TTCCTGCAGGAGTCATTTTAGCTTAGTCTTCTGAAGGACACAGATACCATGGAAGAG357 MetGluGlu AACACGTTTGGGGTTCAGCAGATCCAACCCAATGTAATTTCTGTTCGT405 AsnThrPheGlyValGlnGlnIleGlnProAsnValIleSerValArg 51015 CTCTTCAAACGCAAAGTGGGAGGTCTGGGCTTCCTGGTGAAGGAACGG453

LeuPheLysArgLysValGlyGlyLeuGlyPheLeuValLysGluArg 20253035 GTCAGCAAGCCTCCCGTGATCATCTCAGACCTGATTCGAGGAGGTGCT501 ValSerLysProProValIleIleSerAspLeuIleArgGlyGlyAla 404550 GCGGAGCAGAGCGGCCTTATCCAAGCTGGAGACATCATTCTCGCAGTC549 AlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIleLeuAlaVal 556065 AACGATCGGCCCTTGGTAGACCTCAGCTATGACAGTGCCCTGGAGGTT597 AsnAspArgProLeuValAspLeuSerTyrAspSerAlaLeuGluVal 707580 CTCAGGGGCATTGCCTCTGAGACCCACGTGGTCCTCATTCTGAGGGGC645 LeuArgGlyIleAlaSerGluThrHisValValLeuIleLeuArgGly 859095 CCTGAGGGCTTCACTACACATCTGGAGACCACCTTCACAGGGGATGGA693 ProGluGlyPheThrThrHisLeuGluThrThrPheThrGlyAspGly 100105110115 ACCCCCAAGACCATCCGGGTGACCCAGCCCCTCGGTCCTCCCACCAAA741 ThrProLysThrIleArgValThrGlnProLeuGlyProProThrLys 120125130 GCCGTCGATCTGTCTCACCAGCCTTCAGCCAGCAAAGACCAGTCATTA789 AlaValAspLeuSerHisGlnProSerAlaSerLysAspGlnSerLeu 135140145 GCAGTAGACAGAGTCACAGGTCTGGGTAATGGCCCTCAGCATGCCCAA837 AlaValAspArgValThrGlyLeuGlyAsnGlyProGlnHisAlaGln 150155160 GGCCATGGGCAGGGAGCTGGCTCAGTCTCCCAAGCTAATGGTGTGGCC885 GlyHisGlyGlnGlyAlaGlySerValSerGlnAlaAsnGlyValAla 165170175 ATTGACCCCACGATGAAAAGCACCAAGGCCAACCTCCAGGACATCGGG933 IleAspProThrMetLysSerThrLysAlaAsnLeuGlnAspIleGly 180185190195 GAACATGATGAACTGCTCAAAGAGATAGAACCTGTGCTGAGCATCCTC981 GluHisAspGluLeuLeuLysGluIleGluProValLeuSerIleLeu 200205210 AACAGTGGGAGCAAAGCCACCAACAGAGGGGGACCAGCCAAAGCAGAG1029 AsnSerGlySerLysAlaThrAsnArgGlyGlyProAlaLysAlaGlu 215220225 ATGAAAGACACAGGAATCCAGGTGGACAGAGACCTCGATGGCAAATCG1077 MetLysAspThrGlyIleGlnValAspArgAspLeuAspGlyLysSer 230235240 CACAAAGCTCCGCCCCTGGGCGGGGACAATGACCGCGTCTTCAATGAC1125 HisLysAlaProProLeuGlyGlyAspAsnAspArgValPheAsnAsp 245250255 CTGTGGGGGAAGGACAACGTTCCTGTGATCCTTAACAACCCGTATTCA1173 LeuTrpGlyLysAspAsnValProValIleLeuAsnAsnProTyrSer 260265270275 GAGAAGGAACAGTCCCCTACCTCGGGGAAACAGTCTCCCACCAAGAAC1221 GluLysGluGlnSerProThrSerGlyLysGlnSerProThrLysAsn 280285290 GGCAGCCCTTCCAGGTGCCCCCGTTTCCTCAAGGTCAAGAACTGGGAG1269 GlySerProSerArgCysProArgPheLeuLysValLysAsnTrpGlu 295300305 ACGGACGTGGTCCTCACCGACACCCTGCACCTGAAGAGCACACTGGAA1317 ThrAspValValLeuThrAspThrLeuHisLeuLysSerThrLeuGlu 310315320 ACGGGGTGCACAGAGCACATTTGCATGGGCTCGATCATGCTGCCTTCC1365 ThrGlyCysThrGluHisIleCysMetGlySerIleMetLeuProSer 325330335 CAGCACACGCGGAAGCCAGAAGATGTCCGCACAAAGGACCAGCTCTTC1413 GlnHisThrArgLysProGluAspValArgThrLysAspGlnLeuPhe 340345350355 CCTCTAGCCAAAGAATTTCTCGACCAATACTACTCATCCATTAAGAGA1461 ProLeuAlaLysGluPheLeuAspGlnTyrTyrSerSerIleLysArg 360365370 TTTGGCTCCAAGGCCCACATGGACAGGCTGGAGGAGGTGAACAAGGAG1509 PheGlySerLysAlaHisMetAspArgLeuGluGluValAsnLysGlu 375380385 ATTGAAAGCACCAGCACCTACCAGCTCAAGGACACCGAGCTCATCTAT1557 IleGluSerThrSerThrTyrGlnLeuLysAspThrGluLeuIleTyr 390395400 GGCGCCAAGCATGCCTGGCGGAACGCCTCTCGATGTGTGGGCAGGATC1605 GlyAlaLysHisAlaTrpArgAsnAlaSerArgCysValGlyArgIle 405410415 CAGTGGTCCAAGCTGCAGGTGTTCGATGCCCGAGACTGCACCACAGCC1653 GlnTrpSerLysLeuGlnValPheAspAlaArgAspCysThrThrAla 420425430435 CACGGCATGTTCAACTACATCTGTAACCATGTCAAGTATGCCACCAAC1701 HisGlyMetPheAsnTyrIleCysAsnHisValLysTyrAlaThrAsn 440445450 AAAGGGAATCTCAGGTCGGCCATCACGATATTCCCTCAGAGGACTGAC1749 LysGlyAsnLeuArgSerAlaIleThrIlePheProGlnArgThrAsp 455460465 GGCAAACATGACTTCCGAGTGTGGAACTCGCAGCTCATCCGCTACGCG1797 GlyLysHisAspPheArgValTrpAsnSerGlnLeuIleArgTyrAla 470475480 GGCTACAAGCAGCCAGATGGCTCTACCTTGGGGGATCCAGCCAATGTG1845 GlyTyrLysGlnProAspGlySerThrLeuGlyAspProAlaAsnVal 485490495 CAGTTCACGGAGATCTGTATACAGCAGGGCTGGAAAGCCCCAAGAGGC1893 GlnPheThrGluIleCysIleGlnGlnGlyTrpLysAlaProArgGly 500505510515 CGCTTCGACGTGCTGCCTCTCCTGCTTCAGGCCAATGGCAATGACCCT1941 ArgPheAspValLeuProLeuLeuLeuGlnAlaAsnGlyAsnAspPro 520525530 GAGCTCTTCCAGATCCCCCCAGAGCTGGTGCTGGAAGTGCCCATCAGG1989 GluLeuPheGlnIleProProGluLeuValLeuGluValProIleArg 535540545 CACCCCAAGTTCGACTGGTTTAAGGACCTGGGGCTCAAATGGTATGGC2037 HisProLysPheAspTrpPheLysAspLeuGlyLeuLysTrpTyrGly 550555560 CTCCCCGCTGTGTCCAACATGCTGCTGGAGATCGGGGGCCTGGAGTTC2085 LeuProAlaValSerAsnMetLeuLeuGluIleGlyGlyLeuGluPhe 565570575 AGCGCCTGTCCCTTCAGCGGCTGGTACATGGGCACAGAGATCGGCGTC2133 SerAlaCysProPheSerGlyTrpTyrMetGlyThrGluIleGlyVal 580585590595 CGTGACTACTGTGACAACTCTCGATACAACATCCTGGAGGAAGTAGCC2181 ArgAspTyrCysAspAsnSerArgTyrAsnIleLeuGluGluValAla 600605610 AAGAAGATGGATTTGGACATGAGGAAGACCTCGTCCCTCTGGAAGGAC2229 LysLysMetAspLeuAspMetArgLysThrSerSerLeuTrpLysAsp 615620625 CAAGCACTGGTGGAGATCAACATTGCTGTTCTATATAGCTTCCAGAGT2277 GlnAlaLeuValGluIleAsnIleAlaValLeuTyrSerPheGlnSer 630635640 GACAAGGTGACCATCGTTGACCACCACTCTGCCACGGAGTCCTTCATC2325 AspLysValThrIleValAspHisHisSerAlaThrGluSerPheIle 645650655 AAACACATGGAGAATGAATACCGCTGCAGAGGGGGCTGCCCCGCCGAC2373 LysHisMetGluAsnGluTyrArgCysArgGlyGlyCysProAlaAsp 660665670675 TGGGTGTGGATTGTGCCTCCCATGTCGGGCAGCATCACCCCTGTCTTC2421 TrpValTrpIleValProProMetSerGlySerIleThrProValPhe 680685690 CACCAGGAGATGCTCAACTATAGACTCACCCCGTCCTTTGAATACCAG2469 HisGlnGluMetLeuAsnTyrArgLeuThrProSerPheGluTyrGln 695700705 CCTGATCCATGGAACACCCACGTGTGGAAGGGCACCAACGGGACCCCC2517 ProAspProTrpAsnThrHisValTrpLysGlyThrAsnGlyThrPro 710715720 ACGAAGCGGCGAGCTATCGGCTTTAAGAAATTGGCAGAGGCCGTCAAG2565 ThrLysArgArgAlaIleGlyPheLysLysLeuAlaGluAlaValLys 725730735 TTCTCAGCCAAGCTAATGGGGCAGGCCATGGCCAAGAGGGTCAAGGCG2613 PheSerAlaLysLeuMetGlyGlnAlaMetAlaLysArgValLysAla 740745750755 ACCATTCTCTACGCCACAGAGACAGGCAAATCACAAGCCTATGCCAAG2661 ThrIleLeuTyrAlaThrGluThrGlyLysSerGlnAlaTyrAlaLys 760765770 ACCCTGTGTGAGATCTTCAAGCACGCCTTCGATGCCAAGGCAATGTCC2709 ThrLeuCysGluIlePheLysHisAlaPheAspAlaLysAlaMetSer 775780785 ATGGAGGAGTATGACATCGTGCACCTGGAGCACGAAGCCCTGGTCTTG2757 MetGluGluTyrAspIleValHisLeuGluHisGluAlaLeuValLeu 790795800 GTGGTCACCAGCACCTTTGGCAATGGAGACCCCCCTGAGAACGGGGAG2805 ValValThrSerThrPheGlyAsnGlyAspProProGluAsnGlyGlu 805810815 AAATTCGGCTGTGCTTTAATGGAGATGAGGCACCCCAACTCTGTGCAG2853 LysPheGlyCysAlaLeuMetGluMetArgHisProAsnSerValGln 820825830835 GAGGAGAGAAAGAGCTACAAGGTCCGATTCAACAGCGTCTCCTCCTAT2901 GluGluArgLysSerTyrLysValArgPheAsnSerValSerSerTyr 840845850 TCTGACTCCCGAAAGTCATCGGGCGACGGACCCGACCTCAGAGACAAC2949 SerAspSerArgLysSerSerGlyAspGlyProAspLeuArgAspAsn 855860865 TTTGAAAGTACTGGACCCCTGGCCAATGTGAGGTTCTCAGTGTTCGGC2997 PheGluSerThrGlyProLeuAlaAsnValArgPheSerValPheGly 870875880 CTCGGCTCTCGGGCGTACCCCCACTTCTGTGCCTTTGGGCATGCGGTG3045 LeuGlySerArgAlaTyrProHisPheCysAlaPheGlyHisAlaVal 885890895 GACACCCTCCTGGAGGAACTGGGAGGGGAGAGGATTCTGAAGATGAGG3093 AspThrLeuLeuGluGluLeuGlyGlyGluArgIleLeuLysMetArg 900905910915 GAGGGGGATGAGCTTTGCGGACAGGAAGAAGCTTTCAGGACCTGGGCC3141 GluGlyAspGluLeuCysGlyGlnGluGluAlaPheArgThrTrpAla 920925930 AAGAAAGTCTTCAAGGCAGCCTGTGATGTGTTCTGCGTGGGGGATGAC3189 LysLysValPheLysAlaAlaCysAspValPheCysValGlyAspAsp 935940945 GTCAACATCGAGAAGCCGAACAACTCCCTCATTAGCAATGACCGAAGC3237 ValAsnIleGluLysProAsnAsnSerLeuIleSerAsnAspArgSer 950955960 TGGAAGAGGAACAAGTTCCGCCTCACGTATGTGGCGGAAGCTCCAGAT3285 TrpLysArgAsnLysPheArgLeuThrTyrValAlaGluAlaProAsp 965970975 CTGACCCAAGGTCTTTCCAATGTTCACAAAAAACGAGTCTCGGCTGCT3333 LeuThrGlnGlyLeuSerAsnValHisLysLysArgValSerAlaAla 980985990995 CGACTCCTCAGCCGCCAAAACCTGCAAAGCCCTAAGTTCAGCCGATCG3381 ArgLeuLeuSerArgGlnAsnLeuGlnSerProLysPheSerArgSer 100010051010 ACCATCTTCGTGCGTCTCCACACCAACGGGAATCAGGAGCTGCAGTAC3429 ThrIlePheValArgLeuHisThrAsnGlyAsnGlnGluLeuGlnTyr 101510201025 CAGCCAGGGGACCACCTGGGTGTCTTCCCCGGCAACCACGAGGACCTC3477 GlnProGlyAspHisLeuGlyValPheProGlyAsnHisGluAspLeu 103010351040 GTGAATGCACTCATTGAACGGCTGGAGGATGCACCGCCTGCCAACCAC3525 ValAsnAlaLeuIleGluArgLeuGluAspAlaProProAlaAsnHis 104510501055 GTGGTGAAGGTGGAGATGCTGGAGGAGAGGAACACTGCTCTGGGTGTC3573 ValValLysValGluMetLeuGluGluArgAsnThrAlaLeuGlyVal 1060106510701075 ATCAGTAATTGGAAGGATGAATCTCGCCTCCCACCCTGCACCATCTTC3621 IleSerAsnTrpLysAspGluSerArgLeuProProCysThrIlePhe 108010851090 CAGGCCTTCAAGTACTACCTGGACATCACCACGCCGCCCACGCCCCTG3669 GlnAlaPheLysTyrTyrLeuAspIleThrThrProProThrProLeu 109511001105 CAGCTGCAGCAGTTCGCCTCTCTGGCCACTAATGAGAAAGAGAAGCAG3717 GlnLeuGlnGlnPheAlaSerLeuAlaThrAsnGluLysGluLysGln 111011151120 CGGTTGCTGGTCCTCAGCAAGGGGCTCCAGGAATATGAGGAGTGGAAG3765 ArgLeuLeuValLeuSerLysGlyLeuGlnGluTyrGluGluTrpLys 112511301135 TGGGGCAAGAACCCCACAATGGTGGAGGTGCTGGAGGAGTTCCCGTCC3813 TrpGlyLysAsnProThrMetValGluValLeuGluGluPheProSer 1140114511501155 ATCCAGATGCCGGCTACACTTCTCCTCACTCAGCTGTCGCTGCTGCAG3861 IleGlnMetProAlaThrLeuLeuLeuThrGlnLeuSerLeuLeuGln 116011651170 CCTCGCTACTACTCCATCAGCTCCTCTCCAGACATGTACCCCGACGAG3909 ProArgTyrTyrSerIleSerSerSerProAspMetTyrProAspGlu 117511801185 GTGCACCTCACTGTGGCCATCGTCTCCTACCACACCCGAGACGGAGAA3957 ValHisLeuThrValAlaIleValSerTyrHisThrArgAspGlyGlu 119011951200 GGACCAGTCCACCACGGGGTGTGCTCCTCCTGGCTCAACAGAATACAG4005 GlyProValHisHisGlyValCysSerSerTrpLeuAsnArgIleGln 120512101215 GCTGACGATGTAGTCCCCTGCTTCGTGAGAGGTGCCCCTAGCTTCCAC4053 AlaAspAspValValProCysPheValArgGlyAlaProSerPheHis 1220122512301235 CTGCCTCGAAACCCCCAGGTGCCTTGCATCCTGGTTGGCCCAGGCACT4101 LeuProArgAsnProGlnValProCysIleLeuValGlyProGlyThr 124012451250 GGCATCGCACCCTTCCGAAGCTTCTGGCAACAGCGACAATTTGACATC4149 GlyIleAlaProPheArgSerPheTrpGlnGlnArgGlnPheAspIle 125512601265 CAACACAAAGGAATGAATCCGTGCCCCATGGTTCTGGTCTTCGGGTGT4197 GlnHisLysGlyMetAsnProCysProMetValLeuValPheGlyCys 127012751280 CGACAATCCAAGATAGATCATATCTACAGAGAGGAGACCCTGCAGGCT4245 ArgGlnSerLysIleAspHisIleTyrArgGluGluThrLeuGlnAla 128512901295 AAGAACAAGGGCGTCTTCAGAGAGCTGTACACTGCCTATTCCCGGGAA4293 LysAsnLysGlyValPheArgGluLeuTyrThrAlaTyrSerArgGlu 1300130513101315 CCGGACAGGCCAAAGAAATATGTACAGGACGTGCTGCAGGAACAGCTG4341 ProAspArgProLysLysTyrValGlnAspValLeuGlnGluGlnLeu 132013251330 GCTGAGTCTGTGTACCGCGCCCTGAAGGAGCAAGGAGGCCACATTTAT4389 AlaGluSerValTyrArgAlaLeuLysGluGlnGlyGlyHisIleTyr 133513401345 GTCTGTGGGGACGTTACCATGGCCGCCGATGTCCTCAAAGCCATCCAG4437 ValCysGlyAspValThrMetAlaAlaAspValLeuLysAlaIleGln 135013551360

CGCATAATGACCCAGCAGGGGAAACTCTCAGAGGAGGACGCTGGTGTA4485 ArgIleMetThrGlnGlnGlyLysLeuSerGluGluAspAlaGlyVal 136513701375 TTCATCAGCAGGCTGAGGGATGACAACCGGTACCACGAGGACATCTTT4533 PheIleSerArgLeuArgAspAspAsnArgTyrHisGluAspIlePhe 1380138513901395 GGAGTCACCCTCAGAACGTATGAAGTGACCAACCGCCTTAGATCTGAG4581 GlyValThrLeuArgThrTyrGluValThrAsnArgLeuArgSerGlu 140014051410 TCCATCGCCTTCATCGAAGAGAGCAAAAAAGACGCAGATGAGGTTTTC4629 SerIleAlaPheIleGluGluSerLysLysAspAlaAspGluValPhe 141514201425 AGCTCCTAACTGGATCCTCCTGCCCCCGTGCGTGCGATGTGGCGGCTGCCCCAAGT4685 SerSer 143 GCCCAAGTAAGGGCGGCCGCAGGTTGACTAAATTCGGACACACACGGCTGAACCGAGTGG4745 CCCTGCTCTGCCTCTTGTCCTGTTGCTGTGTCCTGGTCCTTCTTCCTGCTCTGGGCTCTC4805 TCAACCCCACCCCTGGGTTTTCTCCTTGACTCTTGGGCTACGATGCATCACCCTTGTACC4865 CTGCAGTGGCTCTCACAAAACCGCATCCTCCCCACCCCCACCCGATTGCTGCCAAGGGCA4925 GGTTGCGGTGCATGGCTGTTGCTCCTGTTGTTGGGGTCTGAAGGTGGCTGGCGCTGGGCC4985 TCAGGTCACCCTGAACCAGTCCCTTGGCCACTTAAGCCCCCTTCCACCCTCTTTTTATGA5045 TGGTGTGTTTGT5057 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1429 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: MetGluGluAsnThrPheGlyValGlnGlnIleGlnProAsnValIle 151015 SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal 202530 LysGluArgValSerLysProProValIleIleSerAspLeuIleArg 354045 GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle 505560 LeuAlaValAsnAspArgProLeuValAspLeuSerTyrAspSerAla 65707580 LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle 859095 LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr 100105110 GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro 115120125 ProThrLysAlaValAspLeuSerHisGlnProSerAlaSerLysAsp 130135140 GlnSerLeuAlaValAspArgValThrGlyLeuGlyAsnGlyProGln 145150155160 HisAlaGlnGlyHisGlyGlnGlyAlaGlySerValSerGlnAlaAsn 165170175 GlyValAlaIleAspProThrMetLysSerThrLysAlaAsnLeuGln 180185190 AspIleGlyGluHisAspGluLeuLeuLysGluIleGluProValLeu 195200205 SerIleLeuAsnSerGlySerLysAlaThrAsnArgGlyGlyProAla 210215220 LysAlaGluMetLysAspThrGlyIleGlnValAspArgAspLeuAsp 225230235240 GlyLysSerHisLysAlaProProLeuGlyGlyAspAsnAspArgVal 245250255 PheAsnAspLeuTrpGlyLysAspAsnValProValIleLeuAsnAsn 260265270 ProTyrSerGluLysGluGlnSerProThrSerGlyLysGlnSerPro 275280285 ThrLysAsnGlySerProSerArgCysProArgPheLeuLysValLys 290295300 AsnTrpGluThrAspValValLeuThrAspThrLeuHisLeuLysSer 305310315320 ThrLeuGluThrGlyCysThrGluHisIleCysMetGlySerIleMet 325330335 LeuProSerGlnHisThrArgLysProGluAspValArgThrLysAsp 340345350 GlnLeuPheProLeuAlaLysGluPheLeuAspGlnTyrTyrSerSer 355360365 IleLysArgPheGlySerLysAlaHisMetAspArgLeuGluGluVal 370375380 AsnLysGluIleGluSerThrSerThrTyrGlnLeuLysAspThrGlu 385390395400 LeuIleTyrGlyAlaLysHisAlaTrpArgAsnAlaSerArgCysVal 405410415 GlyArgIleGlnTrpSerLysLeuGlnValPheAspAlaArgAspCys 420425430 ThrThrAlaHisGlyMetPheAsnTyrIleCysAsnHisValLysTyr 435440445 AlaThrAsnLysGlyAsnLeuArgSerAlaIleThrIlePheProGln 450455460 ArgThrAspGlyLysHisAspPheArgValTrpAsnSerGlnLeuIle 465470475480 ArgTyrAlaGlyTyrLysGlnProAspGlySerThrLeuGlyAspPro 485490495 AlaAsnValGlnPheThrGluIleCysIleGlnGlnGlyTrpLysAla 500505510 ProArgGlyArgPheAspValLeuProLeuLeuLeuGlnAlaAsnGly 515520525 AsnAspProGluLeuPheGlnIleProProGluLeuValLeuGluVal 530535540 ProIleArgHisProLysPheAspTrpPheLysAspLeuGlyLeuLys 545550555560 TrpTyrGlyLeuProAlaValSerAsnMetLeuLeuGluIleGlyGly 565570575 LeuGluPheSerAlaCysProPheSerGlyTrpTyrMetGlyThrGlu 580585590 IleGlyValArgAspTyrCysAspAsnSerArgTyrAsnIleLeuGlu 595600605 GluValAlaLysLysMetAspLeuAspMetArgLysThrSerSerLeu 610615620 TrpLysAspGlnAlaLeuValGluIleAsnIleAlaValLeuTyrSer 625630635640 PheGlnSerAspLysValThrIleValAspHisHisSerAlaThrGlu 645650655 SerPheIleLysHisMetGluAsnGluTyrArgCysArgGlyGlyCys 660665670 ProAlaAspTrpValTrpIleValProProMetSerGlySerIleThr 675680685

ProValPheHisGlnGluMetLeuAsnTyrArgLeuThrProSerPhe 690695700 GluTyrGlnProAspProTrpAsnThrHisValTrpLysGlyThrAsn 705710715720 GlyThrProThrLysArgArgAlaIleGlyPheLysLysLeuAlaGlu 725730735 AlaValLysPheSerAlaLysLeuMetGlyGlnAlaMetAlaLysArg 740745750 ValLysAlaThrIleLeuTyrAlaThrGluThrGlyLysSerGlnAla 755760765 TyrAlaLysThrLeuCysGluIlePheLysHisAlaPheAspAlaLys 770775780 AlaMetSerMetGluGluTyrAspIleValHisLeuGluHisGluAla 785790795800 LeuValLeuValValThrSerThrPheGlyAsnGlyAspProProGlu 805810815 AsnGlyGluLysPheGlyCysAlaLeuMetGluMetArgHisProAsn 820825830 SerValGlnGluGluArgLysSerTyrLysValArgPheAsnSerVal 835840845 SerSerTyrSerAspSerArgLysSerSerGlyAspGlyProAspLeu 850855860 ArgAspAsnPheGluSerThrGlyProLeuAlaAsnValArgPheSer 865870875880 ValPheGlyLeuGlySerArgAlaTyrProHisPheCysAlaPheGly 885890895 HisAlaValAspThrLeuLeuGluGluLeuGlyGlyGluArgIleLeu 900905910 LysMetArgGluGlyAspGluLeuCysGlyGlnGluGluAlaPheArg 915920925 ThrTrpAlaLysLysValPheLysAlaAlaCysAspValPheCysVal 930935940 GlyAspAspValAsnIleGluLysProAsnAsnSerLeuIleSerAsn 945950955960 AspArgSerTrpLysArgAsnLysPheArgLeuThrTyrValAlaGlu 965970975 AlaProAspLeuThrGlnGlyLeuSerAsnValHisLysLysArgVal 980985990 SerAlaAlaArgLeuLeuSerArgGlnAsnLeuGlnSerProLysPhe 99510001005 SerArgSerThrIlePheValArgLeuHisThrAsnGlyAsnGlnGlu 101010151020 LeuGlnTyrGlnProGlyAspHisLeuGlyValPheProGlyAsnHis 1025103010351040 GluAspLeuValAsnAlaLeuIleGluArgLeuGluAspAlaProPro 104510501055 AlaAsnHisValValLysValGluMetLeuGluGluArgAsnThrAla 106010651070 LeuGlyValIleSerAsnTrpLysAspGluSerArgLeuProProCys 107510801085 ThrIlePheGlnAlaPheLysTyrTyrLeuAspIleThrThrProPro 109010951100 ThrProLeuGlnLeuGlnGlnPheAlaSerLeuAlaThrAsnGluLys 1105111011151120 GluLysGlnArgLeuLeuValLeuSerLysGlyLeuGlnGluTyrGlu 112511301135 GluTrpLysTrpGlyLysAsnProThrMetValGluValLeuGluGlu 114011451150 PheProSerIleGlnMetProAlaThrLeuLeuLeuThrGlnLeuSer 115511601165 LeuLeuGlnProArgTyrTyrSerIleSerSerSerProAspMetTyr 117011751180 ProAspGluValHisLeuThrValAlaIleValSerTyrHisThrArg 1185119011951200 AspGlyGluGlyProValHisHisGlyValCysSerSerTrpLeuAsn 120512101215 ArgIleGlnAlaAspAspValValProCysPheValArgGlyAlaPro 122012251230 SerPheHisLeuProArgAsnProGlnValProCysIleLeuValGly 123512401245 ProGlyThrGlyIleAlaProPheArgSerPheTrpGlnGlnArgGln 125012551260 PheAspIleGlnHisLysGlyMetAsnProCysProMetValLeuVal 1265127012751280 PheGlyCysArgGlnSerLysIleAspHisIleTyrArgGluGluThr 128512901295 LeuGlnAlaLysAsnLysGlyValPheArgGluLeuTyrThrAlaTyr 130013051310 SerArgGluProAspArgProLysLysTyrValGlnAspValLeuGln 131513201325 GluGlnLeuAlaGluSerValTyrArgAlaLeuLysGluGlnGlyGly 133013351340 HisIleTyrValCysGlyAspValThrMetAlaAlaAspValLeuLys 1345135013551360 AlaIleGlnArgIleMetThrGlnGlnGlyLysLeuSerGluGluAsp 136513701375 AlaGlyValPheIleSerArgLeuArgAspAspAsnArgTyrHisGlu 138013851390 AspIlePheGlyValThrLeuArgThrTyrGluValThrAsnArgLeu 139514001405 ArgSerGluSerIleAlaPheIleGluGluSerLysLysAspAlaAsp 141014151420 GluValPheSerSer 1425 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5086 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: human bcl-2 cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1459..2178 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCGCCCGCCCCTCCGCGCCGCCTGCCCGCCCGCCCGCCGCGCTCCCGCCCGCCGCTCTCC60 GTGGCCCCGCCGCGCTGCCGCCGCCGCCGCTGCCAGCGAAGGTGCCGGGGCTCCGGGCCC120 TCCCTGCCGGCGGCCGTCAGCGCTCGGAGCGAACTGCGCGACGGGAGGTCCGGGAGGCGA180 CCGTAGTCGCGCCGCCGCGCAGGACCAGGAGGAGGAGAAAGGGTGCGCAGCCCGGAGGCG240 GGGTGCGCCGGTGGGGTGCAGCGGAAGAGGGGGTCCAGGGGGGAGAACTTCGTAGCAGTC300 ATCCTTTTTAGGAAAAGAGGGAAAAAATAAAACCCTCCCCCACCACCTCCTTCTCCCCAC360 CCCTCGCCGCACCACACACAGCGCGGGCTTCTAGCGCTCGGCACCGGCGGGCCAGGCGCG420 TCCTGCCTTCATTTATCCAGCAGCTTTTCGGAAAATGCATTTGCTGTTCGGAGTTTAATC480 AGAAGACGATTCCTGCCTCCGTCCCCGGCTCCTTCATCGTCCCATCTCCCCTGTCTCTCT540 CCTGGGGAGGCGTGAAGCGGTCCCGTGGATAGAGATTCATGCCTGTGTCCGCGCGTGTGT600 GCGCGCGTATAAATTGCCGAGAAGGGGAAAACATCACAGGACTTCTGCGAATACCGGACT660 GAAAATTGTAATTCATCTGCCGCCGCCGCTGCCAAAAAAAAACTCGAGCTCTTGAGATCT720 CCGGTTGGGATTCCTGCGGATTGACATTTCTGTGAAGCAGAAGTCTGGGAATCGATCTGG780 AAATCCTCCTAATTTTTACTCCCTCTCCCCCCGACTCCTGATTCATTGGGAAGTTTCAAA840 TCAGCTATAACTGGAGAGTGCTGAAGATTGATGGGATCGTTGCCTTATGCATTTGTTTTG900 GTTTTACAAAAAGGAAACTTGACAGAGGATCATGCTGTACTTAAAAAATACAAGTAAGTC960 TCGCACAGGAAATTGGTTTAATGTAACTTTCAATGGAAACCTTTGAGATTTTTTACTTAA1020 AGTGCATTCGAGTAAATTTAATTTCCAGGCAGCTTAATACATTGTTTTTAGCCGTGTTAC1080 TTGTAGTGTGTATGCCCTGCTTTCACTCAGTGTGTACAGGGAAACGCACCTGATTTTTTA1140 CTTATTAGTTTGTTTTTTCTTTAACCTTTCAGCATCACAGAGGAAGTAGACTGATATTAA1200 CAATACTTACTAATAATAACGTGCCTCATGAAATAAAGATCCGAAAGGAATTGGAATAAA1260 AATTTCCTGCGTCTCATGCCAAGAGGGAAACACCAGAATCAAGTGTTCCGCGTGATTGAA1320 GACACCCCCTCGTCCAAGAATGCAAAGCACATCCAATAAAATAGCTGGATTATAACTCCT1380 CTTCTTTCTCTGGGGGCCGTGGGGTGGGAGCTGGGGCGAGAGGTGCCGTTGGCCCCCGTT1440 GCTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGAACGGGGTACGACAAC1491 MetAlaHisAlaGlyArgThrGlyTyrAspAsn 1510 CGGGAGATAGTGATGAAGTACATCCATTATAAGCTGTCGCAGAGGGGC1539 ArgGluIleValMetLysTyrIleHisTyrLysLeuSerGlnArgGly 152025 TACGAGTGGGATGCGGGAGATGTGGGCGCCGCGCCCCCGGGGGCCGCC1587 TyrGluTrpAspAlaGlyAspValGlyAlaAlaProProGlyAlaAla 303540 CCCGCACCGGGCATCTTCTCCTCCCAGCCCGGGCACACGCCCCATCCA1635 ProAlaProGlyIlePheSerSerGlnProGlyHisThrProHisPro 455055 GCCGCATCCCGCGACCCGGTCGCCAGGACCTCGCCGCTGCAGACCCCG1683 AlaAlaSerArgAspProValAlaArgThrSerProLeuGlnThrPro 60657075 GCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCACCT1731 AlaAlaProGlyAlaAlaAlaGlyProAlaLeuSerProValProPro 808590 GTGGTCCACCTGGCCCTCCGCCAAGCCGGCGACGACTTCTCCCGCCGC1779 ValValHisLeuAlaLeuArgGlnAlaGlyAspAspPheSerArgArg 95100105 TACCGCGGCGACTTCGCCGAGATGTCCAGCCAGCTGCACCTGACGCCC1827 TyrArgGlyAspPheAlaGluMetSerSerGlnLeuHisLeuThrPro 110115120 TTCACCGCGCGGGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGG1875 PheThrAlaArgGlyArgPheAlaThrValValGluGluLeuPheArg 125130135 GACGGGGTGAACTGGGGGAGGATTGTGGCCTTCTTTGAGTTCGGTGGG1923 AspGlyValAsnTrpGlyArgIleValAlaPhePheGluPheGlyGly 140145150155 GTCATGTGTGTGGAGAGCGTCAACCGGGAGATGTCGCCCCTGGTGGAC1971 ValMetCysValGluSerValAsnArgGluMetSerProLeuValAsp 160165170 AACATCGCCCTGTGGATGACTGAGTACCTGAACCGGCACCTGCACACC2019 AsnIleAlaLeuTrpMetThrGluTyrLeuAsnArgHisLeuHisThr 175180185 TGGATCCAGGATAACGGAGGCTGGGATGCCTTTGTGGAACTGTACGGC2067 TrpIleGlnAspAsnGlyGlyTrpAspAlaPheValGluLeuTyrGly 190195200 CCCAGCATGCGGCCTCTGTTTGATTTCTCCTGGCTGTCTCTGAAGACT2115 ProSerMetArgProLeuPheAspPheSerTrpLeuSerLeuLysThr 205210215 CTGCTCAGTTTGGCCCTGGTGGGAGCTTGCATCACCCTGGGTGCCTAT2163 LeuLeuSerLeuAlaLeuValGlyAlaCysIleThrLeuGlyAlaTyr 220225230235 CTGAGCCACAAGTGAAGTCAACATGCCTGCCCCAAACAAATATGCAAAAGGT2215 LeuSerHisLys 240 TCACTAAAGCAGTAGAAATAATATGCATTGTCAGTGATGTACCATGAAACAAAGCTGCAG2275 GCTGTTTAAGAAAAAATAACACACATATAAACATCACACACACAGACAGACACACACACA2335 CACAACAATTAACAGTCTTCAGGCAAAACGTCGAATCAGCTATTTACTGCCAAAGGGAAA2395 TATCATTTATTTTTTACATTATTAAGAAAAAAGATTTATTTATTTAAGACAGTCCCATCA2455 AAACTCCGTCTTTGGAAATCCGACCACTAATTGCCAAACACCGCTTCGTGTGGCTCCACC2515 TGGATGTTCTGTGCCTGTAAACATAGATTCGCTTTCCATGTTGTTGGCCGGATCACCATC2575 TGAAGAGCAGACGGATGGAAAAAGGACCTGATCATTGGGGAAGCTGGCTTTCTGGCTGCT2635 GGAGGCTGGGGAGAAGGTGTTCATTCACTTGCATTTCTTTGCCCTGGGGGCGTGATATTA2695 ACAGAGGGAGGGTTCCCGTGGGGGGAAGTCCATGCCTCCCTGGCCTGAAGAAGAGACTCT2755 TTGCATATGACTCACATGATGCATACCTGGTGGGAGGAAAAGAGTTGGGAACTTCAGATG2815 GACCTAGTACCCACTGAGATTTCCACGCCGAAGGACAGCGATGGGAAAAATGCCCTTAAA2875 TCATAGGAAAGTATTTTTTTAAGCTACCAATTGTGCCGAGAAAAGCATTTTAGCAATTTA2935 TACAATATCATCCAGTACCTTAAACCCTGATTGTGTATATTCATATATTTTGGATACGCA2995 CCCCCCAACTCCCAATACTGGCTCTGTCTGAGTAAGAAACAGAATCCTCTGGAACTTGAG3055 GAAGTGAACATTTCGGTGACTTCCGATCAGGAAGGCTAGAGTTACCCAGAGCATCAGGCC3115 GCCACAAGTGCCTGCTTTTAGGAGACCGAAGTCCGCAGAACCTACCTGTGTCCCAGCTTG3175 GAGGCCTGGTCCTGGAACTGAGCCGGGCCCTCACTGGCCTCCTCCAGGGATGATCAACAG3235 GGTAGTGTGGTCTCCGAATGTCTGGAAGCTGATGGATGGAGCTCAGAATTCCACTGTCAA3295 GAAAGAGCAGTAGAGGGGTGTGGCTGGGCCTGTCACCCTGGGGCCCTCCAGGTAGGCCCG3355 TTTTCACGTGGAGCATAGGAGCCACGACCCTTCTTAAGACATGTATCACTGTAGAGGGAA3415 GGAACAGAGGCCCTGGGCCTTCCTATCAGAAGGACATGGTGAAGGCTGGGAACGTGAGGA3475 GAGGCAATGGCCACGGCCCATTTTGGCTGTAGCACATGGCACGTTGGCTGTGTGGCCTTG3535 GCCACCTGTGAGTTTAAAGCAAGGCTTTAAATGACTTTGGAGAGGGTCACAAATCCTAAA3595 AGAAGCATTGAAGTGAGGTGTCATGGATTAATTGACCCCTGTCTATGGAATTACATGTAA3655 AACATTATCTTGTCACTGTAGTTTGGTTTTATTTGAAAACCTGACAAAAAAAAAGTTCCA3715 GGTGTGGAATATGGGGGTTATCTGTACATCCTGGGGCATTAAAAAAAAATCAATGGTGGG3775 GAACTATAAAGAAGTAACAAAAGAAGTGACATCTTCAGCAAATAAACTAGGAAATTTTTT3835 TTTCTTCCAGTTTAGAATCAGCCTTGAAACATTGATGGAATAACTCTGTGGCATTATTGC3895 ATTATATACCATTTATCTGTATTAACTTTGGAATGTACTCTGTTCAATGTTTAATGCTGT3955 GGTTGATATTTCGAAAGCTGCTTTAAAAAAATACATGCATCTCAGCGTTTTTTTGTTTTT4015 AATTGTATTTAGTTATGGCCTATACACTATTTGTGAGCAAAGGTGATCGTTTTCTGTTTG4075 AGATTTTTATCTCTTGATTCTTCAAAAGCATTCTGAGAAGGTGAGATAAGCCCTGAGTCT4135 CAGCTACCTAAGAAAAACCTGGATGTCACTGGCCACTGAGGAGCTTTGTTTCAACCAAGT4195 CATGTGCATTTCCACGTCAACAGAATTGTTTATTGTGACAGTTATATCTGTTGTCCCTTT4255 GACCTTGTTTCTTGAAGGTTTCCTCGTCCCTGGGCAATTCCGCATTTAATTCATGGTATT4315 CAGGATTACATGCATGTTTGGTTAAACCCATGAGATTCATTCAGTTAAAAATCCAGATGG4375 CGAATGACCAGCAGATTCAAATCTATGGTGGTTTGACCTTTAGAGAGTTGCTTTACGTGG4435 CCTGTTTCAACACAGACCCACCCAGAGCCCTCCTGCCCTCCTTCCGCGGGGGCTTTCTCA4495 TGGCTGTCCTTCAGGGTCTTCCTGAAATGCAGTGGTCGTTACGCTCCACCAAGAAAGCAG4555 GAAACCTGTGGTATGAAGCCAGACCTCCCCGGCGGGCCTCAGGGAACAGAATGATCAGAC4615 CTTTGAATGATTCTAATTTTTAAGCAAAATATTATTTTATGAAAGGTTTACATTGTCAAA4675 GTGATGAATATGGAATATCCAATCCTGTGCTGCTATCCTGCCAAAATCATTTTAATGGAG4735 TCAGTTTGCAGTATGCTCCACGTGGTAAGATCCTCCAAGCTGCTTTAGAAGTAACAATGA4795 AGAACGTGGACGTTTTTAATATAAAGCCTGTTTTGTCTTTTGTTGTTGTTCAAACGGGAT4855 TCACAGAGTATTTGAAAAATGTATATATATTAAGAGGTCACGGGGGCTAATTGCTAGCTG4915 GCTGCCTTTTGCTGTGGGGTTTTGTTACCTGGTTTTAATAACAGTAAATGTGCCCAGCCT4975 CTTGGCCCCAGAACTGTACAGTATTGTGGCTGCACTTGCTCTAAGAGTAGTTGATGTTGC5035 ATTTTCCTTATTGTTAAAAACATGTTAGAAGCAATGAATGTATATAAAAGC5086 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 239 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: MetAlaHisAlaGlyArgThrGlyTyrAspAsnArgGluIleValMet 151015 LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla 202530 GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyIle 354045 PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp 505560 ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla 65707580 AlaAlaGlyProAlaLeuSerProValProProValValHisLeuAla 859095 LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgGlyAspPhe 100105110 AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly

115120125 ArgPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp 130135140 GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu 145150155160 SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp 165170175 MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn 180185190 GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro 195200205 LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla 210215220 LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuSerHisLys 225230235 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1846 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: bcl-2 fusion gene; Seto, et al., EMBO J 7:123 (1988) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 887..1606 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ACCACCTCCTTCTCCCCACCCCTCGCCGCACCACACACAGCGCGGGCTTCTGGCGCTCGG60 CACCGGCGGGCCAGGCGCGTCCTGTCTTCATTTATCCAGCAGCTTTTCGGAAAATCCATT120 TGGTGTTCGGAGTTTAATCAGAAGAGGATTCCTGCCTCCGTCCCCGGCTCCTTCATCGTC180 CCCTCTCCCCTGTCTCTCTCCTGGGGAGGCGTGAAGAGAGATTCATGCCTGTGCCCGCGC240 GTGTGTGCGCGCGTATAAATTGCCGAGAAGGGGAAAACATCACAGGACTTCTGCGAATAC300 CGGACTGAAAATTGTAGCTCATCTGCCGCCGCCGCTGCCTTTTTTTTTTCTCGAGCTCTT360 GAGATCTCCGGTTGGGACTCCTGCGGATTGACATTTCTGTGAAGCAGAAGTCTGGGAATC420 GATCTGGAAATCCTCCTAATTTTTACTCCCTCTCCCCCCGACTCCTGATTCATTGGGAAG480 TTTCAAATCAGCTATAACTGGAGAGAGCTGAAGATTGATGGGATCGTTGCCTTATGCCTT540 TGTTTTGGTTTTACAAAAAGGAAACTTGACAGAGGATCATGCTATACTTAAAAAATACAA600 CATCGCAGAGGAAGTAGACTCATATTAAAAATACTTACTAATAATAACGTGCCTCATGAA660 GTAAAGATCCGAAAGGAATTGGAATAAAACTTTCCTGCATCTCAAGCCAAGGGGGAAACA720 CCAGAATCAAGTGTTCCGCGTGATTGAAGACACCCCCTCGTCCAAGAATGCAAAGCACAT780 CCAATAAAAGAGCTGGATTATAACTCCTCTTCTTTCTCTGGGGGCCGTGGGGTAGGGGCT840 GGGGCGAGAGGTGCCGTTGGCCCCCGTTGCTTTTCCTCTGGGAGGGATGGCGCAC895 MetAlaHis 1 GCTGGGAGAAGTGGTTACGATAACCGGGAGATAGTGATGAAGTACATC943 AlaGlyArgSerGlyTyrAspAsnArgGluIleValMetLysTyrIle 51015 CATTATAAGCTGTCGCAGAGGGGCTACGAGTGGGATGCGGGAGATGTG991 HisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAlaGlyAspVal 20253035 GGCGCCGCGCCCCCGGGGGCCGCCCCCGCACCGGGCTTCTTCTCCTCC1039 GlyAlaAlaProProGlyAlaAlaProAlaProGlyPhePheSerSer 404550 CAGCCCGGGCACACGCCCCATCCAGCCGCATCCCGGGACCCGGTCGCC1087 GlnProGlyHisThrProHisProAlaAlaSerArgAspProValAla 556065 AGGACCTCGCCACTACAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGG1135 ArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAlaGly 707580 CCTGCGCTCAGCCCGGTGCCACCTGTGGTCCACCTGACCCTCCGCCAG1183 ProAlaLeuSerProValProProValValHisLeuThrLeuArgGln 859095 GCCGGCGACGACTTCTCCCGCCGCTACCGCCGCGACTTCGCCGAGATG1231 AlaGlyAspAspPheSerArgArgTyrArgArgAspPheAlaGluMet 100105110115 TCCAGCCAGCTGCACCTGACGCCCTTCACCGCGCGGGGATGCTTTGCC1279 SerSerGlnLeuHisLeuThrProPheThrAlaArgGlyCysPheAla 120125130 ACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGGGAGGATT1327 ThrValValGluGluLeuPheArgAspGlyValAsnTrpGlyArgIle 135140145 GTGGCCTTCTTTGAGTTCGGTGGGGTCATGTGTGTGGAGAGCGTCAAC1375 ValAlaPhePheGluPheGlyGlyValMetCysValGluSerValAsn 150155160 CGGGAGATGTCGCCCCTGGTGGACAACATCGCCCTGTGGATGACTGAG1423 ArgGluMetSerProLeuValAspAsnIleAlaLeuTrpMetThrGlu 165170175 TACCTGAACCGGCACCTGCACACCTGGATCCAGGATAACGGAGGCTGG1471 TyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsnGlyGlyTrp 180185190195 GATGCCTTTGTGGAACTGTACGGCCCCAGCATGCGGCCTCTGTTTGAT1519 AspAlaPheValGluLeuTyrGlyProSerMetArgProLeuPheAsp 200205210 TTCTCCTGGCTGTCTCTGAAGACTCTGCTCAGTTTGGCCCTGGTGGGA1567 PheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAlaLeuValGly 215220225 GCTTGCATCACCCTGGGTGCCTATCTGGGCCACAAGTGAAGTCAAC1613 AlaCysIleThrLeuGlyAlaTyrLeuGlyHisLys 230235240 ATGCCTGCCCCAAACAAATATGCAAAAGGTTCACTAAAGCAGTAGAAATAATATGCATTG1673 TCAGTGATGTACCATGAAACAAAGCTGCAGGCTGTTTAAGAAAAAATAACACACATATAA1733 ACATCACACACACAGACAGACACACACACACACAACAATTAACAGTCTTCAGGCAAAACG1793 TCGAATCAGCTATTTACTGCCAAAGGGAAATATCATTTATTTTTTACATTATT1846 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 239 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet 151015 LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla 202530 GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyPhe 354045 PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp 505560 ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla 65707580 AlaAlaGlyProAlaLeuSerProValProProValValHisLeuThr 859095 LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgArgAspPhe 100105110 AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly 115120125 CysPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp 130135140 GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu 145150155160 SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp 165170175 MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn 180185190 GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro 195200205 LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla 210215220 LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuGlyHisLys 225230235 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4353 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Human NOS-1 gene, Fujisawa, et al, J. Neurochem 63:140 1994 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..4305 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATGGAGGATCACATGTTCGGTGTTCAGCAAATCCAGCCCAATGTCATT48 MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle 151015 TCTGTTCGTCTCTTCAAGCGCAAAGTTGGGGGCCTGGGATTTCTGGTG96 SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal 202530 AAGGAGCGGGTCAGTAAGCCGCCCGTGATCATCTCTGACCTGATTCGT144 LysGluArgValSerLysProProValIleIleSerAspLeuIleArg 354045 GGGGGCGCCGCAGAGCAGAGTGGCCTCATCCAGGCCGGAGACATCATT192 GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle 505560 CTTGCGGTCAACGGCCGGCCCTTGGTGGACCTGAGCTATGACAGCGCC240 LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla 65707580 CTGGAGGTACTCAGAGGCATTGCCTCTGAGACCCACGTGGTCCTCATT288 LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle 859095 CTGAGGGGCCCTGAAGGTTTCACCACGCACCTGGAGACCACCTTTACA336 LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr 100105110 GGTGATGGGACCCCCAAGACCATCCGGGTGACACAGCCCCTGGGTCCC384 GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro 115120125 CCCACCAAAGCCGTGGATCTGTCCCACCAGCCACCGGCCGGCAAAGAA432 ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu 130135140 CAGCCCCTGGCAGTGGATGGGGCCTCGGGTCCCGGGAATGGGCCTCAG480 GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln 145150155160 CATGCCTACGATGATGGGCAGGAGGCTGGCTCACTCCCCCATGCCAAC528 HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn 165170175 GGCCTGGCCCCCAGGCCCCCAGGCCAGGACCCCGCGAAGAAAGCAACC576 GlyLeuAlaProArgProProGlyGlnAspProAlaLysLysAlaThr 180185190 AGAGTCAGCCTCCAAGGCAGAGGGGAGAACAATGAACTGCTCAAGGAG624 ArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGlu 195200205 ATAGAGCCTGTGCTGAGCCTTCTCACCAGTGGGAGCAGAGGGGTCAAG672 IleGluProValLeuSerLeuLeuThrSerGlySerArgGlyValLys 210215220 GGAGGGGCACCTGCCAAGGCAGAGATGAAAGATATGGGAATCCAGGTG720 GlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnVal 225230235240 GACAGAGATTTGGACGGCAAGTCACACAAACCTCTGCCCCTCGGCGTG768 AspArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyVal 245250255 GAGAACGACCGAGTCTTCAATGACCTATGGGGGAAGGGCAATGTGCCT816 GluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValPro 260265270 GTCGTCCTCAACAACCCATATTCAGAGAAGGAGCAGCCCCCCACCTCA864 ValValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSer 275280285 GGAAAACAGTCCCCCACAAAGAATGGCAGCCCCTCCAAGTGTCCACGC912 GlyLysGlnSerProThrLysAsnGlySerProSerLysCysProArg 290295300 TTCCTCAAGGTCAAGAACTGGGAGACTGAGGTGGTTCTCACTGACACC960 PheLeuLysValLysAsnTrpGluThrGluValValLeuThrAspThr 305310315320 CTCCACCTTAAGAGCACATTGGAAACGGGATGCACTGAGTACATCTGC1008 LeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCys 325330335 ATGGGCTCCATCATGCATCCTTCTCAGCATGCAAGGAGGCCTGAAGAC1056 MetGlySerIleMetHisProSerGlnHisAlaArgArgProGluAsp 340345350 GTCCGCACAAAAGGACAGCTCTTCCCTCTCGCCAAAGAGTTTATTGAT1104 ValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAsp 355360365 CAATACTATTCATCAATTAAAAGATTTGGCTCCAAAGCCCACATGGAA1152 GlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGlu 370375380 AGGCTGGAAGAGGTGAACAAAGAGATCGACACCACTAGCACTTACCAG1200 ArgLeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGln 385390395400 CTCAAGGACACAGAGCTCATCTATGGGGCCAAGCACGCCTGGCGGAAT1248 LeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsn 405410415 GCCTCGCGCTGTGTGGGCAGGATCCAGTGGTCCAAGCTGCAGGTATTC1296 AlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPhe 420425430 GATGCCCGTGACTGCACCACGGCCCACGGGATGTTCAACTACATCTGT1344 AspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCys 435440445 AACCATGTCAAGTATGCCACCAACAAAGGGAACCTCAGGTCTGCCATC1392 AsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIle 450455460 ACCATATTCCCCCAGAGGACAGACGGCAAGCACGACTTCCGAGTCTGG1440 ThrIlePheProGlnArgThrAspGlyLysHisAspPheArgValTrp 465470475480 AACTCCCAGCTCATCCGCTACGCTGGCTACAAGCAGCCTGACGGCTCC1488 AsnSerGlnLeuIleArgTyrAlaGlyTyrLysGlnProAspGlySer 485490495 ACCCTGGGGGACCCAGCCAATGTGCAGTTCACAGAGATATGCATACAG1536 ThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGln 500505510 CAGGGCTGGAAACCGCCTAGAGGCCGCTTCGATGTCCTGCCGCTCCTG1584 GlnGlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeu 515520525 CTTCAGGCCAACGGCAATGACCCTGAGCTCTTCCAGATTCCTCCAGAG1632 LeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGlu

530535540 CTGGTGTTGGAAGTTCCCATCAGGCACCCCAAGTTTGAGTGGTTCAAG1680 LeuValLeuGluValProIleArgHisProLysPheGluTrpPheLys 545550555560 GACCTGGGGCTGAAGTGGTACGGCCTCCCCGCCGTGTCCAACATGCTC1728 AspLeuGlyLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeu 565570575 CTAGAGATTGGCGGCCTGGAGTTCAGCGCCTGTCCCTTCAGTGGCTGG1776 LeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrp 580585590 TACATGGGCACAGAGATTGGTGTCCGCGACTACTGTGACAACTCCCGC1824 TyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArg 595600605 TACAATATCCTGGAGGAAGTGGCCAAGAAGATGAACTTAGACATGAGG1872 TyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArg 610615620 AAGACGTCCTCCCTGTGGAAGGACCAGGCGCTGGTGGAGATCAATATC1920 LysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIle 625630635640 GCGGTTCTCTATAGCTTCCAGAGTGACAAAGTGACCATTGTTGACCAT1968 AlaValLeuTyrSerPheGlnSerAspLysValThrIleValAspHis 645650655 CACTCCGCCACCGAGTCCTTCATTAAGCACATGGAGAATGAGTACCGC2016 HisSerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArg 660665670 TGCCGGGGGGGCTGCCCTGCCGACTGGGTGTGGATCGTGCCCCCCATG2064 CysArgGlyGlyCysProAlaAspTrpValTrpIleValProProMet 675680685 TCCGGAAGCATCACCCCTGTGTTCCACCAGGAGATGCTCAACTACCGG2112 SerGlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArg 690695700 CTCACCCCCTCCTTCGAATACCAGCCTGATCCCTGGAACACGCATGTC2160 LeuThrProSerPheGluTyrGlnProAspProTrpAsnThrHisVal 705710715720 TGGAAAGGCACCAACGGGACCCCCACAAAGCGGCGAGCCATCGGCTTC2208 TrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPhe 725730735 AAGAAGCTAGCAGAAGCTGTCAAGTTCTCGGCCAAGCTGATGGGGCAG2256 LysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGln 740745750 GCTATGGCCAAGAGGGTGAAAGCGACCATCCTCTATGCCACAGAGACA2304 AlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThr 755760765 GGCAAATCGCAAGCTTATGCCAAGACCTTGTGTGAGATCTTCAAACAC2352 GlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHis 770775780 GCCTTTGATGCCAAGGTGATGTCCATGGAAGAATATGACATTGTGCAC2400 AlaPheAspAlaLysValMetSerMetGluGluTyrAspIleValHis 785790795800 CTGGAACATGAAACTCTGGTCCTTGTGGTCACCAGCACCTTTGGCAAT2448 LeuGluHisGluThrLeuValLeuValValThrSerThrPheGlyAsn 805810815 GGAGATCCCCCTGAGAATGGGGAGAAATTCGGCTGTGCTTTGATGGAA2496 GlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGlu 820825830 ATGAGGCACCCCAACTCTGTGCAGGAAGAAAGGAAGAGCTACAAGGTC2544 MetArgHisProAsnSerValGlnGluGluArgLysSerTyrLysVal 835840845 CGATTCAACAGCGTCTCCTCCTACTCTGACTCCCAAAAATCATCAGGC2592 ArgPheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGly 850855860 GATGGGCCCGACCTCAGAGACAACTTTGAGAGTGCTGGACCCCTGGCC2640 AspGlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAla 865870875880 AATGTGAGGTTCTCAGTTTTTGGCCTCGGCTCACGAGCATACCCTCAC2688 AsnValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHis 885890895 TTTTGCGCCTTCGGACACGCTGTGGACACCCTCCTGGAAGAACTGGGA2736 PheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGly 900905910 GGGGAGAGGATCCTGAAGATGAGGGAAGGGGATGAGCTCTGTGGGCAG2784 GlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGln 915920925 GAAGAGGCTTTCAGGACCTGGGCCAAGAAGGTCTTCAAGGCAGCCTGT2832 GluGluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCys 930935940 GATGTCTTCTGTGTGGGAGATGATGTCAACATTGAAAAGGCCAACAAT2880 AspValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsn 945950955960 TCCCTCATCAGCAATGATCGCAGCTGGAAGAGAAACAAGTTCCGCCTC2928 SerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeu 965970975 ACCTTTGTGGCCGAAGCTCCAGAACTCACACAAGGTCTATCCAATGTC2976 ThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnVal 980985990 CACAAAAAGCGAGTCTCAGCTGCCCGGCTCCTTAGCCGTCAAAACCTC3024 HisLysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeu 99510001005 CAGAGCCCTAAATCCAGTCGGTCAACTATCTTCGTGCGTCTCCACACC3072 GlnSerProLysSerSerArgSerThrIlePheValArgLeuHisThr 101010151020 AACGGGAGCCAGGAGCTGCAGTACCAGCCTGGGGACCACCTGGGTGTC3120 AsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyVal 1025103010351040 TTCCCTGGCAACCACGAGGACCTCGTGAATGCCCTGATCGAGCGGCTG3168 PheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeu 104510501055 GAGGACGCGCCGCCTGTCAACCAGATGGTGAAAGTGGAACTGCTGGAG3216 GluAspAlaProProValAsnGlnMetValLysValGluLeuLeuGlu 106010651070 GAGCGGAACACGGCTTTAGGTGTCATCAGTAACTGGACAGACGAGCTC3264 GluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeu 107510801085 CGCCTCCCACCCTGCACCATCTTCCAGGCCTTCAAGTACTACCTGGAC3312 ArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAsp 109010951100 ATCACCACGCCACCAACGCCCCTGCAGCTGCAGCAGTTTGCCTCCCTA3360 IleThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeu 1105111011151120 GCTACCAGCGAGAAGGAGAAGCAGCGTCTGCTGGTCCTCAGCAAGGGT3408 AlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGly 112511301135 TTGCAGGAGTACGAGGAATGGAAATGGGGCAAGAACCCCACCATCGTG3456 LeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleVal 114011451150 GAGGTGCTGGAGGAGTTCCCATCTATCCAGATGCCGGCCACCCTGCTC3504

GluValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeu 115511601165 CTGACCCAGCTGTCCCTGCTGCAGCCCCGCTACTATTCCATCAGCTCC3552 LeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSer 117011751180 TCCCCAGACATGTACCCTGATGAAGTGCACCTCACTGTGGCCATCGTT3600 SerProAspMetTyrProAspGluValHisLeuThrValAlaIleVal 1185119011951200 TCCTACCGCACTCGAGATGGAGAAGGACCAATTCACCACGGCGTATGC3648 SerTyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCys 120512101215 TCCTCCTGGCTCAACCGGATACAGGCTGACGAACTGGTCCCCTGTTTC3696 SerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPhe 122012251230 GTGAGAGGAGCACCCAGCTTCCACCTGCCCCGGAACCCCCAAGTCCCC3744 ValArgGlyAlaProSerPheHisLeuProArgAsnProGlnValPro 123512401245 TGCATCCTCGTTGGACCAGGCACCGGCATTGCCCCTTTCCGAAGCTTC3792 CysIleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPhe 125012551260 TGGCAACAGCGGCAATTTGATATCCAACACAAAGGAATGAACCCCTGC3840 TrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCys 1265127012751280 CCCATGGTCCTGGTCTTCGGGTGCCGGCAATCCAAGATAGATCATATC3888 ProMetValLeuValPheGlyCysArgGlnSerLysIleAspHisIle 128512901295 TACAGGGAAGAGACCCTGCAGGCCAAGAACAAGGGGGTCTTCAGAGAG3936 TyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGlu 130013051310 CTGTACACGGCTTACTCCCGGGAGCCAGACAAACCAAAGAAGTACGTG3984 LeuTyrThrAlaTyrSerArgGluProAspLysProLysLysTyrVal 131513201325 CAGGACATCCTGCAGGAGCAGCTGGCGGAGTCTGTGTACCGAGCCCTG4032 GlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeu 133013351340 AAGGAGCAAGGGGGCCACATATACGTCTGTGGGGACGTCACCATGGCT4080 LysGluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAla 1345135013551360 GCTGATGTCCTCAAAGCCATCCAGCGCATCATGACCCAGCAGGGGAAG4128 AlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLys 136513701375 CTCTCGGCAGAGGACGCCGGCGTATTCATCAGCCGGATGAGGGATGAC4176 LeuSerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAsp 138013851390 AACCGATACCATGAGGATATTTTTGGAGTCACCCTGCGAACGTACGAA4224 AsnArgTyrHisGluAspIlePheGlyValThrLeuArgThrTyrGlu 139514001405 GTGACCAACCGCCTTAGATCTGAGTCCATTGCCTTCATTGAAGAGAGC4272 ValThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSer 141014151420 AAAAAAGACACCGATGAGGTTTTCAGCTCCTAACTGGACCCTCTTGCCCA4322 LysLysAspThrAspGluValPheSerSer 14251430143 GCCGGCTGCAAGTTTGTAAGCGCGGGACAGA4353 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1434 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle 151015 SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal 202530 LysGluArgValSerLysProProValIleIleSerAspLeuIleArg 354045 GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle 505560 LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla 65707580 LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle 859095 LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr 100105110 GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro 115120125 ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu 130135140 GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln 145150155160 HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn 165170175 GlyLeuAlaProArgProProGlyGlnAspProAlaLysLysAlaThr 180185190 ArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGlu 195200205 IleGluProValLeuSerLeuLeuThrSerGlySerArgGlyValLys 210215220 GlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnVal 225230235240 AspArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyVal 245250255 GluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValPro 260265270 ValValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSer 275280285 GlyLysGlnSerProThrLysAsnGlySerProSerLysCysProArg 290295300 PheLeuLysValLysAsnTrpGluThrGluValValLeuThrAspThr 305310315320 LeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCys 325330335 MetGlySerIleMetHisProSerGlnHisAlaArgArgProGluAsp 340345350 ValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAsp 355360365 GlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGlu 370375380 ArgLeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGln 385390395400 LeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsn 405410415 AlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPhe 420425430 AspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCys 435440445 AsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIle 450455460 ThrIlePheProGlnArgThrAspGlyLysHisAspPheArgValTrp 465470475480 AsnSerGlnLeuIleArgTyrAlaGlyTyrLysGlnProAspGlySer 485490495 ThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGln 500505510 GlnGlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeu 515520525 LeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGlu 530535540 LeuValLeuGluValProIleArgHisProLysPheGluTrpPheLys 545550555560 AspLeuGlyLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeu 565570575 LeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrp 580585590 TyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArg 595600605 TyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArg 610615620 LysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIle 625630635640 AlaValLeuTyrSerPheGlnSerAspLysValThrIleValAspHis 645650655 HisSerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArg 660665670 CysArgGlyGlyCysProAlaAspTrpValTrpIleValProProMet 675680685 SerGlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArg 690695700 LeuThrProSerPheGluTyrGlnProAspProTrpAsnThrHisVal 705710715720 TrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPhe 725730735 LysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGln 740745750 AlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThr 755760765 GlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHis 770775780 AlaPheAspAlaLysValMetSerMetGluGluTyrAspIleValHis 785790795800 LeuGluHisGluThrLeuValLeuValValThrSerThrPheGlyAsn 805810815 GlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGlu 820825830 MetArgHisProAsnSerValGlnGluGluArgLysSerTyrLysVal 835840845 ArgPheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGly 850855860 AspGlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAla 865870875880 AsnValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHis 885890895 PheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGly 900905910 GlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGln 915920925 GluGluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCys 930935940 AspValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsn 945950955960 SerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeu 965970975 ThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnVal 980985990 HisLysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeu 99510001005 GlnSerProLysSerSerArgSerThrIlePheValArgLeuHisThr 101010151020 AsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyVal 1025103010351040 PheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeu 104510501055 GluAspAlaProProValAsnGlnMetValLysValGluLeuLeuGlu 106010651070 GluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeu 107510801085 ArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAsp 109010951100 IleThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeu 1105111011151120 AlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGly 112511301135 LeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleVal 114011451150 GluValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeu 115511601165 LeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSer 117011751180 SerProAspMetTyrProAspGluValHisLeuThrValAlaIleVal 1185119011951200 SerTyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCys 120512101215 SerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPhe 122012251230 ValArgGlyAlaProSerPheHisLeuProArgAsnProGlnValPro 123512401245 CysIleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPhe 125012551260 TrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCys 1265127012751280 ProMetValLeuValPheGlyCysArgGlnSerLysIleAspHisIle 128512901295 TyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGlu 130013051310 LeuTyrThrAlaTyrSerArgGluProAspLysProLysLysTyrVal 131513201325 GlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeu 133013351340 LysGluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAla 1345135013551360 AlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLys 136513701375 LeuSerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAsp 138013851390 AsnArgTyrHisGluAspIlePheGlyValThrLeuArgThrTyrGlu 139514001405 ValThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSer 141014151420 LysLysAspThrAspGluValPheSerSer 14251430 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4780 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Human NOS-SN gene, Nakane, et al, FEBS Lett 316:175 (1993) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 431..4732 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GAGCGGACGGGCTCATGATGCCTCAGATCTGATCCGCATCTAACAGGCTGGCAATGAAGA60 TACCCAGAGAATAGTTCACATCTATCATGCGTCACTTCTAGACACAGCCATCAGACGCAT120 CTCCTCCCCTTTCTGCCTGACCTTAGGACACGTCCCACCGCCTCTCTTGACGTCTGCCTG180 GTCAACCATCACTTCCTTAGAGAATAAGGAGAGAGGCGGATGCAGGAAATCATGCCACCG240 ACGGGCCACCAGCCATGAGTGGGTGACGCTGAGCTGACGTCAAAGACAGAGAGGGCTGAA300 GCCTTGTCAGCACCTGTCACCCCGGCTCCTGCTCTCCGTGTAGCCTGAAGCCTGGATCCT360 CCTGGTGAAATCATCTTGGCCTGATAGCATTGTGAGGTCTTCAGACAGGACCCCTCGGAA420 GCTAGTTACCATGGAGGATCACATGTTCGGTGTTCAGCAAATCCAGCCC469 MetGluAspHisMetPheGlyValGlnGlnIleGlnPro 1510 AATGTCATTTCTGTTCGTCTCTTCAAGCGCAAAGTTGGGGGCCTGGGA517 AsnValIleSerValArgLeuPheLysArgLysValGlyGlyLeuGly 152025 TTTCTGGTGAAGGAGCGGGTCAGTAAGCCGCCCGTGATCATCTCTGAC565 PheLeuValLysGluArgValSerLysProProValIleIleSerAsp 30354045 CTGATTCGTGGGGGCGCCGCAGAGCAGAGTGGCCTCATCCAGGCCGGA613 LeuIleArgGlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGly 505560 GACATCATTCTTGCGGTCAACGGCCGGCCCTTGGTGGACCTGAGCTAT661 AspIleIleLeuAlaValAsnGlyArgProLeuValAspLeuSerTyr 657075 GACAGCGCCCTGGAGGTACTCAGAGGCATTGCCTCTGAGACCCACGTG709 AspSerAlaLeuGluValLeuArgGlyIleAlaSerGluThrHisVal 808590 GTCCTCATTCTGAGGGGCCCTGAAGGTTTCACCACGCACCTGGAGACC757 ValLeuIleLeuArgGlyProGluGlyPheThrThrHisLeuGluThr 95100105 ACCTTTACAGGTGATGGGACCCCCAAGACCATCCGGGTGACACAGCCC805 ThrPheThrGlyAspGlyThrProLysThrIleArgValThrGlnPro 110115120125 CTGGGTCCCCCCACCAAAGCCGTGGATCTGTCCCACCAGCCACCGGCC853 LeuGlyProProThrLysAlaValAspLeuSerHisGlnProProAla 130135140 GGCAAAGAACAGCCCCTGGCAGTGGATGGGGCCTCGGGTCCCGGGAAT901 GlyLysGluGlnProLeuAlaValAspGlyAlaSerGlyProGlyAsn 145150155 GGGCCTCAGCATGCCTACGATGATGGGCAGGAGGCTGGCTCACTCCCC949 GlyProGlnHisAlaTyrAspAspGlyGlnGluAlaGlySerLeuPro 160165170 CATGCCAACGGCTGGCCCCAGGCCCCCAGGCAGGACCCCGCGAAGAAA997 HisAlaAsnGlyTrpProGlnAlaProArgGlnAspProAlaLysLys 175180185 GCAACCAGAGTCAGCCTCCAAGGCAGAGGGGAGAACAATGAACTGCTC1045 AlaThrArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeu 190195200205 AAGGAGATAGAGCCTGTGCTGAGCCTTCTCACCAGTGGGAGCAGAGGG1093 LysGluIleGluProValLeuSerLeuLeuThrSerGlySerArgGly 210215220 GTCAAGGGAGGGGCACCTGCCAAGGCAGAGATGAAAGATATGGGAATC1141 ValLysGlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIle 225230235 CAGGTGGACAGAGATTTGGACGGCAAGTCACACAAACCTCTGCCCCTC1189 GlnValAspArgAspLeuAspGlyLysSerHisLysProLeuProLeu 240245250 GGCGTGGAGAACGACCGAGTCTTCAATGACCTATGGGGGAAGGGCAAT1237 GlyValGluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsn 255260265 GTGCCTGTCGTCCTCAACAACCCATATTCAGAGAAGGAGCAGCCCCCC1285 ValProValValLeuAsnAsnProTyrSerGluLysGluGlnProPro 270275280285 ACCTCAGGAAAACAGTCCCCCACAAAGAATGGCAGCCCCTCCAAGTGT1333 ThrSerGlyLysGlnSerProThrLysAsnGlySerProSerLysCys 290295300 CCACGCTTCCTCAAGGTCAAGAACTGGGAGACTGAGGTGGTTCTCACT1381 ProArgPheLeuLysValLysAsnTrpGluThrGluValValLeuThr 305310315 GACACCCTCCACCTTAAGAGCACATTGGAAACGGGATGCACTGAGTAC1429 AspThrLeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyr 320325330 ATCTGCATGGGCTCCATCATGCATCCTTCTCAGCATGCAAGGAGGCCT1477 IleCysMetGlySerIleMetHisProSerGlnHisAlaArgArgPro 335340345 GAAGACGTCCGCACAAAAGGACAGCTCTTCCCTCTCGCCAAAGAGTTT1525 GluAspValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPhe 350355360365 ATTGATCAATACTATTCATCAATTAAAAGATTTGGCTCCAAAGCCCAC1573 IleAspGlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHis 370375380 ATGGAAAGGCTGGAAGAGGTGAACAAAGAGATCGACACCACTAGCACT1621 MetGluArgLeuGluGluValAsnLysGluIleAspThrThrSerThr 385390395 TACCAGCTCAAGGACACAGAGCTCATCTATGGGGCCAAGCACGCCTGG1669 TyrGlnLeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrp 400405410 CGGAATGCCTCGCGCTGTGTGGGCAGGATCCAGTGGTCCAAGCTGCAG1717 ArgAsnAlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGln 415420425 GTATTCGATGCCCGTGACTGCACCACGGCCCACGGGATGTTCAACTAC1765 ValPheAspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyr 430435440445 ATCTGTAACCATGTCAAGTATGCCACCAACAAAGGGAACCTCAGGTCT1813 IleCysAsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSer 450455460 GCCATCACCATATTCCCCCAGAGGACAGACGGCAAGCACGACTTCCGA1861 AlaIleThrIlePheProGlnArgThrAspGlyLysHisAspPheArg 465470475 GTCTGGAACTCCCAGCTCATCCGCTACGCTGGCTACAAGCACCGTGAC1909 ValTrpAsnSerGlnLeuIleArgTyrAlaGlyTyrLysHisArgAsp 480485490 GGCTCCACCCTGGGGGACCCAGCCAATGTGCAGTTCACAGAGATATGC1957 GlySerThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCys 495500505 ATACAGCAGGGCTGGAAACCGCCTAGAGGCCGCTTCGATGTCCTGCCG2005 IleGlnGlnGlyTrpLysProProArgGlyArgPheAspValLeuPro 510515520525 CTCCTGCTTCAGGCCAACGGCAATGACCCTGAGCTCTTCCAGATTCCT2053 LeuLeuLeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIlePro 530535540 CCAGAGCTGGTGTTGGAACTTCCCATCAGGCACCCCAAGTTTGAGTGG2101 ProGluLeuValLeuGluLeuProIleArgHisProLysPheGluTrp 545550555 TTCAAGGACCTGGCGCTGAAGTGGTACGGCCTCCCCGCCGTGTCCAAC2149 PheLysAspLeuAlaLeuLysTrpTyrGlyLeuProAlaValSerAsn 560565570 ATGCTCCTAGAGATTGGCGGCCTGGAGTTCAGCGCCTGTCCCTTCAGT2197 MetLeuLeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSer 575580585 GGCTGGTACATGGGCACAGAGATTGGTGTCCGCGACTACTGTGACAAC2245 GlyTrpTyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsn 590595600605 TCCCGCTACAATATCCTGGAGGAAGTGGCCAAGAAGATGAACTTAGAC2293 SerArgTyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAsp 610615620 ATGAGGAAGACGTCCTCCCTGTGGAAGGACCAGGCGCTGGTGGAGATC2341 MetArgLysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIle 625630635 AATATCGCGGTTCTCTATAGCTTCCAGAGTGACAAAGTGACCATTGTT2389 AsnIleAlaValLeuTyrSerPheGlnSerAspLysValThrIleVal 640645650 GACCATCACTCCGCCACCGAGTCCTTCATTAAGCACATGGAGAATGAG2437 AspHisHisSerAlaThrGluSerPheIleLysHisMetGluAsnGlu 655660665 TACCGCTGCCGGGGGGGCTGCCCTGCCGACTGGGTGTGGATCGTGCCC2485 TyrArgCysArgGlyGlyCysProAlaAspTrpValTrpIleValPro 670675680685 CCCATGTCCGGAAGCATCACCCCTGTGTTCCACCAGGAGATGCTCAAC2533 ProMetSerGlySerIleThrProValPheHisGlnGluMetLeuAsn 690695700 TACCGGCTCACCCCCTCCTTCGAATACCAGCCTGATCCCTGGAACACG2581 TyrArgLeuThrProSerPheGluTyrGlnProAspProTrpAsnThr 705710715 CATGTCTGGAAAGGCACCAACGGGACCCCCACAAAGCGGCGAGCCATC2629 HisValTrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIle 720725730 GGCTTCAAGAAGCTAGCAGAAGCTGTCAAGTTCTCGGCCAAGCTGATG2677 GlyPheLysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMet 735740745 GGGCAGGCTATGGCCAAGAGGGTGAAAGCGACCATCCTCTATGCCACA2725 GlyGlnAlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThr 750755760765 GAGACAGGCAAATCGCAAGCTTATGCCAAGACCTTGTGTGAGATCTTC2773 GluThrGlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePhe 770775780 AAACACGCCTTTGATGCCAAGGTGATGTCCATGGAAGAATATGACATT2821 LysHisAlaPheAspAlaLysValMetSerMetGluGluTyrAspIle 785790795 GTGCACCTGGAACATGAAACTCTGGTCCTTGTGGTCACCAGCACCTTT2869 ValHisLeuGluHisGluThrLeuValLeuValValThrSerThrPhe 800805810 GGCAATGGAGATCCCCCTGAGAATGGGGAGAAATTCGGCTGTGCTTTG2917 GlyAsnGlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeu 815820825 ATGGAAATGAGGCACCCCAACTCTGTGCAGGAAGAAAGGAAGAGCTAC2965 MetGluMetArgHisProAsnSerValGlnGluGluArgLysSerTyr 830835840845 AAGGTCCGATTCAACAGCGTCTCCTCCTACTCTGACTCCCAAAAATCA3013 LysValArgPheAsnSerValSerSerTyrSerAspSerGlnLysSer 850855860 TCAGGCGATGGGCCCGACCTCAGAGACAACTTTGAGAGTGCTGGACCC3061 SerGlyAspGlyProAspLeuArgAspAsnPheGluSerAlaGlyPro 865870875 CTGGCCAATGTGAGGTTCTCAGTTTTTGGCCTCGGCTCACGAGCATAC3109 LeuAlaAsnValArgPheSerValPheGlyLeuGlySerArgAlaTyr 880885890 CCTCACTTTTGCGCCTTCGGACACGCTGTGGACACCCTCCTGGAAGAA3157 ProHisPheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGlu 895900905 CTGGGAGGGGAGAGGATCCTGAAGATGAGGGAAGGGGATGAGCTCTGT3205 LeuGlyGlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCys 910915920925 GGGCAGGAAGAGGCTTTCAGGACCTGGGCCAAGAAGGTCTTCAAGGCA3253 GlyGlnGluGluAlaPheArgThrTrpAlaLysLysValPheLysAla 930935940 GCCTGTGATGTCTTCTGTGTGGGAGATGATGTCAACATTGAAAAGGCC3301 AlaCysAspValPheCysValGlyAspAspValAsnIleGluLysAla 945950955 AACAATTCCCTCATCAGCAATGATCGCAGCTGGAAGAGAAACAAGTTC3349 AsnAsnSerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPhe 960965970 CGCCTCACCTTTGTGGCCGAAGCTCCAGAACTCACACAAGGTCTATCC3397 ArgLeuThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSer 975980985 AATGTCCACAAAAAGCGAGTCTCAGCTGCCCGGCTCCTTAGCCGTCAA3445 AsnValHisLysLysArgValSerAlaAlaArgLeuLeuSerArgGln 99099510001005 AACCTCCAGAGCCCTAAATCCAGTCGGTCAACTATCTTCGTGCGTCTC3493 AsnLeuGlnSerProLysSerSerArgSerThrIlePheValArgLeu 101010151020 CACACCAACGGGAGCCAGGAGCTGCAGTACCAGCCTGGGGACCACCTG3541 HisThrAsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeu 102510301035 GGTGTCTTCCCTGGCAACCACGAGGACCTCGTGAATGCCCTGATCGAG3589 GlyValPheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGlu 104010451050 CGGCTGGAGGACGCGCCGCCTGTCAACCAGATGGTGAAAGTGGAACTG3637 ArgLeuGluAspAlaProProValAsnGlnMetValLysValGluLeu 105510601065 CTGGAGGAGCGGAACACGGCTTTAGGTGTCATCAGTAACTGGACAGAC3685 LeuGluGluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAsp 1070107510801085 GAGCTCCGCCTCCCGCCCTGCACCATCTTCCAGGCCTTCAAGTACTAC3733 GluLeuArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyr 109010951100 CTGGACATCACCACGCCACCAACGCCTCTGCAGCTGCAGCAGTTTGCC3781 LeuAspIleThrThrProProThrProLeuGlnLeuGlnGlnPheAla 110511101115 TCCCTAGCTACCAGCGAGAAGGAGAAGCAGCGTCTGCTGGTCCTCAGC3829 SerLeuAlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSer 112011251130 AAGGGTTTGCAGGAGTACGAGGAATGGAAATGGGGCAAGAACCCCACC3877 LysGlyLeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThr 113511401145 ATCGTGGAGGTGCTGGAGGAGTTCCCATCTATCCAGATGCCGGCCACC3925 IleValGluValLeuGluGluPheProSerIleGlnMetProAlaThr 1150115511601165 CTGCTCCTGACCCAGCTGTCCCTGCTGCAGCCCCGCTACTATTCCATC3973 LeuLeuLeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIle 117011751180 AGCTCCTCCCCAGACATGTACCCTGATGAAGTGCACCTCACTGTGGCC4021 SerSerSerProAspMetTyrProAspGluValHisLeuThrValAla 118511901195 ATCGTTTCCTACCGCACTCGAGATGGAGAAGGACCAATTCACCACGGC4069 IleValSerTyrArgThrArgAspGlyGluGlyProIleHisHisGly 120012051210 GTATGCTCCTCCTGGCTCAACCGGATACAGGCTGACGAACTGGTCCCC4117 ValCysSerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValPro 121512201225 TGTTTCGTGAGAGGAGCACCCAGCTTCCACCTGCCCCGGAACCCCCAA4165 CysPheValArgGlyAlaProSerPheHisLeuProArgAsnProGln 1230123512401245 GTCCCCTGCATCCTCGTTGGACCAGGCACCGGCATTGCCCCTTTCCGA4213 ValProCysIleLeuValGlyProGlyThrGlyIleAlaProPheArg 125012551260 AGCTTCTGGCAACAGCGGCAATTTGATATCCAACACAAAGGAATGAAC4261

SerPheTrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsn 126512701275 CCCTGCCCCATGGTCCTGGTCTTCGGGTGCCGGCAATCCAAGATAGAT4309 ProCysProMetValLeuValPheGlyCysArgGlnSerLysIleAsp 128012851290 CATATCTACAGGGAAGAGACCCTGCAGGCCAAGAACAAGGGGGTCTTC4357 HisIleTyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPhe 129513001305 AGAGAGCTGTACACGGCTTACTCCCGGGAGCCAGACAAACCAAAGAAG4405 ArgGluLeuTyrThrAlaTyrSerArgGluProAspLysProLysLys 1310131513201325 TACGTGCAGGACATCCTGCAGGAGCAGCTGGCGGAGTCTGTGTACCGA4453 TyrValGlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArg 133013351340 GCCCTGAAGGAGCAAGGGGGCCACATATACGTCTGTGGGGACGTCACC4501 AlaLeuLysGluGlnGlyGlyHisIleTyrValCysGlyAspValThr 134513501355 ATGGCTGCTGATGTCCTCAAAGCCATCCAGCGCATCATGACCCAGCAG4549 MetAlaAlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGln 136013651370 GGGAAGCTCTCGGCAGAGGACGCCGGCGTATTCATCAGCCGGATGAGG4597 GlyLysLeuSerAlaGluAspAlaGlyValPheIleSerArgMetArg 137513801385 GATGACAACCGATACCATGAGGATATTTTTGGAGTCACCCTGCGAACG4645 AspAspAsnArgTyrHisGluAspIlePheGlyValThrLeuArgThr 1390139514001405 ATCGAAGTGACCAACCGCCTTAGATCTGAGTCCATTGCCTTCATTGAA4693 IleGluValThrAsnArgLeuArgSerGluSerIleAlaPheIleGlu 141014151420 GAGAGCAAAAAAGACACCGATGAGGTTTTCAGCTCCTAACTGGACC4739 GluSerLysLysAspThrAspGluValPheSerSer 14251430 CTCTTGCCCAGCCGGCTGCAAGTTTGTAAGCGCGGGACAGA4780 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1433 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle 151015 SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal 202530 LysGluArgValSerLysProProValIleIleSerAspLeuIleArg 354045 GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle 505560 LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla 65707580 LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle 859095 LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr 100105110 GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro 115120125 ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu 130135140 GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln 145150155160 HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn 165170175 GlyTrpProGlnAlaProArgGlnAspProAlaLysLysAlaThrArg 180185190 ValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGluIle 195200205 GluProValLeuSerLeuLeuThrSerGlySerArgGlyValLysGly 210215220 GlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnValAsp 225230235240 ArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyValGlu 245250255 AsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValProVal 260265270 ValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSerGly 275280285 LysGlnSerProThrLysAsnGlySerProSerLysCysProArgPhe 290295300 LeuLysValLysAsnTrpGluThrGluValValLeuThrAspThrLeu 305310315320 HisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCysMet 325330335 GlySerIleMetHisProSerGlnHisAlaArgArgProGluAspVal 340345350 ArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAspGln 355360365 TyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGluArg 370375380 LeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGlnLeu 385390395400 LysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsnAla 405410415 SerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPheAsp 420425430 AlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCysAsn 435440445 HisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIleThr 450455460 IlePheProGlnArgThrAspGlyLysHisAspPheArgValTrpAsn 465470475480 SerGlnLeuIleArgTyrAlaGlyTyrLysHisArgAspGlySerThr 485490495 LeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGlnGln 500505510 GlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeuLeu 515520525 GlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGluLeu 530535540 ValLeuGluLeuProIleArgHisProLysPheGluTrpPheLysAsp 545550555560 LeuAlaLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeuLeu 565570575 GluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrpTyr 580585590 MetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArgTyr 595600605

AsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArgLys 610615620 ThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIleAla 625630635640 ValLeuTyrSerPheGlnSerAspLysValThrIleValAspHisHis 645650655 SerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArgCys 660665670 ArgGlyGlyCysProAlaAspTrpValTrpIleValProProMetSer 675680685 GlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArgLeu 690695700 ThrProSerPheGluTyrGlnProAspProTrpAsnThrHisValTrp 705710715720 LysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPheLys 725730735 LysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGlnAla 740745750 MetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThrGly 755760765 LysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHisAla 770775780 PheAspAlaLysValMetSerMetGluGluTyrAspIleValHisLeu 785790795800 GluHisGluThrLeuValLeuValValThrSerThrPheGlyAsnGly 805810815 AspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGluMet 820825830 ArgHisProAsnSerValGlnGluGluArgLysSerTyrLysValArg 835840845 PheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGlyAsp 850855860 GlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAlaAsn 865870875880 ValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHisPhe 885890895 CysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGlyGly 900905910 GluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGlnGlu 915920925 GluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCysAsp 930935940 ValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsnSer 945950955960 LeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeuThr 965970975 PheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnValHis 980985990 LysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeuGln 99510001005 SerProLysSerSerArgSerThrIlePheValArgLeuHisThrAsn 101010151020 GlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyValPhe 1025103010351040 ProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeuGlu 104510501055 AspAlaProProValAsnGlnMetValLysValGluLeuLeuGluGlu 106010651070 ArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeuArg 107510801085 LeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAspIle 109010951100 ThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeuAla 1105111011151120 ThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGlyLeu 112511301135 GlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleValGlu 114011451150 ValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeuLeu 115511601165 ThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSerSer 117011751180 ProAspMetTyrProAspGluValHisLeuThrValAlaIleValSer 1185119011951200 TyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCysSer 120512101215 SerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPheVal 122012251230 ArgGlyAlaProSerPheHisLeuProArgAsnProGlnValProCys 123512401245 IleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPheTrp 125012551260 GlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCysPro 1265127012751280 MetValLeuValPheGlyCysArgGlnSerLysIleAspHisIleTyr 128512901295 ArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGluLeu 130013051310 TyrThrAlaTyrSerArgGluProAspLysProLysLysTyrValGln 131513201325 AspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeuLys 133013351340 GluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAlaAla 1345135013551360 AspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLysLeu 136513701375 SerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAspAsn 138013851390 ArgTyrHisGluAspIlePheGlyValThrLeuArgThrIleGluVal 139514001405 ThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSerLys 141014151420 LysAspThrAspGluValPheSerSer 14251430 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 256 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: EPO-1 HRE element (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GAACTGAAACCACCAATATGACTCTTGGCTTTTCTGTTTTCTGGGAACCTCCAAATCCCC60 TGGCTCTGTCCCACTCCTGGCAGCAGTGCAGCAGGTCCAGGTCCGGGAAATGAGGGGTGG120 AGGGGGCTGGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCC180 TAGGCCACAAGCTCTGCCTACGCTGGTCAATAAGGTGTCTCCATTCAAGGCCTCACCGCA240 GTAAGGCAGCTGCCAA256 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 42 bp EPO 3'hypoxia response enhancer element (Madan, et al, PNAS 90:3928, 1993) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGA42 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 86 nucleotide fragment from .alpha.MHC promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GTCCCAGCAGATGACTCCAAATTTAGGCAGCAGGCACGTGGAATGAGCTATAAAGGGGCT60 GGAGCGCTGAGAGCTGTCAGACCGAG86 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2423 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: mouse catalase gene GenBank #L25069 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 88..1671 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: ATTGCCTTCTCCGGGTGGAGACCAGACCGCTGCGTCCGTCCCTGCTGTCTCACGTTCCGC60 AGCTCTGCAGCTCCGCAATCCTACACCATGTCGGACAGTCGGGACCCAGCC111 MetSerAspSerArgAspProAla 15 AGCGACCAGATGAAGCAGTGGAAGGAGCAGCGGGCCTCGCAGAGACCT159 SerAspGlnMetLysGlnTrpLysGluGlnArgAlaSerGlnArgPro 101520 GATGTCCTGACCACCGGAGGCGGGAACCCAATAGGAGATAAACTTAAT207 AspValLeuThrThrGlyGlyGlyAsnProIleGlyAspLysLeuAsn 25303540 ATCATGACCGCGGGGTCCCGAGGGCCCCTCCTCGTTCAGGATGTGGTT255 IleMetThrAlaGlySerArgGlyProLeuLeuValGlnAspValVal 455055 TTCACTGACGAGATGGCACACTTTGACAGAGAGCGGATTCCTGAGAGA303 PheThrAspGluMetAlaHisPheAspArgGluArgIleProGluArg 606570 GTGGTACACGCAAAAGGAGCAGGTGCTTTTGGATACTTTGAGGTCACC351 ValValHisAlaLysGlyAlaGlyAlaPheGlyTyrPheGluValThr 758085 CACGATATCACCAGATACTCCAAGGGAAAGGTGTTTGAGCATATTGGA399 HisAspIleThrArgTyrSerLysGlyLysValPheGluHisIleGly 9095100 AAGAGGACCCCTATTGCCGTTCGGTTCTCCACAGTCGCTGGAGAGTCA447 LysArgThrProIleAlaValArgPheSerThrValAlaGlyGluSer 105110115120 GGCTCAGCTGACACAGTTCGTGACCCTCGGGGGTTTGCAGTGAAATTT495 GlySerAlaAspThrValArgAspProArgGlyPheAlaValLysPhe 125130135 TACACTGAAGATGGTAACTGGGATCTTGTGGGAAACAACACCCCTATT543 TyrThrGluAspGlyAsnTrpAspLeuValGlyAsnAsnThrProIle 140145150 TTCTTCATCAGGGATGCCATATTGTTTCCATCCTTTATCCATAGCCAG591 PhePheIleArgAspAlaIleLeuPheProSerPheIleHisSerGln 155160165 AAGAGAAACCCACAGACTCACCTGAAGGATCCTGACATGGTCTGGGAC639 LysArgAsnProGlnThrHisLeuLysAspProAspMetValTrpAsp 170175180 TTCTGGAGTCTTCGTCCCGAGTCTCTCCATCAGGTTTCTTTCTTGTTC687 PheTrpSerLeuArgProGluSerLeuHisGlnValSerPheLeuPhe 185190195200 AGTGACCGAGGGATTCCCGATGGTCACCGGCACATGAATGGCTATGGA735 SerAspArgGlyIleProAspGlyHisArgHisMetAsnGlyTyrGly 205210215 TCACACACCTTCAAGTTGGTTAATGCAGATGGAGAGGCAGTCTATTGC783 SerHisThrPheLysLeuValAsnAlaAspGlyGluAlaValTyrCys 220225230 AAGTTCCATTACAAGACCGACCAGGGCATCAAAAACTTGCCTGTTGGA831 LysPheHisTyrLysThrAspGlnGlyIleLysAsnLeuProValGly 235240245 GAGGCAGGAAGGCTTGCTCAGGAAGATCCGGATTATGGCCTCCGAGAT879 GluAlaGlyArgLeuAlaGlnGluAspProAspTyrGlyLeuArgAsp 250255260 CTTTTCAATGCCATCGCCAATGGCAATTACCCGTCCTGGACGTTTTAC927 LeuPheAsnAlaIleAlaAsnGlyAsnTyrProSerTrpThrPheTyr 265270275280 ATCCAGGTCATGACTTTTAAGGAGGCAGAAACTTTCCCATTTAATCCA975 IleGlnValMetThrPheLysGluAlaGluThrPheProPheAsnPro 285290295 TTTGATCTGACCAAGGTTTGGCCTCACAAGGACTACCCTCTTATACCA1023 PheAspLeuThrLysValTrpProHisLysAspTyrProLeuIlePro 300305310 GTTGGCAAAGTGGTTTTAAACAAAAATCCAGTTAATTACTTTGCTGAA1071 ValGlyLysValValLeuAsnLysAsnProValAsnTyrPheAlaGlu 315320325 GTTGAACAGATGGCTTTTGACCCAAGCAATATGCCCCCTGGCATCGAG1119 ValGluGlnMetAlaPheAspProSerAsnMetProProGlyIleGlu 330335340 CCCAGCCCTGACAAAAAGCTTCAGGGCCGCCTTTTTGCCTACCCGGAC1167 ProSerProAspLysLysLeuGlnGlyArgLeuPheAlaTyrProAsp 345350355360 ACTCACCGCCACCGCCTGGGACCCAACTATCTGCAGATACCTGTGAAC1215 ThrHisArgHisArgLeuGlyProAsnTyrLeuGlnIleProValAsn 365370375 TGTCCCTACCGCGCTCGAGTGGCCAACTACCAGCGTGATGGCCCCATG1263 CysProTyrArgAlaArgValAlaAsnTyrGlnArgAspGlyProMet 380385390 TGCATGCATGACAACCAGGGTGGTGCCCCCAACTATTACCCCAACAGC1311 CysMetHisAspAsnGlnGlyGlyAlaProAsnTyrTyrProAsnSer 395400405 TTCAGCGCACCAGAGCAGCAGCGCTCAGCCCTGGAGCACAGCGTCCAG1359 PheSerAlaProGluGlnGlnArgSerAlaLeuGluHisSerValGln 410415420 TGCGCTGTAGATGTGAAACGCTTCAACAGTGCTAATGAAGACAATGTC1407 CysAlaValAspValLysArgPheAsnSerAlaAsnGluAspAsnVal 425430435440 ACTCAGGTGCGGACATTCTACACAAAGGTGTTGAATGAGGAGGAGAGG1455

ThrGlnValArgThrPheTyrThrLysValLeuAsnGluGluGluArg 445450455 AAACGCCTGTGTGAGAACATTGCCGGCCACCTGAAGGACGCTCAGCTT1503 LysArgLeuCysGluAsnIleAlaGlyHisLeuLysAspAlaGlnLeu 460465470 TTCATTCAGAAGAAAGCGGTCAAGAATTTCACTGACGTCCACCCTGAC1551 PheIleGlnLysLysAlaValLysAsnPheThrAspValHisProAsp 475480485 TATGGGGCCCGCATCCAGGCTCTTCTGGACAAGTACAACGCTGAGAAG1599 TyrGlyAlaArgIleGlnAlaLeuLeuAspLysTyrAsnAlaGluLys 490495500 CCTAAGAACGCAATTCACACCTACACGCAGGCCGGCTCTCACATGGCT1647 ProLysAsnAlaIleHisThrTyrThrGlnAlaGlySerHisMetAla 505510515520 GCGAAGGGAAAAGCTAACCTGTAACTCCGGTGCTCAGCCTCCGCTGAGGAG1698 AlaLysGlyLysAlaAsnLeu 525 ACCTCTCGTGAAGCCGAGCCTGAGGATCACCTGTAATCAACGCTGGATGGATTCTCCCCC1758 GCCGGAGCGCAGACTCACGCTGATGACTTTAAAACGATAATCCGGGCTTCTAGAGTGAAT1818 GATAACCATGCTTTTGATGCCGTTTCCTGAAGGGAAATGAAAGGTTAGGGCTTAGCAATC1878 ATTTAACAGAAACATGGATCTAATAGGACTTCTGTTTGGATTATTCATTTAAATGACTAC1938 ATTTAAAATGATTACAAGAAAGGTGTTCTAGCCAGAAACATGACTTGATTAGACAAGATA1998 AAAATCTTGGCGAGAATAGTGTATTCTCCTATTACCTCATGGTCTGGTATATATACAATA2058 CAACACACATACCACACACACACACACATGCAATACACACACTACACACACATACACACA2118 CTCACACACACTCATACACACACATGAAGAGATGATAAAGATGGCCCACTCAGAATTTTT2178 TTTTTATTTTTCTAAGGTCCTTATAAGCAAAACCATACTTGCATCATGTCTTCCAAAAGT2238 AACTTTAGCACTGTTGAAACTTAATGTTTATTCCTGTGCTGTGCGGTGCTGTGCTGTGCT2298 GTGCTGTGCAGCTAATCAGATTCTTGTTTTTTCCCACTTGGATTATGTTGATGCTAATAC2358 GCAGTGATTTCACATAGGATGATTTGTACTTGCTTACATTTTTACAATAAAATGATCTAC2418 ATGGA2423 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 527 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: MetSerAspSerArgAspProAlaSerAspGlnMetLysGlnTrpLys 151015 GluGlnArgAlaSerGlnArgProAspValLeuThrThrGlyGlyGly 202530 AsnProIleGlyAspLysLeuAsnIleMetThrAlaGlySerArgGly 354045 ProLeuLeuValGlnAspValValPheThrAspGluMetAlaHisPhe 505560 AspArgGluArgIleProGluArgValValHisAlaLysGlyAlaGly 65707580 AlaPheGlyTyrPheGluValThrHisAspIleThrArgTyrSerLys 859095 GlyLysValPheGluHisIleGlyLysArgThrProIleAlaValArg 100105110 PheSerThrValAlaGlyGluSerGlySerAlaAspThrValArgAsp 115120125 ProArgGlyPheAlaValLysPheTyrThrGluAspGlyAsnTrpAsp 130135140 LeuValGlyAsnAsnThrProIlePhePheIleArgAspAlaIleLeu 145150155160 PheProSerPheIleHisSerGlnLysArgAsnProGlnThrHisLeu 165170175 LysAspProAspMetValTrpAspPheTrpSerLeuArgProGluSer 180185190 LeuHisGlnValSerPheLeuPheSerAspArgGlyIleProAspGly 195200205 HisArgHisMetAsnGlyTyrGlySerHisThrPheLysLeuValAsn 210215220 AlaAspGlyGluAlaValTyrCysLysPheHisTyrLysThrAspGln 225230235240 GlyIleLysAsnLeuProValGlyGluAlaGlyArgLeuAlaGlnGlu 245250255 AspProAspTyrGlyLeuArgAspLeuPheAsnAlaIleAlaAsnGly 260265270 AsnTyrProSerTrpThrPheTyrIleGlnValMetThrPheLysGlu 275280285 AlaGluThrPheProPheAsnProPheAspLeuThrLysValTrpPro 290295300 HisLysAspTyrProLeuIleProValGlyLysValValLeuAsnLys 305310315320 AsnProValAsnTyrPheAlaGluValGluGlnMetAlaPheAspPro 325330335 SerAsnMetProProGlyIleGluProSerProAspLysLysLeuGln 340345350 GlyArgLeuPheAlaTyrProAspThrHisArgHisArgLeuGlyPro 355360365 AsnTyrLeuGlnIleProValAsnCysProTyrArgAlaArgValAla 370375380 AsnTyrGlnArgAspGlyProMetCysMetHisAspAsnGlnGlyGly 385390395400 AlaProAsnTyrTyrProAsnSerPheSerAlaProGluGlnGlnArg 405410415 SerAlaLeuGluHisSerValGlnCysAlaValAspValLysArgPhe 420425430 AsnSerAlaAsnGluAspAsnValThrGlnValArgThrPheTyrThr 435440445 LysValLeuAsnGluGluGluArgLysArgLeuCysGluAsnIleAla 450455460 GlyHisLeuLysAspAlaGlnLeuPheIleGlnLysLysAlaValLys 465470475480 AsnPheThrAspValHisProAspTyrGlyAlaArgIleGlnAlaLeu 485490495 LeuAspLysTyrAsnAlaGluLysProLysAsnAlaIleHisThrTyr 500505510 ThrGlnAlaGlySerHisMetAlaAlaLysGlyLysAlaAsnLeu 515520525 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 969 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: human manganese superoxide dismutase EMBL #X59445 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 61..729 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TGGCTTCGGCAGCGGCTTCAGCAGATCGGCGGCATCAGCGGTAGCACCAGCACTAGCAGC60 ATGTTGAGCCGGGCAGTGTGCGGCACCAGCAGGCAGCTGGCTCCGGCT108 MetLeuSerArgAlaValCysGlyThrSerArgGlnLeuAlaProAla 151015 TTGGGGTATCTGGGCTCCAGGCAGAAGCACAGCCTCCCCGACCTGCCC156 LeuGlyTyrLeuGlySerArgGlnLysHisSerLeuProAspLeuPro 202530 TACGACTACGGCGCCCTGGAACCTCACATCAACGCGCAGATCATGCAG204 TyrAspTyrGlyAlaLeuGluProHisIleAsnAlaGlnIleMetGln 354045 CTGCACCACAGCAAGCACCACGCGGCCTACGTGAACAACCTGAACGTC252 LeuHisHisSerLysHisHisAlaAlaTyrValAsnAsnLeuAsnVal 505560 AACGAGGAGAAGTACCAGGAGGCGTTGGCCAAGGGAGATGTTACAGCC300 AsnGluGluLysTyrGlnGluAlaLeuAlaLysGlyAspValThrAla 65707580 CAGATAGCTCTTCAGCCTGCACTGAAGTTCAATGGTGGTGGTCATATC348 GlnIleAlaLeuGlnProAlaLeuLysPheAsnGlyGlyGlyHisIle 859095 AATCATAGCATTTTCTGGACAAACCTCAGCCCTAACGGTGGTGGAGAA396 AsnHisSerIlePheTrpThrAsnLeuSerProAsnGlyGlyGlyGlu 100105110 CCCAAAGGGGAGTTGCTGGAAGCCATCAAACGTGACTTTGGTTCCTTT444 ProLysGlyGluLeuLeuGluAlaIleLysArgAspPheGlySerPhe 115120125 GACAAGTTTAAGGAGAAGCTGACGGCTGCATCTGTTGGTGTCCAAGGC492 AspLysPheLysGluLysLeuThrAlaAlaSerValGlyValGlnGly 130135140 TCAGGTTGGGGTTGGCTTGGTTTCAATAAGGAACGGGGACACTTACAA540 SerGlyTrpGlyTrpLeuGlyPheAsnLysGluArgGlyHisLeuGln 145150155160 ATTGCTGCTTGTCCAAATCAGGATCCACTGCAAGGAACAACAGGCCTT588 IleAlaAlaCysProAsnGlnAspProLeuGlnGlyThrThrGlyLeu 165170175 ATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTACTACCTTCAG636 IleProLeuLeuGlyIleAspValTrpGluHisAlaTyrTyrLeuGln 180185190 TATAAAAATGTCAGGCCTGATTATCTAAAAGCTATTTGGAATGTAATC684 TyrLysAsnValArgProAspTyrLeuLysAlaIleTrpAsnValIle 195200205 AACTGGGAGAATGTAACTGAAAGATACATGGCTTGCAAAAAGTAAACCACGA736 AsnTrpGluAsnValThrGluArgTyrMetAlaCysLysLys 210215220 TCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAGTGGTATAGAGTACTGC796 AGAATACAGTAAGCTGCTCTATTGTAGCATTTCTTGATGTTGCTTAGTCACTTATTTCAT856 AAACAACTTAATGTTCTGAATAATTTCTTACTAAACATTTTGTTATTGGGCAAGTGATTG916 AAAATAGTAAATGCTTTGTGTGATTGAAAAAAAAAAAAAAAAAAAAAAAAAAA969 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 222 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: MetLeuSerArgAlaValCysGlyThrSerArgGlnLeuAlaProAla 151015 LeuGlyTyrLeuGlySerArgGlnLysHisSerLeuProAspLeuPro 202530 TyrAspTyrGlyAlaLeuGluProHisIleAsnAlaGlnIleMetGln 354045 LeuHisHisSerLysHisHisAlaAlaTyrValAsnAsnLeuAsnVal 505560 AsnGluGluLysTyrGlnGluAlaLeuAlaLysGlyAspValThrAla 65707580 GlnIleAlaLeuGlnProAlaLeuLysPheAsnGlyGlyGlyHisIle 859095 AsnHisSerIlePheTrpThrAsnLeuSerProAsnGlyGlyGlyGlu 100105110 ProLysGlyGluLeuLeuGluAlaIleLysArgAspPheGlySerPhe 115120125 AspLysPheLysGluLysLeuThrAlaAlaSerValGlyValGlnGly 130135140 SerGlyTrpGlyTrpLeuGlyPheAsnLysGluArgGlyHisLeuGln 145150155160 IleAlaAlaCysProAsnGlnAspProLeuGlnGlyThrThrGlyLeu 165170175 IleProLeuLeuGlyIleAspValTrpGluHisAlaTyrTyrLeuGln 180185190 TyrLysAsnValArgProAspTyrLeuLysAlaIleTrpAsnValIle 195200205 AsnTrpGluAsnValThrGluArgTyrMetAlaCysLysLys 210215220 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 691 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: human enolase gene (EMBL #X56832) fragment containinig nucleotides -628 to +63 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 629..691 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CCTGGGGGTGGAGGTAGTAAAGGGTGAGCATGGTATTGGCTTGGAGGAAGTGGGGGACAT60 TTCTGCTTTTTTTCCTCCTGGGACTGGAGATGCTTGAAAAAGCTGGGGGAAGGGGCGGCT120 GGAGCAAGCAGATGGGACAAACTCTGGGAACACCGAAGGATCTAGGGAAAGGAGGCTGTG180 AGGAGGGCAGCAGGGATGGATAGAAAAGGGCAGCTAGAGCTGGAACCTGATAGGGAATTG240 GGGGCCCAAGGAGATTTCGGAGCAGGAAAATGAGAACCAGAAAGGATTTGAAGGCCACCA300 GCCATGGAGAACAGACTGCTTGACCAGAGGGGTGGAAGGAGAAGGCCTAAGTGGAGGCTT360 GGGGGAGGTGGGGGCTTGGTGAGCGGTGGCATCCCAGGAGCTATAGATAAGAGGCCCCTG420 GATTCTTAGGATGGGAGGGTGGAATAAGAGCTGTTCTGAGTGGGGGAGGGGGCTGCGCCT480 GCCTCTTTGGTCTGTGACCTTTTTGTAGGGTATTTTTAGCTCCAGCACCTGCCTTCTTGG540 AGTGGGGAAGAATCTTAAAGGGCAAGGGATTTCTGGTTCCTTAAGAGATCAACTGTCTAC600 ACTCACTCACACCTCCTGTCCTGCAGCCATGGCCATGCAGAAAATCTTTGCC652 MetAlaMetGlnLysIlePheAla 15 CGGGAAATCTTGGACTCCAGGGGCAACCCCACGGTGGAG691 ArgGluIleLeuAspSerArgGlyAsnProThrValGlu 101520 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: MetAlaMetGlnLysIlePheAlaArgGluIleLeuAspSerArgGly 151015 AsnProThrValGlu 20 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: PKM/ENO3 consensus sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GAGAGGCGGGCTNNCTG17 __________________________________________________________________________