Tissue specific hypoxia regulated therapeutic constructs

Methods and compositions relating to chimeric genes containing (i) a tissue-specific promoter and (ii) a hypoxia response enhancer element, both of which are operably linked to a selected gene, such as a reporter gene, therapeutic gene (e.g., bcl-2, NOS, catalase and SOD), or deleterious gene are disclosed. Expression of the selected gene is enhanced in the target tissue under hypoxic conditions, such as conditions encountered during episodes of ischemia and reperfusion. The methods and compositions may be used as therapeutics and/or diagnostics.

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 
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