Note: Descriptions are shown in the official language in which they were submitted.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
HYPOXIA-INDUCIBLE HUMAN GENES, PROTEINS, AND USES THEREOF
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention relates to hypoxia-inducible genes, and fragments
thereof, and to the use of these sequences in the diagnosis and treatment of
disease
conditions involving hypoxia, including stroke, heart attack, and cancer.
b) Description of Related Art
Hypoxia is responsible for regulating a number of cellular and systemic
processes, including angiogenesis, erythropoiesis, and glycolysis. Hypoxic
insult
and hypoxia-induced gene expression also play a role in a variety of severe
pathological conditions including ischemia, retinopathy, neonatal distress,
and
cancer.
Hypoxia-induced gene expression is associated with ischemia (and
reperfusion) in many tissues including the liver, heart, eyes, and brain. Many
of
the hypoxia-induced genes are believed to be involved in the protection or
repair
of the cells exposed to hypoxia. Enhancement of the body's protective
expression
2 0 of some stress-induced genes is therefore likely to be beneficial in many
ischemia/reperfusion-related conditions such as liver transplantation, bypass
operations, cardiac arrest, and stroke. For instance, in the brain, the
response to
brain ischemia includes the enhanced expression of growth factors and anti-
apoptosis genes (Koistinaho et al. (1997) Neuroreport 20:i-viii).
2 5 However, the ischemic induction of gene expression is not always
favorable. For example, brain ischemia can also result in the expression of
apoptosis genes or other genes which promote degeneration of the neuronal
hells.
Ischemia can also induce an extreme inflammatory reaction in the injured brain
via the upregulation of proinflammatory cytokines, chemokines, and endothelial-
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
2
leukocyte adhesion molecules (Feuerstein et al ( 1997) Ann. N. Y. Acad.Sci. 15
:179-
93). There is some evidence that this hypoxia-induced inflammatory response is
a
major cause of brain damage.
Eye diseases associated with neovascularization also involve hypoxia.
These eye diseases include diabetic retinopathy, retinopathy of prematurity,
and
sickle cell retinopathy. All can be serious enough to lead to blindness. The
feasibility of treatment of retinopathy of prematurity by antisense inhibition
of a
hypoxia-induced gene, vascular endothelial growth factor (VEGF), has been
demonstrated (Robinson, Patent No. 5,661,135).
o The process of wound healing also involves the induction of gene
expression by hypoxia (Anderson et al., Patent No. 5,681,706). TNF-a (tumor
necrosis factor-a) expression and secretion by macrophages is one response
involved in wound healing that is induced by low oxygen. Other hypoxia-induced
effects include the formation of scar tissue.
In addition to playing a major regulatory role in the body's response to
stress in postnatal life, tissue hypoxia is responsible for regulating
expression of
genes in the developing embryo, particularly with regard to angiogenesis and
vasoformation (Iyer et al. ( 1998) Genes and Development 12:149-162; Maltepe
et
al. (1997) Nature 386:403-407). Hypoxia also plays a role in neonatal stress
and
2 o pregnancy-related diseases. For instance, oxygen tension appears to
regulate
cytotrophoblast proliferation and differentiation within the uterus (Genbacev
et al.
( 1997) Science 277:1669-1672). Some disease conditions related to pregnancy,
such as preeclampsia, are associated with abnormal cytotrophoblast
differentiation
and behavior. A number of studies have shown that an increased concentration
of
2 s a hypoxia-induced gene product, insulin-like Growth Binding Protein (IGFBP-
1 ),
is associated with preeclampsia once manifest in the third trimester, even
though
US Patent No. 5,712,103 teaches that reduced levels of IGFBP-1 in maternal
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
3
blood in the first and second trimester, especially during the middle of the
second
trimester, can be used as a predictive indicator of preeclampsia.
Hypoxia has also been established to play a key role in neoplastic tissues.
The progression of human tumors to malignancy is an evolutionary process
involving the differential expression of multiple genes in response to unique
microenvironments. Low oxygen conditions create a dominant tumor
microenvironment which directly favors processes driving malignant
progression,
such as angiogenesis or elimination of p53 tumor suppressor activity.
In addition to promoting further tumor growth, the abnormally low oxygen
levels that are found in nearly all solid tumors negatively impact therapeutic
efforts. Hypoxic tumors often demonstrate resistance to radiation therapy and
chemotherapy.
The connection between tumor hypoxia and the treatment of cancer is
further exemplified by a study of cervical cancer that showed that the oxygen
level
of a tumor was an independent prognostic factor (Hoeckel et al. ( 1996) Semin.
Radiat. Oncol. 6:1-8). The prognostic value of the oxygen level of a tumor was
found to be more significant than all other indicators such as the age of the
patient,
clinical stage, or tumor size.
A number of oxygen-regulated genes have been identified in the art.
2 o Expression of many of these genes is induced by the interaction of hypoxia
inducible factor-1 (HIF-1 ), a transcription factor complex, with the factor's
DNA
recognition site on the gene, the hypoxia-responsive element (HRE). HIF-1 has
been cloned and found to not be activated by stressors such as heat shock and
ionizing radiation.
Differeritial-display polymerase chain reaction (PCR) has been used to
identify additional genes induced by hypoxia (O'Rourke et al. (1996) Eur. J.
Biochem. 241:403-410). Six hypoxia-induced genes were identified, three of
which were of known function. In addition to the known genes, two expressed
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
4
sequence tags (ESTs), and one full-length sequence were identified. The
differential-display PCR method used by O'Rourke et al. to screen for
hypoxically
induced genes was found to be limited in its ability to identify hypoxically-
induced genes.
s In addition to the identification of hypoxia-induced genes, the
identification
of the stress-responsive regulatory elements of those genes is also of
interest. The
identification of such regulatory elements may provide for an inherently tumor-
specific form of gene therapy. The HRE from a previously identified
hypoxically
induced gene, mouse phosphoglycerate kinase-l, has been used to control
1 o expression of heterologous genes both in vitro and in vivo (within a
tumor) under
hypoxic conditions (Dachs et al. (1997) Nature Medicine 3: 515-520).
Similarly,
a method for utilizing an anoxia-responsive element to effect controlled
expression of a heterologous protein has been reported (Anderson et al.,
Patent
No. 5,6$1,706).
SUIVfMARY OF THE INVENTION
The present invention relates to genes whose expression is induced under
hypoxic conditions.
One aspect of the present invention provides the isolated polynucleotide
2 o having the sequence shown as SEQ ID NO:1 (Fig. lA), comprising the cDNA of
the hypoxia-induced human gene HIGl, and encoding the polypeptide sequence of
SEQ ID N0:2 (HIG 1; Fig. 1 B). Polynucleotides with sequences complementary
to SEQ ID NO:1, fragments of SEQ ID NO:1 which are at least twelve nucleotides
in length, and sequences which hybridize to SEQ ID NO:1 are also contemplated
2 s by the present invention. In particular, one aspect of the invention
concerns the
fragment of the sequence set forth in SEQ ID NO:1 comprising nucleotides 62-
343, the nucleotides representing the coding sequence of human HIGI. The
complements to the coding sequence, at least twelve nucleotide-long fragments
of
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
the coding sequence, and sequences which hybridize to the coding sequence of
HIGl are also provided by the invention.
Another aspect of the present invention provides the isolated
polynucleotide having the sequence shown as SEQ ID N0:3 (Fig. 2A), comprising
5 the cDNA of the hypoxia induced gene HIG2, and encoding the polypeptide
sequence of SEQ ID N0:4 (HIG2; Fig. 2B). The complements to SEQ ID N0:3,
as well as at least twelve nucleotide-long fragments thereof and sequences
which
hybridize thereto are also provided. The invention refers in particular to a
polynucleotide having a sequence corresponding to nucleotides 274-465 of the
1 o sequence set forth in SEQ ID N0:3, or complements thereof, or at least
twelve
nucleotide-long fragments thereof, or sequences which hybridize thereto.
Nucleotides 274-465 represent the coding sequence of human HIG2.
The present invention also encompasses expression vectors and delivery
vehicles which contain polynucleotides of the present invention and host cells
that
are genetically engineered with polynucleotides of the present invention.
In another embodiment, the invention provides for an oligonucleotide probe
comprising fragments, preferably at least about 15 nucleotides long, of the
polynucleotides of SEQ ID NO: l or SEQ ID N0:3, or the complement thereto.
Polypeptides of the sequences set forth in SEQ ID N0:2 (HIG 1 ) and SEQ
2 o ID N0:4 (HIG2), or biochemically equivalent fragments of the polypeptides
of
either sequence, are further contemplated by the present invention.
Antibodies that are specifically immunoreactive to the hypoxia-induced
polypeptides HIG 1 or HIG2 of the present invention are also provided.
In still another embodiment, the present invention provides for arrays of
2 5 polynucleotides or polypeptides corresponding to at least two different
hypoxia-
inducible genes, hypoxia-induced polypeptides, or antibodies immunoreactive
with hypoxia-induced polypeptides.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
6
Hypoxia-iriducible genes suitable for use in the arrays, diagnostic methods,
and treatment methods of the invention described herein are not limited to
HIGI
and HIG2, or derivatives thereof, but also include a number of known genes now
determined to be hypoxia-inducible. Additional hypoxia-induced genes useful in
the methods and arrays of the present invention include, but are not limited
to, the
genes of annexin V, lipocortin 2, hnRNP A1, Ku autoantigen,
phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,
fibroblast growth factor-3, EPH receptor ligand, plasminogen activator
inhibitor-
I, macrophage migration inhibitory factor, fibronectin receptor, lysyl
1 o hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent
and
tunicamycin-responsive protein, CDGlike kinase-1, quiescin, growth arrest DNA
damage-inducible protein 45, DECI, low density lipoprotein receptor related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-I, fructose
bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3LlNix, and Pim-1.
In one aspect, the present invention provides diagnostic and prognostic
tools for assaying for the expression of hypoxia-inducible genes in a tissue
of an
animal, for determining the presence of hypoxia in a tissue in an animal, and
for
evaluating a hypoxia-related condition in an animal particularly in order to
tailor
2 o therapy to a known hypoxic state. The detection of expression products,
such as
mRNA transcripts or proteins, of the hypoxia-inducible genes of HIGI, HIG2,
annexin V, lipocortin 2, hnRNP Al, Ku autoantigen,
phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,
fibroblast growth factor-3, EPH receptor ligand, plasminogen activator
inhibitor-
l, macrophage migration inhibitory factor, fibronectin receptor, lysyl
hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and
tunicamycin-responsive protein, CDGlike kinase-l, quiescin, growth arrest DNA
damage-inducible protein 45, DECI, low density lipoprotein receptor related
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
7
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose
bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3LlNix, or Pim-1, or combinations
.
thereof, to determine the presence of hypoxia in a tissue or evaluate a
hypoxia-
related condition in an animal is encompassed by the present invention.
Methods
of diagnosing and treating hypoxia-related conditions via such methods are
also
encompassed by the present invention.
Other methods of assaying for expression of hypoxia-inducible genes,
determining the presence of hypoxia in a tissue in an animal, or evaluating a
1 o hypoxia-related condition in an animal involves the use of the arrays of
the
invention. First, a polynucleotide array or antibody array of the invention
may be
contacted with polynucleotides or polypeptides, respectively, either from or
derived from a sample of body fluid or tissue obtained from the animal. Next,
the
amount and position of polynucleotide or polypeptide from the animal's sample
~ 5 which binds to the sites of the array is determined. Optionally, the gene
expression pattern observed may be correlated with an appropriate treatment.
Other aspects of the invention concern treating a tissue which is a tumor by
first determining the presence of hypoxia in the tumor and, second, treating
the
tumor with an established form of therapy for cancers such as radiation
therapy,
2 o chemotherapy, and surgery.
In other aspects, the invention provides for methods of attenuating the
hypoxic response of a tissue by blocking expression of a hypoxia-inducible
gene
HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen,
phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,
2 5 fibroblast growth factor-3, EPH receptor ligand, plasminogen activator
inhibitor-
I, macrophage migration inhibitory factor, fibronectin receptor, lysyl
hydroxylase-2, endothelia-2, B-cell translocation gene-l, reducing agent and
tunicamycin-responsive protein, CDC-like kinase-l, quiescin, growth arrest DNA
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
8
damage-inducible protein 45, DECI, low density lipoprotein receptor related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-l, fructose
bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3LlNix, or Pim-1 in the cell or by
neutralizing the polypeptide expression products of these genes in the tissue.
The
invention also provides for methods of treating hypoxia-related conditions by
attenuating the hypoxic response of a tissue in an animal such as a human.
Methods for enhancing the response of tissue to hypoxia are provided in
other embodiments of the present invention. These methods involve
1 o administering expression vectors comprising the hypoxia-inducible genes of
the
present invention or administering polypeptide expression products of hypoxia-
inducible genes to the tissue.
Methods for identifying stress-inducible and stress repressible genes are
also provided.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the human HIG I cDNA and protein sequences. The nucleotide
sequence for the human HIGI gene is shown in Figure IA from 5' to 3'(SEQ ID
2 o NO:1 ). The coding sequence is underlined. The other regions are
untranslated
regions (5' and 3' UTR) of the gene. The protein sequence of human HIG I is
shown in Figure 1B (SEQ ID N0:2).
Figure 2 shows the human HIG2 cDNA and protein sequences. The nucleotide
sequence for the human HIG2 gene is shown in Figure 2A from 5' to 3' (SEQ ID
N0:3). The coding sequence is underlined. The other regions are untranslated
regions (5' and 3' UTR) of the gene. The protein sequence of human HIG2 is
shown in Figure 2B (SEQ ID N0:4).
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
9
Figure 3 shows the marine HIG 1 cDNA and protein sequences. The nucleotide
sequence for the marine HIGl gene is shown in Figure 3A from 5' to 3' (SEQ ID
NO:S). The coding sequence is underlined. The other regions are untranslated
regions (5' and 3' UTR) of the gene. The protein sequence of marine HIG 1 is
shown in Figure 3B (SEQ ID N0:6).
Figure 4 shows the HIG1 cDNA and protein sequences of seriola quinqueradiata.
The nucleotide sequence for this fish HIGI is shown in Figure 4A from 5' to 3'
l o (SEQ ID N0:7). The coding sequence is underlined. The other regions are
untranslated regions (5' and 3' UTR) of the gene. The protein sequence of fish
HIG 1 is shown in Figure 4B (SEQ ID N0:8).
Figure S shows the marine HIG2 cDNA and protein sequences. The nucleotide
sequence for the marine HIG2 gene is shown in Figure SA from 5' to 3' (SEQ ID
N0:9). The coding sequence is underlined. The other regions are untranslated
regions of the gene (5' and 3' UTR). The protein sequence of marine HIG2 is
shown in Figure SB (SEQ ID NO:10).
2 o Figure 6 shows the alignment of human HIG 1 and HIG2 protein sequences
with
the HIG1 and HIG2 sequences of other species. The HIG1 homologues from
humans (hHIG 1 ), mice (mHIG 1 ), and fish (seriola quinqueradiate) (fHIG 1 or
GHL 1 ) are aligned in Figure 6A; the HIG2 homologues from humans (hHIG2)
and mice (mHIG2) are aligned in figure 6B.
Figure 7 schematically illustrates the addition of linkers to cDNA library
fragments. The linker addition is followed by PCR amplification.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
Figure 8 illustrates how the subtraction protocol is used to enrich the tester
cDNA
library with sequences unique to the tester cDNAs.
DETAILED DESCRIPTION OF THE INVENTION
a) Definitions and General Parameters
The following definitions are set forth to illustrate and define the meaning
and scope of the various terms used to describe the invention herein.
1 o By the term "hypoxia" (or "hypoxic") is meant, for the purposes of the
specification and claims, an environment of reduced oxygen tension such that
the
oxygen content is less than or equal to about S%. In most cases, hypoxic
tissue
will have an oxygen content that is less than or equal to about 2%.
"Normoxic" or "oxic" conditions are conditions comprising a normal level
is of oxygen for that particular environment. Normoxic or oxic tissue
typically has
an oxygen content above about 5%.
The terms "hypoxia-induced" or "hypoxia-inducible" when referring to a
gene means that the gene is expressed at a higher level when the host cell is
exposed to hypoxic conditions than when exposed to normoxic conditions.
2 o Typically, the number of mRNA transcripts of a hypoxia-induced gene would
is at
least about 20% higher in a hypoxic cell versus a normoxic cell. Preferably,
expression of the hypoxia-induced gene is at least about 2-fold higher in
hypoxic
versus normoxic cells. Most preferably, expression of the hypoxia-inducible
gene
is at least about 5-fold higher in hypoxic cells versus normoxic cells.
2 s A "hypoxia-related condition" in an animal is a condition where hypoxia or
altered (typically, enhanced) levels of expression of hypoxia-inducible genes
in a
tissue of the animal is involved. The hypoxia or altered expression of hypoxia-
inducible genes may either be a symptom or play a role in the cause,
development,
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
11
progression, amelioration, or cure of the condition. A hypoxia-related
condition
may optionally be a disease or pathological condition. Hypoxia-related
conditions include, but are not limited to, cancer, ischemia, reperfusion,
retinopathy, neonatal distress, preeclampsia, cardiac arrest, stroke, and
wound
healing.
The term "hypoxia-induced protein" or "hypoxia-induced gene product"
means a protein encoded by a gene whose expression is induced by hypoxia.
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
1 o example, naturally-occurring polynucleotides or polypeptides present in a
living
animal are not isolated, but the same polynucleotides or polypeptides could be
part
of a vector or composition, and be isolated in that such vector or composition
is
not part of its natural environment.
A "sample obtained from a patient" or a "sample obtained from an animal"
is may be a sample of tissue or a sample of body fluid. The term "tissue" is
used
herein to refer to any biological matter made up of one cell, multiple cells,
an
agglomeration of cells, or an entire organ. The term tissue, as used herein,
encompasses a cell or cells which can be either normal or abnormal (i.e. a
tumor).
A "body fluid" may be any liquid substance extracted, excreted, or secreted
from
2 o an organism or a tissue of an organism. The body fluid need not
necessarily
contain cells. Body fluids of relevance to the present invention include, but
are
not limited to, whole blood, serum, plasma, urine, cerebral spinal fluid,
tears, and
amniotic fluid.
The term "biochemically equivalent variations" means protein or nucleic
2 s acid sequences which differ in some respect from the specific sequences
disclosed
herein, but nonetheless exhibit the same, or substantially the same,
functionality.
In the case of cDNA, for example, this means that modified sequences which
contain other nucleic acids than those specifically disclosed are encompassed,
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
12
provided that the alternate cDNA encodes mRNA which in turn encodes a protein
of this invention. Such modifications may involve the substitution of only a
few
bases, or many. The modifications may involve substitution of degenerate
coding
sequences or replacement of one coding sequence with another; introduction of
non-natural nucleic acids is contemplated. It is not necessary for the
alternate
DNA to hybridize with that disclosed herein provided that the functional
criterion
is met. Preferably, the modified nucleic acid sequence hybridizes to and is at
least
95% complementary to the sequence of interest.
Similarly, in the case of the proteins of this invention, alterations in the
1 o amino acid sequence which do not affect functionality may be made. Such
variations may involve replacement of one amino acid with another, use of side
chain modified or non-natural amino acids, and truncation. The skilled artisan
will recognize which sites are most amenable to alteration without affecting
the
basic function.
A "polynucleotide", "oligonucleotide", or "nucleic acid" includes, but is
not limited to, mRNA, cDNA, genomic DNA, and synthetic DNA and RNA
sequences, comprising the natural nucleoside bases adenine, guanine, cytosine,
thymine, and uracil. The term also encompasses sequences having one or more
modified nucleosides. The terms "polynucleotide" and "oligonucleotide" are
used
2 o interchangeably herein. No limitation as to length or to synthetic origin
are
suggested by the use of either of these terms herein.
The term "polypeptide" means a poly(amino acid) comprising at least two
amino acids linked by peptide bonds. A "protein" is a polypeptide which is
encoded by a gene.
2 5 "Neutralizing" a polypeptide or protein means inhibiting, partially or
wholly, the bioactivity of the polypeptide or protein. This inhibition of
activity
may mean inhibition of catalytic activity, prevention of binding to a receptor
or
ligand, blockage or dimer formation, or the like.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
13
The term "sequences which hybridize thereto" means polynucleotide
sequences which are capable of forming Watson-Crick hydrogen bonds with
another polynucleotide sequence under normal hybridization conditions, such as
in
buffered (pH. 7.0-7.5) aqueous, saline solutions (for instance, 1 to 500 mM
NaCI)
at room temperature. Although normal hybridization conditions will depend on
the length of the polynucleotides involved, typically they include the
presence of
at least one cation such as Na+, K+, Mg2+, or Ca2+, a near neutral pH, and
temperatures less than 55°C. Although the sequences which hybridize to
a
polynucleotide may be about 90%-100% complementary to the polynucleotide, if
1 o the sequences are of sufficient length, in solutions with high salt
concentrations,
and/or under low temperature conditions, polynucleotides with complementarity
of 70% or above, or even just 50% or above, may hybridize to the
polynucleotide.
Sequences which hybridize thereto typically comprise at least 12 nucleotides,
and
preferably at least about 15 nucleotides, which are complementary to the
target
polynucleotide.
A "coding sequence" is a polynucleotide or nucleic acid sequence which is
transcribed and translated (in the case of DNA) or translated (in the case of
mRNA) into a polypeptide in vitro or in vivo when placed under the control of
appropriate regulatory sequences. The boundaries of the coding sequence are
2 o determined by a translation start codon at the 5' (amino) terminus and a
translation
stop codon at the 3' (carboxy) terminus. A transcription termination sequence
will
usually be located 3' to the coding sequence.
Nucleic acid "control sequences" refer to translational start and stop
codons, promoter sequences, ribosome binding sites, polyadenylation signals,
2 5 transcription termination sequences, upstream regulatory domains,
enhancers, and
the Like, as necessary and sufficient for the transcription and translation of
a given
coding sequence in a defined host cell. Examples of control sequences suitable
for
eucaryotic cells are promoters, polyadenylation signals, and enhancers. All of
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
14
these control sequences need not be present in a recombinant vector so long as
those necessary and sufficient for the transcription and translation of the
desired
gene are present.
"Operably or operatively linked" refers to the configuration of the coding
s and control sequences so as to perform the desired function. Thus, control
sequences operably linked to a coding sequence are capable of effecting the
expression of the coding sequence. A coding sequence is operably linked to or
under the control of transcriptional regulatory regions in a cell when RNA
polymerase will bind the promoter sequence and transcribe the coding sequence
1 o into mRNA that can be translated into the encoded protein. The control
sequences
need not be contiguous with the coding sequence, so long as they function to
direct the expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence and the
coding
sequence and the promoter sequence can still be considered "operably linked"
to
1 s the coding sequence.
The expression products described herein may consist of proteinaceous
material having a defined chemical structure. However, the precise structure
depends on a number of factors, particularly chemical modifications common to
proteins. For example, since all proteins contain ionizable amino and carboxyl
2 o groups, the protein may be obtained in acidic or basic salt form, or in
neutral form.
The primary amino acid sequence may be derivatized using sugar molecules
(glycosylation} or by other chemical derivatizations involving covalent or
ionic
attachment with, for example, lipids, phosphate, acetyl groups and the like,
often
occurring through association with saccharides. These modifications may occur
in
2 5 vitro, or in vivo, the latter being performed by a host cell through
posttranslational
processing systems. Such modifications may increase or decrease the biological
activity of the molecule, and such chemically modified molecules are also
intended to come within the scope of the invention.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
I5
"Vector" means a polynucleotide comprised of single strand, double strand,
or circular DNA or RNA. An "expression vector" is comprised of the following
elements operatively linked at appropriate distances for allowing functional
gene
expression: replication origin, promoter, enhancer, 5' mRNA leader sequence,
ribosomal binding site, nucleic acid cassette, termination and polyadenylation
sites, and selectable marker sequences. One or more of these elements may be
omitted in specific applications. The nucleic acid cassette can include a
restriction
site for insertion of the nucleic acid sequence to be expressed. In a
functional
vector the nucleic acid cassette contains the nucleic acid sequence to be
expressed
1 o including translation initiation and termination sites. An expression
vector is
constructed so that the particular coding sequence is located in the vector
with the
appropriate regulatory sequences, the positioning and orientation of the
coding
sequence with respect to the control sequences being such that the coding
sequence is transcribed under the "control" of the control sequences.
Modification
i 5 of the sequences encoding the particular protein of interest may be
desirable to
achieve this end. For example, in some cases it may be necessary to modify the
sequence so that it may be attached to the control sequences with the
appropriate
orientation; or to maintain the reading frame. The control sequences and other
regulatory sequences may be ligated to the coding sequence prior to insertion
into
2 o a vector. Alternatively, the coding sequence can be cloned directly into
an
expression vector which already contains the control sequences and an
appropriate
restriction site which is in reading frame with and under regulatory control
of the
control sequences.
A "regulatory element" is a segment of DNA to which a transcription
2 5 factors) binds and alters the activity of a gene's promoter either
positively
(induction) or negatively (repression).
A "stress-responsive element" or "stress-responsive regulatory element" is
a regulatory element which binds transcription factors activated by the cell
in
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
16
response to environmental stress. Environmental stressors may include one or
more of the following: oxygen depletion; radiation; heat shock; pH change;
hypothermia; or glucose starvation.
A "delivery vehicle", as used herein, refers to a means of delivering a
polypeptide or a polynucleotide to a cell. The delivery vehicle is preferably
used
to deliver an expression vector to a cell or a cell in an organism. A delivery
vehicle may be a virus, such as a retrovirus, an adenovirus, an adeno-
associated
virus, a herpes simplex virus, or a vaccinia virus.
Other possible delivery vehicles are non-viral. For instance, one of the
1 o many liposome formulations known to those skilled in the art, such as
Lipofectin,
may serve as a delivery vehicle. Liposomes are hollow spherical vesicles
composed of lipids arranged in a similar fashion as those lipids which make up
the
cell membrane. They have internal aqueous space useful for entrapping water
soluble compounds such as polynucleotides. Recognition molecules can be
attached to their surface for the targeting of the delivery vehicles to
specific
tissues.
As used herein, an "antibody" refers to a protein consisting of one or more
polypeptides substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes. Antibodies may exist as intact immunoglobulins or as a
2 o number of fragments, including those well-characterized fragments produced
by
digestion with various peptidases. While various antibody fragments are
defined
in terms of the digestion of an intact antibody, one of skill will appreciate
that
antibody fragments may be synthesized de novo either chemically or by
utilizing
recombinant DNA methodology. Thus, the term antibody, as used herein also
includes antibody fragments either produced by the modification of whole
antibodies or synthesized de novo using recombinant DNA methodologies.
Antibody fragments encompassed by the use of the term "antibodies" include,
but
are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd
fragments.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
17
The phrase "specifically binds to a polypeptide" or "specifically
immunoreactive with", when referring to an antibody refers to a binding
reaction
which is determinative of the presence of the polypeptide (or protein) in the
presence of a heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies bind to a
particular protein and do not bind in a significant amount to other proteins
present
in the sample. Specific binding to a protein under such conditions may require
an
antibody that is selected for its specificity for a particular protein or
polypeptide.
A variety of immunoassay formats may be used to select anitbodies specifically
1 o immunoreactive with a particular protein. For example, solid-phase ELISA
immunoassays are rountinely used to select monoclonal antibodies specifically
immunoreactive with a protein.
b) Hypoxia-Inducible Genes and Expression Products
We have discovered a novel human gene, herein referred to as HIGI,
whose expression is induced by cellular response to hypoxia (see the specific
examples, Examples 1-6 below). We have isolated a cDNA of the human HIGI
gene (SEQ ID NO:1; Fig. lA) and identified the coding sequence to be
nucleotides 62-343 of SEQ ID NO:1. The protein encoded by HIGl comprises the
2o amino acid sequence shown in Figure 1B (SEQ ID N0:2). Polynucleotides with
sequences complementary to SEQ ID NO:1, polynucleotides that are fragments of
SEQ ID NO:1 of at least twelve nucleotides in length and polynucleotides which
hybridize to SEQ ID NO:1 are also within the scope of the present invention.
The
fragments of SEQ ID NO:1 are preferably at least 1 S nucleotides long.
2 5 In particular, polynucleotides comprising the nucleotides 62-343 of SEQ ID
NO:1, or complements thereto, or at least twelve nucleotide long fragments
thereof, or sequences which hybridize thereto are preferred. Fragments of the
coding sequence of HIGI are preferably at least fifteen nucleotides in length.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
18
We have also discovered a second, novel human gene, herein referred to as
HIG2, whose expression is induced by cellular response to hypoxia. We have
isolated a cDNA clone of this gene. The cDNA sequence of the HIG2 gene is
shown in Fig. 2A (SEQ ID N0:3). The coding sequence of HIG2 comprises
nucleotides 274-465 of SEQ ID N0:3. Fragments of the HIG2 sequence, and of
the HIG2 coding sequence in particular, of at least twelve, and preferably
fifteen,
nucleotides in length are provided by the present invention as well.
Polynucleotides of sequence which is complementary to SEQ ID N0:3 (especially
to nucleotides 274-465) or polynucleotides which hybridize to polynucleotides
of
1 o the sequence set forth in SEQ ID N0:3 (especially to nucleotides 274-465),
are
also contemplated.
Polypeptides encoded by the polynucleotides of HIGI (SEQ ID N0:2; Fig.
1B) and HIG2 (SEQ ID N0:4; Fig. 2B), or biochemically equivalent variations of
either protein, are also provided by the present invention. Fragments of these
polypeptides which consist of at least eight amino acids are provided as well.
Preferably, the fragments are at least 15 amino acids in length.
All biochemically equivalent variations of the aforementioned
polynucleotides and polypeptides are considered to be fully within the scope
of
this invention. The mouse and fish HIG1 polynucleotide and polypeptide
2 o sequences (Figs. 3, 4, and 6) can be considered biochemically equivalent
variations of the human HIG1. The mouse HIG2 polynucleotide and polypeptide
sequences (Figs. 5 and 6) are likewise understood to be biochemically
equivalent
variations of the human HIG 1.
The polynucleotides of this invention may readily be incorporated within
2 5 expression vectors by one of ordinary skill in the art. In a preferred
embodiment,
the polynucleotide sequence comprising nucleotides 62-343 of SEQ ID NO:1 (the
coding sequence of HIG 1 ) or nucleotides 274-465 of SEQ ID N0:2 (the coding
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
19
sequence of HIG2) is operably linked with appropriate control sequences, such
as
a promoter.
Alternatively, larger fragments of the polynucleotides of SEQ ID NO: l or
SEQ ID N0:2 which comprise portions of the untranslated regions of the genes
may be used in an expression vector instead. This may be particularly useful
when hypoxia-induciblity is desired, since the untranslated regions may
contain
critical regulatory regions such as hypoxia-responsive elements.
The polynucleotides of this invention may also be incorporated within a
host cell. In one embodiment, transfection may be used to introduce an
expression
1 o vector containing one of the polynucleotides of the invention into the
cell. The
polynucleotide of the transfected vector may also be operably linked with
control
sequences including regulatory elements to effect the expression within the
cell of
exogenous protein or polypeptide sequences encoded by the polynucleotides of
the
present invention. Methods of cloning, amplification, expression, and
purification
1 s will be apparent to the skilled artisan. Representative methods are
disclosed in
Molecular Cloning: a Laboratory Manual, 2nd Ed., Yol. I-3, eds. Sairibrook et
al.,
Cold Spring Harbor Laboratory ( 1989).
A HIGI or HIG2 polynucleotide may be introduced into an animal either
by first incorporating the vector into a cell and then transferring the cell
to the
2 o animal (ex vivo) or by incorporating the vector into a cell within an
animal directly
(in vivo).
The introduction of a HIGI or HIG2 polynucleotide into a cell may be
achieved by directly injecting the nucleic acid into the cell or by first
mixing the
nucleic acid with polylysine or cationic lipids which will help facilitate
passage
2 s across the cell membrane. However, introduction of the polynucleotide into
the
cell is preferably achieved through the use of a delivery vehicle such as a
liposome
or a virus. Viruses which may be used to introduce a HIGI or HIG2
polynucleotide or expression vector into a cell include, but are not limited
to,
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses,
and
vaccinia viruses.
Antisense oligonucleotides complementary to HIGI and HIG2, particularly
those which are capable of blocking expression of HIGI or HIG2 are provided by
s the present invention. The antisense oligonucleotide is preferably an
oligonucleotide having a sequence complementary to at least a portion
(preferably
at least about 12 nucleotides in length) of SEQ ID NO:1 or SEQ ID N0:3. The
antisense oligonucleotide is preferably between about 15 and about 22
nucleotides
in length. Modifications of the sequence or bases of the antisense
oligonucleotide
1 o may be desirable to facilitate transfer into a cell, stability, or tight
binding to the
HIGI or HIG2 mRNA.
An oligonucleotide probe is provided by another embodiment of the
invention. The probe consists of one of the polynucleotides of this invention,
or
an at least 12 nucleotide-long fragment thereof. The probe may be used to
assay
15 for, and if the probe is properly labeled, quantitate, the hypoxia-induced
expression of HIGI or HIG2 in a cell. In a preferred embodiment, the probe is
at
least about 15 nucleotides in length. In a particularly preferred embodiment,
the
probe is between 15 and 22 nucleotides in length.
Antibodies specifically immunoreactive with the HIG 1 or HIG2
2 o polypeptides represent still another embodiment of the invention. These
antibodies may be monoclonal or polyclonal. The antibodies may optionally be
recombinant or purely synthetic. The antibody may be an intact antibody or
fragment. The preparation of antibodies specific to the HIG 1 and HIG2
polypeptides would be routine for those skilled in the art.
2 s In addition to the identification of the new genes HIG 1 and HIG2 which
were found to be hypoxia-inducible, we have also established for the first
time that
several previously known genes are hypoxia-inducible in humans (see the
specific
examples, Examples 2 and 9, below). These genes include annexin V, lipocortin
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
21
2, hnRNP AI, Ku autoantigen, phosphoribosylpyrophosphate synthetase,
acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH
receptor
ligand, plasminogen activator inhibitor-l, macrophage migration inhibitory
factor, frbronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell
translocation gene-l, reducing agent and tunicamycin-responsive protein, CDG
like kinase-I, quiescin, growth arrest DNA damage-inducible protein 45, DECI,
low density lipoprotein receptor related protein, hamster hairy gene
homologue,
adipophilin, cyclooxygenase-I, fructose bisphosphatase, creative transporter,
fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer,
Nip3LlNix, and Pim-1. Furthermore, expression of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), expression previously known to be hypoxia-inducible
only in endothelial cells (Graven et al. (1998) Am. J. Physiol., 274{2 Pt
1):C347-
355), is now shown by our work to be greatly induced in transformed cells.
Additionally, a multitude of EST sequences from the databases have now been
identified as being hypoxia-inducible (Table 3, Example 8 and Table 5, Example
9).
c) Polynucleotide, Polypeptide, and Antibody Arrays.
Another aspect of the invention involves the presentation of multiple (at
2 0 least two, and preferably more than four) hypoxia-inducible gene
sequences,
polynucleotide probes complementary to the hypoxia-inducible gene sequences,
hypoxia-induced polypeptides, or antibodies (immunoreactive with hypoxia-
induced polypeptides) on an array. In particularly preferred arrays, more than
about 10 different polynucleotides, polypeptides, or antibodies are presented
on
2 5 the array. In an alternative preferred embodiment, the number of different
polynucleotides, proteins, or antibodies on the array is greater than about
25, or
even greater than about 100.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
22
One aspect of the invention provides an array of polynucleotides which
comprises at least two different hypoxia-inducible genes, or complements
thereto,
or at least twelve nucleotide-long fragments thereof, or sequences which
hybridize
thereto. The hypoxia-inducible genes or their fragments may optionally be
selected from HIG 1, HIG2, any of the hypoxia-inducible genes listed in Table
1
(below}, Table 3 (Example 8, below), and Table 5 (Example 9, below). However,
it is understood that all of the hypoxia-inducible gene sequences on the array
need
not be derived only from those hypoxia-inducible listed herein. The
polynucleotides on the array are typically single-stranded.
1 o For instance, in one embodiment of the polynucleotide array, on of the
multiple polynucleotides on the array is derived from either the HIG I or HIG2
gene sequences. The polynucleotides of the array may comprise the entire
sequence of one strand of the gene, or may comprise at least 12 nucleotide
long
fragments thereof, or sequences which hybridized thereto. In an alternative
~ 5 embodiment, one of the polynucleotides of the array comprises a
polynucleotide
corresponding to nucleotides 62-343 of SEQ ID NO: I (HIGl ) or nucleotides 274-
465 of SEQ ID N0:2 (HIG2), or complements. to one of the coding sequences, or
at least twelve nucleotide-long fragments of one of the coding sequences, or
sequences which hybridize to one of the coding sequences. In another
2 o embodiment of the polynucleotide array, at least one of the polynucleotide
sequences of HIGI, HIG2, annexin V, lipocortin 2, hn.RNP Al, Ku autoantigen,
phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,
fibroblast growth factor-3, EPH receptor ligand, plasminogen activator
inhibitor-
l, macrophage migration inhibitory factor, ftbronectin receptor, lysyl
25 hydroxylase-2, endothelin-2, B-cell translocation gene-l, reducing agent
and
tunicamycin-responsive protein, CDGlike kinase-1, quiescin, growth arrest DNA
damage-inducible protein 45, DECI, low density lipoprotein receptor related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-I, fructose
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
23
bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl 2-interacting killer, Nip3LlNix, and Pim-I is represented
on
the array in combination with a second, different polynucleotide sequence from
a
hypoxia-inducible gene. The second polynucleotide sequence may be selected
from HIGI, HIG2, any of the hypoxia-inducible genes represented in Table 1,
shown below, any of the expressed sequence tags of hypoxia-inducible genes
shown in Table 3 (see Example 8), or any other hypoxia-inducible gene or
expressed sequence tag from a hypoxia-inducible gene. It is understood that
regardless of which genes are represented on the array, the gene sequences do
not
have to be represented in their entirety.
The polynucleotide sequences that are immobilized on the array are most
preferably, single-stranded and complementary to the mRNA transcripts of the
relevant hypoxia-inducible genes. The immobilized polynucleotides may be
fragments or complementary sequences of the gene or EST sequence that contain
at least twelve nucleotides and preferably at least fifteen nucleotides.
Alternatively, longer gene fragments including EST fragments of at least 50 or
at
least 100 nucleotides may be used. In a preferred embodiment of the array, the
array is made up of many different gene sequences.
In another embodiment of the polynucleotide array, only polynucleotides
2 o correlating to hypoxia-inducible genes expressing gene products of a
similar
function are included on the array. At least two, but preferentially more than
two,
different hypoxia-induced genes encoding proteins from a single functional
category are represented on the array. Examples of seven functional categories
of
hypoxia-inducible proteins are as follows: ( 1 ) glycolytic enzymes/proteins;
(2)
angiogenesis/tissue remodeling proteins; (3) erythropoiesis/vascular
regulatory
proteins; (4) metabolic/homeostatic proteins; (5) apoptosis proteins; (6) DNA
repair proteins; and (7) cell-cycle proteins. These categories are shown in
Table 1,
below, along with some representative members of each of the categories. It is
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
24
understood that the members of each of the seven functional categories of
hypoxia-inducible proteins are not limited to the lists shown in Table 1. It
is
further understood, that the list of functional categories of hypoxia-
inducible
genes is not.limited to the seven categories listed in Table 1. Again, a
preferred
s embodiment of this array comprises polynucleotide sequences complementary to
the mRNA transcripts of the relevant hypoxia inducible genes. A particularly
preferred embodiment of an array displays multiple polynucleotide sequences,
each of which is complementary to a different gene which encodes a protein
involved in angiogenesis andlor tissue remodeling.
to
Table 1. Seven Functional Categories of Hypoxia-Inducible Genes
GLYCOLYTIC ENZYMES/PROTEINS
lactate dehydrogenase (LDH)
phosphoglycerate kinase (PGK)
aldolase A
L-phosphofructokinase (PFKL)
glucose transporter isoform 3 (Glut-3)
interleukin-2
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
adenylate kinase isoenzyme 3 (AK-3)
ANGIOGENESIS/TISSUE REMODELING PROTEINS
vascular endothelial growth factor (VEGF)
platelet-derived growth factor ~i (PDGF~i)
transforming growth factor ~i (TGF~3)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
tumor necrosis factor a (TNFa)
interleukin-6 (IL-6)
interleukin-2 (IL-2)
tissue factor
fibroblast growth factor (FGF-3)
EPH receptor ligand
plasminogen activator inhibitor-1 (PAI-1)
macrophage migration inhibitory factor (MIF)
fibronectin receptor
lysyl hydroxylase-2
endothelia-2
ERYTHROPOIEISIS/VASCULAR REGULATORY PROTEINS
erythropoietin (EPO)
tyrosine hydroxylase
heme oxygenase
alpha-fetoprotein (AFP)
endothelia
METABOLICIHOMEOSTATIC PROTEINS
insulin-like growth factor binding protein-1 (IGFBP-1 }
metallothionein
creatine kinase
inducible nitric oxide synthase (i-NOS-1)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
26
epidermal growth factor receptor (EGFR)
huntingtin-associated protein 1 (HAP-1)
glucose-regulated protein 78 (GRP78)
glucose-regulated protein 90 (GRP90)
thioredoxin
annexin V
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
heterogeneous nuclear ribonucleoprotein A 1 (hnRNP A 1 )
gamma-glutamyl cysteine synthetase heavy subunit
phosphoribosylpyrophosphate synthetase (PRPP synthetase)
acetoacetylCoA thiolase
fructose bisphosphatase
creatine transporter
fatty acid binding protein
glucose transporter isoform 3 (Glut-3)
adenylate kinase isoenzyme 3 (AK-3)
lactate dehyrogenase (LDH)
APOPTOSIS PROTEINS
insulin-like growth factor binding protein-3 (IGFBP-3)
c-myc
c-jun
Bcl-2-interacting killer (BIK)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
27
19 kDa-interacting protein 3 long/Nip3-like protein X (NipP3L/Nix)
Pim-1
DNA-REPAIR PROTEINS
Ku (70)
CELL-CYCLE PROTEINS
B-cell translocation gene-1 (BTG-1)
reducing agent and tunicamycin responsive protein (RTP)
CDC-like kinase-1 (clk-1 )
quiescin (Q6)
growth arrest DNA damage-inducible protein 45 (GADD45)
In an alternative emnoniment of the polynucleotide array, polynucleotides
correlating to the gene sequences encoding proteins belonging to at least two
different functional categories of hypoxia-inducible genes are displayed on a
single array. Although at least two different polynucleotide sequences are
s required to form the array, in a preferred embodiment many more than two are
used. Again, a preferred embodiment of this array comprises polynucleotide
sequences complementary to the mRNA transcripts of the relevant hypoxia
inducible genes of at least 12 nucleotides in length, and preferably fifteen.
The present invention also provides for polypeptide arrays analogous to the
polynucleotide arrays discussed above, except that the polypeptide sequences
of
the hypoxia-inducible genes, or fragments thereof, are displayed in an array.
The
polypeptide array comprises the polypeptide expression products of at least
two
hypoxia-inducible genes, or biochemically equivalent fragments thereof. For
instance, the polypeptide array my comprise the protein HIG 1 or HIG2 and at
least
1 s one other protein which is a hypoxia induced gene product. Alternatively,
the
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
28
polypeptide array may instead comprise at least one protein selected from the
group consisting ofHIGI, HIG2, annexin V, lipocortin 2, hnRNP A1, Ku
autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase,
ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen
activator inhibitor-I, macrophage migration inhibitory factor, fibronectin
receptor,
lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-l, reducing agent
and
tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA
damage-inducible protein 45, DECI, low density lipoprotein receptor related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose
1 o bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-l, or a
biochemically
equivalent fragment thereof; and at least one of a second polypeptide which is
a
second hypoxia-induced gene product, or a biochemically equivalent fragment
thereof.
Another aspect of the invention concerns a polypeptide array comprising at
least two different hypoxia-induced proteins, or biochemically equivalent
fragments thereof, wherein each hypoxia-induced protein belongs to a different
functional category. Alternatively, the polypeptide array comprises at least
two
different hypoxia-induced proteins or biochemically equivalent fragments
thereof,
2 o wherein said hypoxia-induced proteins are all proteins belonging to a
single
functional category. Optionally, the functional category may be selected from
the
group consisting of glycolytic enzymes/proteins, metabolic/homeostatic
proteins,
apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling
proteins,
cell-cycle proteins, and erythropoiesis/vascular regulatory proteins. (See
Table I,
2 5 above).
Yet another alternative embodiment of the invention, is an array analogous
to a polypeptide array described above, except that antibodies immunoreactive
with the hypoxia-induced polypeptides are immobilized to form the array,
rather
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
29
than the polypeptide sequences themselves. Each array comprises at least two
different antibodies, each of which is immunoreactive with a different hypoxia-
induced protein. Each of the two antibodies is specifically immunoreactive
with
the polypeptide expression products of hypoxia-inducible genes, such as, but
not
s limited to, HIG 1 or HIG2. For instance, in one embodiment, the antibody
array
comprises at least one antibody immunoreactive with a protein selected from
the
group consisting of HIG 1, HIG2, annexin V, lipocortin 2, hnKNP A 1, Ku
autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase,
ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen
activator inhibitor-1, macrophage migration inhibitory factor, fibronectin
receptor,
lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent
and
tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA
damage-inducible protein 45, DEC1, low density lipoprotein receptor related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose
1 s bisphosphatase, creatine transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. The antibody
array further comprises at least one of a second antibody, wherein said second
antibody specifically binds a second hypoxia-induced gene product or a
biochemically equivalent fragment thereof.
2 o The antibodies on the array may be monoclonal or polyclonal. They may be
intact antibodies or fragments of antibodies that are capable of specifically
binding
the polypeptides of the present invention. As is the case with the
polynucleotide
and polypeptide arrays of the invention, the antibody array preferably
comprises at
least four different antibodies, and preferably more than about 10 different
2 5 antibodies.
Methods of constructing arrays of biomolecules, especially
polynucleotides, have been previously established in the art. For instance,
some
methods for preparing particularly high density polynucleotide arrays are
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
disclosed in Pirrung et al., Patent No. 5,143,854, Pirrung et al., Patent No.
5,405,783, and Fodor et al., Patent No. 5,510,270, all of which are herein
incorporated by reference. The poIypeptides, antibodies, or polynucleotides
may
be immobilized on the array either covalently or noncovalently. Methods for
5 immobilizing biomolecules are well known to those of ordinary skill in the
art.
The material to which the polynucleotides or polypeptides are immobilized in
the
array may vary. Possible substrates for construction of a biomolecule array
include, but are not limited to, cellulose, glass, silicon, silicon oxide,
silicon
nitride, polystyrene, germanium,(poly)tetrafluorethylene, and gallium
phosphide.
to
d) Methods of Use
In all of the methods of use described below, the animal is preferably a
mammal. Most preferably, the mammal is a human.
We have demonstrated that the expression of HIGI or HIG2 and a number
15 of other genes is indicative of a cell's response to hypoxia as shown in
the specific
examples shown below (Examples 1-9). Accordingly, detection of abnormal
levels of the transcripts of hypoxia-inducible genes such as HIGI or HIG2, or
combinations thereof, in the tissues or body fluids of an animal can be used
in
both a diagnostic and prognostic manner for hypoxia-related conditions. The
2 o abnormal levels may be characterized by either increased levels or
decreased
levels, depending upon the hypoxia-related condition being analyzed. In other
cases, either the complete absence or any presence of a hypoxia-inducible gene
transcript may be indicative of an abnormal condition. Similarly, detection of
abnormal levels of the hypoxia-induced polypeptides, or combinations thereof,
2 5 can be used in either a diagnostic or prognostic manner for hypoxia-
related
conditions. The presence of hypoxia in a tissue can be evaluated by testing
for the
presence or absence of the transcripts or polypeptides encoded by the
polynucleotides of the invention in either the tissue or in the body fluids of
the
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
31
animal. Detection of the transcripts or polypeptides can be either qualitative
or
quantitative.
One aspect of the invention, therefore, provides a method of determining
the presence of hypoxia in a tissue in an animal or evaluating a hypoxia-
related
condition in a tissue in an animal. These methods comprise assaying for either
the
messenger RNA (mRNA) transcripts or the polypeptide expression product of at
least one gene selected from the group consisting of HIGl, HIG2, annexin V,
lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate
synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3,
1 o EPH receptor ligand, plasminogen activator inhibitor-1, macrophage
migration
inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelia-2, B-
cell
translocation gene-l, reducing agent and tunicamycin-responsive protein, CDC-
like kinase-l, quiescin, growth arrest DNA damage-inducible protein 45, DECl,
low density lipoprotein receptor related protein, hamster hairy gene
homologue,
adipophilin, cyclooxygenase-l, fructose bisphosphatase, creative transporter,
fatty acid binding protein, glucose transporter-like protein 111, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3LlNix, and Pim-I in a body fluid
or
the tissue of the animal. This method determining the presence of hypoxia in a
tissue may be used to diagnose a hypoxia-related condition in a animal.
2 o The presence of hypoxia in a tissue or the degree of expression of hypoxia-
inducible genes determined by these methods may be used to select an
appropriate
treatment for the animal. For instance, the hypoxia-related condition being
evaluated may be cancer and the tissue which is the target of the evaluation
may
optionally be a tumor. The degree to which the tumor is showing gene
expression
2 5 patterns characteristic of hypoxia or the activation of genes involved in
angiogenesis, for instance, can be usefully correlated with appropriate
treatment of
tumors of that particular type.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
32
The hypoxia-related condition, however, need not necessarily be cancer.
The hypoxia-related condition may instead be any condition in which hypoxic
conditions play a role (favorable or detrimental to the animal). Such
conditions
include, but are not limited to, ischemia, reperfusion, retinopathy, neonatal
distress, preeclampsia, cardiac arrest, stroke and wound healing.
The transcripts of hypoxia-inducible genes may be detected by any of
several means known to those skilled in the art. One embodiment of diagnostic
detection involves annealing to the transcript, in vivo or in vitro, a labeled
nucleic
acid probe complementary to the transcript sequence. The labeled probe can be
1 o fluorescent, radioactive, immunoreactive, colormetric or otherwise marked
for
detection. To detect very minute quantities of a transcript, amplification of
the
transcript in a tissue or fluid sample from the animal may first be performed
to aid
subsequent detection of the transcript. Amplification of the hypoxically-
induced
transcripts can be readily achieved using the polynucleotides of the present
invention as primers, using reverse transcriptase to make a cDNA copy of the
transcript, and then using polymerase chain reaction to achieve exponential
amplification.
Detection of expression of the polypeptide products of the HIG 1 or HIG2
genes, or any of the other hypoxia-induced genes could be achieved, for
instance,
2 o by the application of labeled antibodies specifically immunoreactive with
the
polypeptide products. The antibodies can be applied to the tissue in vivo, or
to
tissue or body fluid samples removed from the animal. Various forms of typical
immunoassays known to those skilled in the art would be applicable here. These
assays include both competitive and non-competitive assays. For instance, in
one
2 5 type of assay sometimes referred to as a "sandwich assay", immobilized
antibodies that specifically react with HIG2 polypeptide are contacted with
the
biological tissue or fluid sample. Presence of the immobilized HIG2-antibody
complex could then be achieved by application of a second, labeled antibody
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
33
immunoreactive with either the HIG2 polypeptide or the HIG2-antibody complex.
A Western blot type of assay could also be used in an alternative embodiment
of
the present invention.
If a removed tissue is to be analyzed in vitro, typically, degradation of the
tissue is preferred prior to testing for the presence of either an mRNA
transcript or
a gene product. For instance, if detection of polynucleotides is desired,
proteolytic
degradation is useful (Temsamani et al., Patent No. 5,693,466). Extraction or
isolation of proteins or nucleic acids in the sample is also preferred prior
to
carrying out a diagnostic screen. Numerous methods for the isolation of
proteins
l o or nucleic acids from cells or biological fluids are well established in
the art.
In a preferred embodiment, a diagnostic evaluation of hypoxia-induced
gene expression involves assaying the expression levels of more than one .
hypoxia-inducible inducible genes at a time. The arrays of the invention are
particularly useful for assaying the expression of multiple hypoxia-inducible
genes
~ 5 in parallel. The diagnostic detection methods mentioned above in regard to
in
vitro detection would also apply as methods for detecting the presence of
polynucleotides and polypeptides in a tissue or a body fluid upon
administration of
a sample of the tissue or fluid to one of the arrays of the present invention.
Use of the polynucleotide or antibody arrays of the present invention for
2 o determining the presence of hypoxia in a tissue of an animal or for
evaluating a
hypoxia-related condition in a tissue of an animal allows for an unprecedented
look at the exact nature and stage of the hypoxic response of a tissue, since
the
hypoxia-induced expression of a combination of genes is screened at one time.
Patterns of expression of hypoxia-inducible (or hypoxia-repressible) genes are
25 complex and highly indicative of hypoxia in a tissue, as demonstrated in
the
specific examples shown below, Examples 8 and 9. The pattern of expression of
hypoxia-inducible genes can therefore be used in a diagnostic or prognostic
manner to aid in the treatment of a hypoxia-related condition in an animal.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
34
Information on the pattern of expression of a combination of hypoxia-induced
genes can readily be correlated with the aggressiveness of a tumor for
instance,
thereby providing knowledge critical for establishing the best line of
treatment.
The polypeptide arrays of the present invention also can be used to screen
s for drugs useful in the treatment of hypoxia-related conditions. These drugs
may
be drugs which are capable of inhibiting the hypoxic response of a tissue.
For instance, methods of assaying for expression of hypoxia-inducible
genes in a tissue in an animal, determining the presence of hypoxia in a
tissue in
an animal, or evaluating a hypoxia-related condition in a tissue in an animal
1 o comprise first contacting the proteins or messenger RNA of a sample of
body fluid
or tissue obtained from the animal with an antibody array or polynucleotide
array,
respectively, of the invention. Tissue or fluid samples from an animal may be
contacted directly with an array, and binding of the proteins or mRNA
transcripts
on the array detected. (The cells in a tissue to be assayed would preferably
be
15 lysed prior to application to the array.) Alternatively, the tissue or
fluid sample
may be purified to isolate the proteins or mRNA transcripts prior to
application to
the array. In an alternative embodiment of the method, cDNA is first prepared
from the messenger RNA of the sample by reverse transcription and then the
cDNA is applied to a polynucleotide array. Once the protein, mRNA or cDNA is
2 o delivered to the array, the method comprises detecting the amount and
position of
the protein, mRNA or cDNA which remains bound to the array after removal of
excess or non-bound protein, mRNA, or cDNA.
Additionally, a method of diagnosing a hypoxia-related condition in an
animal may optionally comprise the additional step of correlating the result
of the
2 5 evaluation of the hypoxia-related condition in the tissue in the animal
with an
appropriate treatment for the animal. The hypoxia-related condition which may
be
evaluated, diagnosed or treated by any of the above methods may a condition
such
CA 02322843 2000-09-18
WO 99/48916 PCTNS99/06860
as cancer, ischemia, reperfusion, retinopathy, neonatal distress,
preeclampsia,
cardiac arrest, or stroke.
Another aspect of the invention provides for a method of treating a tumor.
This method involves first determining the presence of hypoxia in a tumor by
any
5 of the methods described above (with or without arrays). The method further
comprises treating said tumor with any combination of an established form of
therapy for cancer such as radiation therapy, chemotherapy, or surgery.
The HIGI or HIG2 polynucleotides or the polynucleotides corresponding
to the gene sequences of other hypoxia-inducible gene sequences, such as those
10 listed in Table 1, may be used to attenuate the response of a tissue to
hypoxia.
These hypoxia-inducible sequences can be targeted within a tissue by the
introduction of antisense oligonucleotides, triple-helix probes, catalytic
nucleic
acids or the like in a manner which inhibits expression of the HIG genes or
other
hypoxia-inducible genes within the tissue.
15 Therefore, in one embodiment, the method of attenuating the hypoxic
response of tissue comprises inhibiting the expression of a gene selected from
the
group consisting of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku
autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase,
ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen
2 o activator inhibitor-l, macrophage migration inhibitory factor, fibronectin
receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-l,
reducing
agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth
arrest DNA damage-inducible protein 45, DECl, low density lipoprotein receptor
related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-l,
2 5 fructose bisphosphatase, creative transporter, fatty acid binding protein,
lactate
dehydrogenase, Bcl-2-interacting killer, Nip3LlNix, and Pim-1 in said cell.
This
inhibition of expression of a hypoxia-inducible gene may optionally be
achieved
by introducing into the cells of said tissue a nucleic acid molecule such as
an
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
36
antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a
ribozyme
which is specific to the hypoxia-inducible gene.
In an alternative embodiment of the invention, the HIG 1 or HIG2 proteins
or other expression products of hypoxia-inducible genes may instead be
targeted
to attenuate the hypoxic response of a tissue. For this purpose, antibodies,
antagonists, inhibitors, or proteases that are specific to the expression
products of
hypoxia-induced genes may be introduced to the tissue.
In one embodiment, a method of attenuating the hypoxic response of a
tissue comprises neutralizing a protein selected from the group consisting of
1 o HIG l, HIG2, annexin V, lipocortin 2, hnRNP A 1, Ku autoantigen,
phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,
fibroblast growth factor-3, EPH receptor Iigand, plasminogen activator
inhibitor-1,
macrophage migration inhibitory factor, fibronectin receptor, lysyl
hydroxylase-2,
endothelia-2, B-cell translocation gene-1, reducing agent and tunicamycin-
responsive protein, CDC-like kinase-l, quiescin, growth arrest DNA damage-
inducible protein 45, DEC1, low density lipoprotein receptor related protein,
hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose
bisphosphatase, creatine transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. In this method
an
2 o agent specifically targeting the protein is optionally introduced into the
cells of the
tissue and can be an antibody, an antagonist, an inhibitor, or a protease.
The methods described above for attenuating the hypoxic response of a
tissue may be used to treat a hypoxia-related condition in an animal. For
instance,
the treatment of a hypoxia-related condition in an animal may be effected by
2 5 targeting the hypoxia-induced gene sequences of the hypoxic (or
potentially
hypoxic) tissue via one or more of the techniques known to those skilled in
the art.
These techniques include, but are not limited; to introduction of antisense
oligonucleotides, triple-helix probes, deoxyribozymes, or ribozymes into the
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
37
subject's tissue of concern. In a preferred embodiment, the animal to be
treated is
a human. The hypoxia-related condition towards which this treatment may be
directed is ischemia, stroke, heart attack, neonatal distress, retinopathy, or
any
other disease condition in which hypoxia plays a significant role. In another
s embodiment, the hypoxia-related condition to be treated is cancer and the
tissue is
a tumor. The disclosed treatment of the tumor may be coupled with any
combination of other cancer therapies such as radiation therapy, chemotherapy,
or
surgery.
Similarly, treatment of the hypoxia-related conditions may also be achieved
1 o by neutralizing the protein expression products of hypoxia-inducible
genes, as
described above. In accordance with this method, antibodies, antagonists,
inhibitors, proteases, or the like which target and neutralize HIG1 and HIG2
polypeptides may be introduced into the animal, preferably human, containing
the
tissue to be treated.
15 The protein expression products of the genes which have been newly
identified as being hypoxia-inducible may be used to identify or screen for
drugs,
such as inhibitors, useful in the treatment of hypoxia-related conditions. For
instance, small molecule drug candidates or peptides may be tested against the
any
of the proteins of HIG1, HIG2, annexin V, iipocortin 2, hnRNP Al, Ku
2 o autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA
thiolase,
ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen
activator inhibitor-1, macrophage migration inhibitory factor, fbronectin
receptor,
lysyl hydroxylase-2, endothelia-2, B-cell translocation gene-l, reducing agent
and
tunicamycin-responsive protein, CDC-like kinase-l, quiescin, growth arrest DNA
2 5 damage-inducible protein 45, DEC 1, low density lipoprotein receptor
related
protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose
bisphosphatase, creative transporter, fatty acid binding protein, lactate
dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, or Pim-1 to see if
inactivation
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
38
of the enzymatic activity or prevention of crucial binding activity of the
hypoxia-
induced protein occurs. Combinatorial libraries of small molecules or
libraries of
peptides such as those produced by phage display may alternatively be screen
against one of the hypoxia-induced proteins described herein.
The expression of some gene products induced by hypoxia can be helpful
in protecting cells from damage or death. Thus, this invention also provides
for
methods of enhancing the hypoxic response of a tissue and thereby and treating
hypoxic tissue (or potentially hypoxic tissue). The method comprises
introducing
an expression vector into the tissue and allowing for expression of the coding
1 o sequence on the vector to take place. The coding sequence of the
expression
vector comprises the sequence of at least one of the genes HIGI, HIG2, annexin
V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate
synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3,
EPH receptor ligand, plasminogen activator inhibitor-l, macrophage migration
inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelia-2, B-
cell
translocation gene-l, reducing agent and tunicamycin-responsive protein, CDC-
like kinase-l, quiescin, growth arrest DNA damage-inducible protein 45, DECl,
low density lipoprotein receptor related protein, hamster hairy gene
homologue,
adipophilin, cyclooxygenase-l, fructose bisphosphatase, creatine transporter,
2 o fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting
killer,
Nip3LlNix, or Pim-1. Expression of the vector's hypoxia-inducible gene within
the tissue should occur at a level which is higher than would occur in the
absence
of the expression vector. Depending on the particular use, the coding sequence
of
the expression vector may be operably linked to its native promoter, another
2 5 hypoxia-inducible promoter, or a constitutive promoter.
Alternatively, the proteins of the hypoxia-inducible genes may be
introduced into the tissue directly to enhance the hypoxic response of the
tissue
and for treatment of hypoxia. Delivery of the proteins may be achieved through
CA 02322843 2000-09-18
WO 99/48916 PCT/US99106860
39
the use of liposomes, hydrogels, controlled-release polymers, or any of the
other
vehicles known in the art to be useful for the delivery of polypeptides as
drugs.
e) Methods for Identifying Stress-Inducible Genes
To facilitate efforts to identify hypoxia-inducible genes, we modified and
improved a PCR subtraction method known as Representational Difference
Analysis (RDA) (see specific example, Example 1, below, and Figures 7 and 8).
The RDA method has been used to distinguish differences between genomic DNA
from two related, but different sources (Wigler et al., Patent No. 5,436,142).
The
RDA technique involves selectively amplifying via polymerise chain reaction
only fragments of those sequences contained within one DNA sample, but not the
other. The selectivity of the amplification step used in this method is not
precise,
but is sufficient to detect differences in the genomes of two human
individuals.
The present invention provides for methods of identifying both stress-
inducible and stress-repressible genes. The methods identify differences
between
~ 5 mRNA from cell populations exposed to different stress conditions. A
representative protocol for the identification of stress-inducible genes is
outlined
in detail in a specific example below (Example 1 ).
The method for identifying stress-inducible or stress-repressible genes and
fragments of .genes involves first subjecting one of two populations of cells
to
2 o stress prior to preparation of two cDNA libraries from the mRNA libraries
of the
two populations. Protocols for the generation of cDNA libraries through
reverse
transcription of mRNA sequences are well known in the art and kits for doing
so
are commercially available (from Gibco BRL, for instance). In a preferred
embodiment of the method, the cDNAs are synthesized by using a mixture of
2 5 oligo-dT primers containing equal proportions of oligomers having a G, A,
or C
residue at the 3'-end ("indexed" or "registered" primers). This approach
ensures
that a given primer will hybridize at the start of a polyA tail sequence of an
mRNA rather than randomly wit~lin the sequence. These oligo-dT primers also
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
have a defined DNA sequence (20 to 24 base pairs in length) that is
incorporated
into each cDNA fragment. This tag permits the use of two PCR primers to
specifically amplify the 3'-end of each cDNA. The two cDNA libraries are
digested separately with restriction enzymes and then linker sequences are
ligated
5 to the ends of the digested cDNA fragments, as shown in Fig. 7. Restriction
digests and Iigation of linkers may be performed in any manner known to those
skilled in the art. Some examples of such methods may be found in Sambrook et
al. .(1989) Molecular Cloning. A Laboratory Manual, 2nd. ed, Cold Spring
Harbor
Laboratory Press, herein incorporated by reference.
1 o The cDNA library from one of the two cell populations is amplified with
tagged oligonucleotide primers by means of the polymerase chain reaction
(PCR).
In a preferred embodiment, the "tag" on the oligonucleotide primers is biotin.
However, any chemical or biological moiety which provides a means of selection
or isolation of the tagged entity (by affinity chromatography, for instance)
is
~ 5 suitable as a tag. In the preferred embodiment, use of biotin as a tag
allows for
removal of the tagged sequences on a streptavidin resin. In an alternative
embodiment, however, oligonucleotides bearing a thiol group, for example, may
instead be used as the tagged primer, since oligonucleotides with attached
thiol
groups can be retained on a variety of affinity resins, such as thiopropyl
sepharose
2 o columns or mercurial resins. The cDNA library PCR-amplified with tagged
primers is referred to herein as "driver" cDNA.
The cDNA library from the stressed cells is amplified with normal, non-
tagged, oligonucleotide primers in a separate polymerase chain reaction. The
cDNA PCR-amplified in this manner is referred to herein as "tester" cDNA.
2 s The non-tagged, amplified, tester cDNA is heated and then reannealed in
the presence of a large excess (typically about 5- to about 100-fold) of the
tagged,
amplified, driver cDNA. See Fig. 8. Next, those DNA strands which either are
themselves tagged or are duplexed with tagged DNA are removed from the
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
41
mixture. This removal is typically done via exposure of the mixture of DNA
strands to a resin or matrix which has affinity for the tag used on the
primers
earlier. In a preferred embodiment, magnetic beads coated with streptavidin
are
used. Other resins, such as streptavidin agarose could be used in conjunction
with
s a biotin tag. Tagged single-stranded or duplex cDNA will be retained on the
affinity resin, and the non-tagged species, which are not retained, can be
found in
the flowthrough or supernatant. In this technique, the cDNA from the non-
stressed cell population is "subtracted" from the cDNA of the stressed cell
population. The remaining, non-tagged cDNA library is said to be "enriched".
1 o The remaining, non-tagged cDNA sequences are then again amplified by means
of
the polymerase chain reaction with non-tagged primers.
After amplification of the remaining non-tagged cDNA sequences, the non-
tagged cDNA library is again heated and reannealed in the presence of a large
excess (typically about 5- to about 100-fold) of the original tagged cDNA
library.
15 Removal of all tagged DNA molecules and reamplification of remaining tagged
sequences again follows. The combination of steps involving heating and
reannealing, removed tagged molecules, and reamplifying remaining, non-tagged
molecules constitutes one round. The methods of the present invention involve
repeating the rounds from zero to many times. In a preferred embodiment, the
2 o method involves a total of approximately 3 to 5 rounds.
In a particularly preferred embodiment, the method involves performing the
steps as described above in parallel with a second set of steps in which the
cDNA
library from the stressed population of cells is instead subtracted from the
cDNA
library from the non-stressed population. This means that in the second set of
2 5 steps, the cDNA library from the stressed cell population is amplified
with tagged
primers and the cDNA library from the non-stressed cell population is
amplified
with non-tagged primers. The original cDNA of the stressed cell population is
repeatedly subtracted from the cDNA of the non-stressed cell population, and
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
42
separately, the original cDNA of the non-stressed cell population is
repeatedly
subtracted from the stressed cell population.
In the final round of the preferred embodiment of the method, one of the
two enriched cDNA libraries obtained from the two sets of steps is subtracted
from the other enriched cDNA library. Which enriched library is subtracted
from
which is entirely dependent upon whether stress-inducible or stress-
repressible
sequences are sought. If stress-inducible sequences are sought, the enriched,
non-
stressed cDNA library is subtracted from the enriched, stressed, cDNA library.
If
stress-repressible sequences are sought, the enriched, stressed-cell cDNA
library is
1 o subtracted from the enriched non-stressed-cell cDNA library.
The final subtraction step of one enriched library against another is
beneficial since the initial subtraction rounds of the procedure tend to
remove only
the cDNAs that are in common and present at high frequency in the two
populations, because cDNA fragments derived from rare messages will initially
be
present at such low concentrations that they might not find a complementary
strand during the hybridization step. After the major sequences in common are
removed by subtraction, the rare sequences will begin to increase in
concentration
so that they can then be effectively subtracted. After multiple cycles of
subtraction are performed, the rarest sequences from both conditions are
enriched
2 o in the libraries, and subtraction of one enriched library from another
yields an
effective isolation of either stress-inducible or stress-repressible genes.
After the desired number of rounds of subtraction have been completed, the
enriched cDNA library may be cloned and sequenced using any one of the
multitude of techniques known to those skilled in the art. A particularly
2 5 convenient method of inserting PCR-amplified DNA strands into vectors
suitable
for cloning and sequencing, known as "T-A cloning", is commercially available
from companies such as Invitrogen and Novagen. Other alternative methods can
CA 02322843 2000-09-18
WO 99/48916 PCT/tJS99/06860
43
be found in Molecular Cloning: A Laboratory Manual, 2nd. ed, Yol. 1-3, eds.
Sambrook et al., Cold Spring Harbor Laboratory Press ( 1989).
In one embodiment, the stress to which one of the two cell populations is
exposed is hypoxia. The method may also be applied to the investigation of
responses to other stresses, such as ionizing radiation, heat, glucose
starvation,
hypothermia, or pH change. Alternatively, the response to a stress such as a
toxin
or a drug may be investigated by employment of the disclosed method.
f) Examples
1 o The following specific examples are intended to illustrate the invention
and
should not be construed as limiting the scope of the claims.
Example 1. Generation of Enrichment cDNA Libraries
Normal human cervical epithelial cells stably immortalized with the human
papillomavirus E6 and E7 oncoproteins (HCE.E6.E7) served as the starting
material for the construction of a cDNA library enriched by representational
difference analysis (RDA). HCE.E6E7 were cultured in synthetic medium PFNiR-
4A (Kim et al. ( 1997) Cancer Res. 57:4200-4).
A total of S ~,g of poly A+ mRNA from both HCE.E6E7 cells cultured
under hypoxic (5% C02/5% H2/90% N2 for 16 hours at 37°C) conditions and
2 o HCE.E6E7 cells cultured under aerobic (5% C02 / 20% 02 / 75% N2 at 37
° C)
conditions were used to generate double-stranded cDNA preparations by using
the
Gibco BRL cDNA Synthesis System.
Hypoxic conditions were generated by the use of an anaerobic chamber
(Sheldon Laboratories, Cornelius OR) that is flushed with a gas mixture of 90%
N2, 5% C02 and 5% H2. Any oxygen that was introduced into the chamber was
consumed over a catalyst with hydrogen. A monitoring oxygen electrode was
used to confirm an environment of 0.05% oxygen or less during experimentation.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
44
One-fifth of the cDNA product (approximately 1-1.5 p.g) from the hypoxic
or oxic cells was digested with 20 units of the Nla III restriction enzyme, 50
mM
potassium acetate, 1 mM DTT, and 100 p,g/ml bovine serum albumin for 60 min
at 37 ° C. The reaction mixture was extracted with phenol and
chloroform,
precipitated with ethanol, redissolved in lOuL of water and lyophilized.
Ethidium
agarose gel electrophoresis was used to verify that the cleavage was
successful.
For each pair of cDNAs used for the RDA procedure (i.e. the "test" and the
"driver"), two different DNA linkers were attached by ligation to the Nla III
cleaved ends. The 3'-end of the linker sequence opposite the ligation site was
terminated with an amine so that it cannot be used as an acceptor or donor for
a
ligase. The linker oligonucleotides used were as follows (where "X" denotes
the
animo-terminated residue at the 3'-end of the shorter of the two strands):
5'-TTTTACCAGCTTATTCAATTCGGTCCTCTCGCACAGGATGCATG-3'
(SEQ ID NO:11)
XATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCTAC-5' (SEQ
ID N0:12)
5'-TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGATGCATG-
3'(SEQ ID N0:13)
2 0 XAACATCTGTAAGATCATAGAGCAGTTCAGCCTTCCTAC-5' (SEQ ID
N0:14)
(The linker pair of SEQ ID N0:13 and SEQ ID N0:14 was used for the
hypoxically incubated cell cDNAs.) The two separate linker strands were
2 5 dissolved in 10 mM Tris-HCl (pH 7.6), 10 mM MgCl2 buffer ( 10 p,M of each
oligomer), then heat-denatured and slowly cooled to room temperature before
use
in a ligation reaction.
CA 02322843 2000-09-18
WO 99/48916 PCT/IJS99/06860
Next, 1 ~,g of the Nla III cleaved cDNA was ligated in a 100 ~L volume of
1 ~iM double-stranded linker, 5% polyethylene glycol, 50 mM Tris-HCl (pH 7.6),
10 mM MgCl2, 1 mM ATP, and 1 mM DTT at 16 ° C for 3 h. Since the
linkers
used to ligate to the cDNA fragments do not have a phosphate at the 3'-end of
the
s Nla III overhang, the resulting ligation products have a single-stranded
nick.
Performing the reaction in this way had the advantage of preventing self
ligation
of the linkers. The excess linkers were removed by gel filtration through a
spin-
column containing Sephacryl S-300HR. The linker-ligated cDNA fragments were
collected in the microfuge tube while the excess unligated linkers were
trapped in
1 o the Sephacryl with other low molecular-weight components. The gel-
filtered,
linker-ligated cDNA fragments were then lyophilized to dryness.
The linker-ligated cDNA fragments were amplified by a single-primer PCR
technique. Again, if the preparation was to be used as the driver cDNA, it was
amplified by using PCR primers with a biotin residue at the 5'-end. If the
15 preparation was to be used as the test cDNA from which the driver is used
to
subtract sequences, then it was amplified by using untagged primers.
The ligated cDNA (0.1 ~,g aliquot) was amplified in a standard PCR buffer
containing 1 ~.M primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2,
and 0.01 % gelatin. Before PCR amplification, the nicked PCR template had to
be
2 o repaired by TAQ polymerise during a 5-min extension reaction at 72
° C. After
this initial incubation, a standard PCR reaction of 3 5 cycles (94 ° C,
30s; 56 ° C,
30s; 72 ° C, 60s) was performed in a Perkin Elmer DNA Thermal Cycler.
The
oligonucleotide primers used in the amplification step were as follows:
2 5 5'-CCAGCTTATTCAATTCGGTCC-3' (SEQ ID NO:15)
5'-GTAGACATTCTAGTATCTCGT-3' (SEQ ID N0:16)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
46
(SEQ ID N0:16 was the primer used to amplify cDNA from the hypoxically
incubated cells.) The entire PCR reaction was passed through a 1 ml Sephacryl
spin column as described above to remove salts, dNTPs, and excess primers. The
yield of the amplification was determined by ethidium agarose gel
electrophoresis.
The product appeared as expected as a smear of DNA fragments ranging from 100
to 2,000 base pairs (bp) in size.
The first round of subtraction was performed by mixing 3 ~.g of the
biotinylated driver cDNA with 0.1 pg of the test cDNA. The mixture was
lyophilized in a 0.5 mL microfuge tube and carefully redissolved in 2 ~.L of
50
to mM HEPES (pH 7.5), 10 mM EDTA, 1.5 mM NaCI, and 2% sodium dodecyl
sulfate (SDS). This very small amount of solution was overlaid with 50 N.I, of
mineral oil to prevent evaporation, and the tube was place in the thermal
cycler
and heated at 95 ° C for I O min. It was then slowly cooled to 68
° C over a period of
1 h, after which the incubation at 68 ° C was continued for a further 4
hours. At
1 s the end of the incubation, 100 ~1 of the same HEPES buffer at 68°C
was added to
the tube. The diluted solution was then cooled to room temperature and the
mineral oil removed.
The biotinlyated cDNAs and any hybridized sequences were removed by
mixing the diluted solution with a 100 ~,I, slurry containing 1 mg of M-280
2 o Streptavidin Dynabeads (Dynal) in the same incubation buffer. The
incubation
was continued at room temperature for 30 min with slow tumbling. The beads
were then pelleted to the bottom of the tube by using a magnet and the
supernatant
was removed and desalted by passing through a 1 mL Sephacryl spin column as
described above. The cDNA solution was then lyophilized and redissolved in 10
2 5 ~,L of water.
The small amount of cDNA remaining after subtraction was reamplified by
PCR using the same primers. A single-stranded binding protein was added to the
PCR reaction mixture used to reamplify the subtracted cDNA fragments: I p.L,
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
47
(one-tenth volume) of the subtracted cDNA preparation was placed in 100 ~,L of
PCR buffer containing 1 ~,g of Escherchia Coli single-stranded binding protein
(Perfect MatchTM, Stratagene). The cDNA was amplified during 25 PCR cycles
(94 ° C, 30 s; 54 ° C, 30 s; 72 ° C, 60 s), and the
product was analyzed by ethidium
agarose gel electrophoresis. The appearance of this reamplified cDNA was
similar to that of the initial material described above.
Multiple rounds of subtraction were performed. The subtraction libraries
were prepared in parallel, so that the library enriched for sequences
expressed
under hypoxic conditions was prepared at the same time as the library enriched
for
1 o sequences expressed under normoxic conditions. In each case, the driver
used for
the initial rounds of subtraction was the original set of cDNA fragments.
After
three rounds of subtraction, the enriched library prepared in parallel was
used as
the driver for the fourth round. In this way, the rarest sequences from both
conditions were enriched in the final library. For instance, to obtain
hypoxically
induced sequences in this final round, the cDNA library enriched for sequences
expressed under normoxic conditions served as the driver library and the cDNA
library enriched for sequences expressed under hypoxic conditions served as
the
test library.
Example 2. Sequence Identification and Northern Blot Analysis of Significant
2o Isolated Expressed Sequence Tags~(ESTs).
Several hundred cDNA fragments were sequenced from each of the two
enrichment libraries produced by the subtraction protocol of Example 1 from
HCE.E6E7 cells cultured under hypoxic and aerobic conditions. Four rounds of
RDA subtraction of the oxic cDNAs from the hypoxic cDNAs generated a
2 5 population of fragments in one of the enrichment libraries representing
genes that
theoretically are induced by hypoxic treatment. Five hundred randomly chosen
clones from the cDNA library were partially sequenced. The obtained sequences
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
48
were analyzed by NCBI-blast to determine the frequency of each of the
genes/ESTs in the enriched population and to identify whether the isolated,
hypoxia-induced ESTs corresponded to previously identified genes or ESTs.
The frequencies of EST sequences among the 500 randomly chosen cDNA
fragments obtained from the cDNA library enriched for sequences expressed
under hypoxic conditions (after all four rounds of subtraction) is shown in
Table 2,
below. The two most frequently occurring ESTs, the HIG 1 EST and the HIG2
EST, corresponded to no known genes. Because these most frequently repeated
clones were unknown, the full-length cDNAs representing HIG1 and HIG2 were
1 o isolated (see Example 3, below).
All the ESTs present in the clones of each library that were represented
more than one time and that did not contain a highly repetitive element were
tested
by Northern blot for induction by hypoxia in Siha cervical carcinoma cells
(and/or
in HCE.E6E7 cells). Selected probes representing ESTs found more than once
were applied to Northern blots of total RNA from cell cultures harvested
following different aerobic and hypoxic exposures to verify hypoxia
iriducibility
or repressibility. For instance, the northern blot assays were used to confirm
that,
a-tubulin mRNA, detected in the HCE.E6E7 aerobic enrichment library,
decreased in response to hypoxia in HCE.E6E7 cells, whereas mRNA
2 o corresponding to the HIG2 EST, found in the hypoxic enrichment library,
strongly
increased under the same hypoxic conditions.
Table 2. Tags isolated from the cDNA library following four rounds of
RDA subtraction of oxic cDNAs from 6ypoxic cDNAs.
# of tag gene to which accession response comment
hits EST #
name corresponds of gene to hypoxia*
106 HIG HIG 1 induced novel gene
1
98 HIG2 HIG2 induced novel gene
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
49
48 HIG3 GAPDH J04038 induced inducibility
pre'
known
11 HIG4 I-INRNP X12671 induced
1 I HIGS Annexin V U01691 induced
8 HIG6 AcetoacetylCoA S70154 induced*
thiolase
7 HIG7 Tissue Factor X67698 induced inducibility
pre'
known
7 5-2A by X76388 not induced
6 unknown gene clone 68 no signal
Alu-like not determined
5 HIGB Lipocortin 2 M 14043 induced
S HIG9 Ribosomal L7 XS7959 induced
4 unknown gene clone 24 not induced
3 Vacuolar ATPase X71490 no signal
3 HIG PRPP synthase D00860 induced
10
3 Alu-like not determined
2 RNA pol l 40Kd AF047441 not induced
subunit
2 HIG11 thioredoxin X77584 induced inducibility
prey
known
2 hSRPl(nuc loc) U28386 not induced
2 HIG Ku(70) J04611 induced
12
2 Sm protein F X85382 not induced
1 168 different
clones
* as
determined
by
Northern
blot
* *
minor
4.2kB
acetoacylCoA
thiolase
message
only
is
induced
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
The procedure for the Northern blot assay was essentially as follows. Total
RNA was isolated with Trizol (Gibco BRL) following the directions of the
manufacturer. 5-10 ~g of total RNA was denatured with glyoxal and size
fractionated on a 1 % agarose phosphate gel. The gel was capillary transferred
to
5 Hybond nylon (Schleicher and Shuell) and UV cross-linked. Probes were
radiolabeled by random priming of gel-purified full length HIG1, or a fragment
of
HIG2 containing only the coding sequence in a Stu 1 fragment (Rediprime,
Amersham). Hybridization was carried out in 0.5 M Na2HP04, 7% SDS, 1 mM
EDTA at 56oC for HIG1 and 65oC for HIG2, washed to 0.2-0.5 x SSC at 56oC or
10 65oC, exposed to a phosphorimager plate, and visualized on a Storm 860
phosphoimager (Molecular Dynamics).
The hypoxia-inducibility of ESTs as determined by Northern blot is
summarized in Table 2, above. The HIG1 and HIG2 sequences both demonstrated
hypoxia-inducibility in the Northern blot assay.
1 s Northern blots of total RNA from various aerobic and hypoxic human cells
[HCE.E6E7s; SiHa cervical squamous carcinoma, MCF-7 breast carcinoma,
H1299 lung carcinoma, Hct116 colonic carcinoma cells; human cervical
fibroblasts (HCFs) and HCF.E6E7s] probed for HIG2 expression demonstrated the
following: ( 1 ) the gene is expressed as a single 1.5 kb transcript (the
original EST
2 o cross-hybridizes with unknown 1.6- and 4-kb transcripts in HCE.E6E7s); (2)
HIG2 mRNA increases from undetectable in 21 % 02 (air) to abundant in 0.02%
02 in HCE.E6E7, SiHa, and MCF-7 cells after 6 h of hypoxia; (3) HIG2 is
moderately expressed in H1299 and Hctl 16 cells after 6 h of hypoxia; (4)
there is
no detectable HIG2 mRNA in HCFs and HCF.E6E7s; (S) in SiHa cells, HIG2
2 5 remains elevated for 48 h of hypoxia but decreases moderately by 72 h of
exposure; and (6) no HIG2 induction is found in SiHa cells b h and 24 h after
treatment with UV-C (20 J/m2), y-irradiation (6 Gy), MMS ( 100 ~,g/mL for 1
h),
serum deprivation (0.1%), or glucose starvation (4%, <1 mM); (7) HIG2
CA 02322843 2000-09-18
WO 99/48916 PCTNS99/06860
51
expression is extinguished after exposure of hypoxic cells to 2 hours of
reoxygenation.
The hypoxia inducibility of HIG 1 has been found to range between about
2-fold and about 5-fold across a variety of different human cell lines
studied. The
s hypoxia-inducibility of HIG2 ranges between about 10- and about 20-fold
across
the various human cell lines studied. (See also Example 4, below).
In addition to the novel genes HIGI and HIG2, several known genes
identified by the subtraction method in Example 1 were confirmed by Northern
blots to be hypoxia inducible. These genes are also listed in Table 2. ESTs
to corresponding to the genes of annexin V, lipocortin 2, hnRNP A1, Ku (70)
autoantigen, glyceraldehyde-3-phosphate dehydrogenase, ribosomal L7,
acetoacetylCoA thiolase, and PRPP synthetase were identified by multiple hits
in
the hypoxia screen. All of these previously known genes were confirmed to be
hypoxia-inducible by Northern blot.
is It should be noted that although acetoacetyl CoA thiolase sequence tag is
listed as induced, the reported, major RNA (1.8 kb) for the gene does not
change.
However, there is a larger, hybridizing, RNA species (4.2 kb) that is induced
after
24-4.8 h hypoxia (data not shown).
ESTs corresponding to glyceraldehyde 3-phosphate dehydrogenase
2 0 (GAPDH) were especially prevalent amongst the cDNA clones. The hypoxia-
induced expression of glyceraidehyde-3-phosphate dehydrogenase had been
previously identified only in normal, non-transformed cells.
Example 3. Isolation and Analysis of FuII-Length HIG1 and HIG2 cDNA
25 Sequence
The HIG2 EST ( 142 bp) was used to probe a conventional cDNA library
constructed from mRNA isolated from SiHa cells exposed to 16 h hypoxia to
obtain the full-length cDNA clone HIG2. This library was probed with
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
52
radiolabelled HIG2 tag using conventional methods. Full length HIGI was
isolated by first identifying overlapping ESTs from the NCBI human EST
database, until a full length sequence was generated { 1.3 S kb). PCR primers
were
then synthesized corresponding S' and 3' UTRs in order to amplify the complete
sequence using RT-PCR of SiHa RNA isolated after a 16 h hypoxia treatment.
The full-length HIGl cDNA was then cloned and sequenced to confirm the
predicted sequence.
The full-length cDNA sequence ofHIGl is shown in Figure lA. The full-
length cDNA sequence of HIG2 is shown in Figure 2A. The translations of the
1 o putative open reading frames from HIGI and HIG2 are listed in Figure 1 B
and 2B,
respectively, and both encode small peptides (95 and 64 as residues
respectively)
without obvious functional motifs.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
53
Example 4. Hypoxic induction of HIGI and HIG2 in cervical cancer cell lines.
Because HIGI and HIG2 represent two novel genes whose functions are
unknown, these genes were investigated in more detail. The expression of HIGI
and HIG2 was examined in a series of human cervical cancer cell lines (SiHa,
CaSki and C33a) under oxic and hypoxic conditions in vitro. (The cell lines
SiHa,
CaSki and C33a were obtained from the ATCC and were cultured in Dulbecco's
modified Eagle's medium (DMEM) or RPMI1640 supplemented with 10% fetal
bovine serum.) Although HIGl is induced moderately within 2 hours of hypoxia
in all the cell lines tested, it remains elevated only in the Siha cells. HIG2
is more
1 o consistently induced from low basal levels in all the cervical cancer
cells tested.
The major HIG2 mRNA species is 1.4 kb in length, but there are two other mRNA
species of minor abundance (8.0 and 9.0 kb) that are induced with identical
kinetics to the major species.
Example 5. Hypoxic induction of HIGl and HIG2 in tumor xenografts.
The hypoxic induction of HIGl and HIG2 in vivo was also tested in tumor
xenografts generated from the C33a cell line by Northern blot analysis of
total
tumor RNA. Gene expression in untreated xenografts was compared to that in
xenografts that were made hypoxic by treatment of the host animal with flavone
2 o acetic acid (FAA) 24 hours prior to explantation and RNA isolation. To
generate
tumor xenografts, 2.5-5 x 106 cells were injected subcutaneously into the
flank of
scid mice and allowed to grow into tumors that reached 1-2 cm in diameter
before
harvest. FAA (Lipha Chemical, NY) was injected IP into the animals at 200
mg/kg in 5% sodium bicarbonate 24 hours prior to tumor harvest. FAA treatment
2 5 resulted in increased tumor hypoxia as measured by ependorff electrode and
increased HIGl and HIG2 expression by 1.2 and 2.4 fold respectively. The
moderate level of HIGI induction in vivo is not unexpected, due to the in
vitro
data. The portion of the human gene used for a probe in these experiments has
CA 02322843 2000-09-18
WO 99/48916 PC'f/US99/06860
54
low homology with mouse RNA and under the conditions used, did not cross-
hybridize.
Example 6. Specificity of the induction of HIGI and HIG2.
We next investigated whether HIG l and HIG2 induction is unique to
hypoxic stress, or if it is elicited by other tumor microenvironment stresses
such as
glucose deprivation, serum starvation, or by~genotoxic stresses such as I1V or
ionizing radiation. We also tested the hypoxia-mimetic, iron-chelating
compound
desferoxamine that has been shown to induce expression from HIF-1 responsive
i o genes. For stress treatments, cells were plated overnight and then treated
the next
day with either 256 nm LTV at 1.2 J/m2/sec, or gamma irradiation from l3~Cs
source at 3.8 Gy/min. Glucose and serum deprivation experiments were
performed by washing the cells three times in phosphate-buffered saline (PBS)
and replacing the indicated media (glucose free RPMI with dialyzed serum, or
7.5 0.1% FBS RPMI).
Northern blot analyses was performed on RNA isolated from C33a cells
exposed to these stresses. HIGI was poorly responsive to hypoxic stress over
this
timecourse, but strongly induced by glucose deprivation. HIG2 was induced
strongly by hypoxia, the hypoxia-mimetic stress desferoxamine (DFO), and
2 o glucose deprivation. LJV light seemed to have little effect upon either
HIGI or
HIG2 expression. In contrast, while ionizing radiation did not change HIGI
expression levels, it did result in a moderate 2.5 fold induction of HIG2 by
24
hours. There were similarities in the pattern of stress responsiveness of HIG2
and
that of the HIF-responsive VEGF gene, suggesting that HIF-1 may be important
in
2 5 HIG2 expression.
Example 7. Identification of HIGI and HIG2 sequences from non-human species
CA 02322843 2000-09-18
WO 99/48916 PC"T/US99/06860
A search of the NCBI-dbEST database for fragments of genes from other
species that might represent evolutionarily conserved orthologues identified
overlapping mouse EST fragments that encode for similar peptides to the human
version of HIGI and HIG2. The marine HIGI and HIG2 orthologues are shown
5 in Figures 3A and SA, respectively. These mouse genes code for predicted
peptides (Figures 3B amd SB, respectively) with 84% and 76% identity to the
human peptides respectively. There also existed a cDNA cloned from fish
(seriola guinqueradiata) in the database that coded for a HIG 1 orthologue
(Figure
4A and 4B). A sequence comparison of the HIG1 homologues is shown in Figure
10 6A. A sequence comparison of the HIG2 homologues is shown in Figure 6B.
We confirmed the existence of marine HIG1 and HIG2 by cloning the
presumed genes and assaying for their expression. We designed oligonucleotide
primers corresponding to sequences in the 5' and 3' untranslated regions that
would amplify these genes. We were able to make primers that amplified the
1 s entire marine HIG2 cDNA, but were only able to make primers that would
amplify the coding sequence for marine HIGI:
mHIGl forward primer (SEQ ID N0:17):
5'-CCGATCTAGAGGAAGGGACCCCGCGTCTCGGA-3'
2 o mHIG 1 reverse primer (SEQ ID N0:18):
5'-GGCGCTCGAGTCTAAACCCACATGTTATTTATTG-3'
mHIG2 forward primer (SEQ ID N0:19):
5'-CCTTACTCCTGCACGACCTGG-3'
mHIG2 reverse primer (SEQ ID N0:20):
2 s 5'-GGCGCTCGAGCACATGTGCATTACACTGGAGA-3'
These primers were then used to amplify the coding sequences of HIGI OR HIG2
from reverse-transcribed RNA isolated from the marine squamous cell tumor cell
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
56
line SCCVII (cultured in DMEM supplemented with 10% FBS). The amplified
fragments were cloned and sequenced, confirming the predicted sequence.
The cloned genes were then used as probes for Northern blot analysis of
RNA isolated from SCCVII cells. Both mHIGI (marine HIGl ) and mHIG2
(marine HIG2) have hypoxia-inducible species of RNA by this analysis. Marine
HIGI has two major RNA species that strongly hybridize to the probe, at
approximately 1.2-1.4 kb in length. The larger message is modestly induced,
while the smaller message is strongly induced to approximately S fold by a 12h
exposure to hypoxia. Marine HIG2 also has two RNA species at approximately
l 0 1.4 and 2.2 kb. Both the marine HIG2 mRNAs seem to be mildly hypoxia-
inducible with 2-3 fold induction by 6-12 hours. For comparison, the same blot
was probed with vascular endothelial growth factor (VEGF) and this message
shows an approximately 5-fold induction by 6h.
Example 8. Analysis of Gene Expression under Hypoxia using Gene Discovery
Arrays (GDA).
Nylon filters containing GDA arrays were purchased from Genome
Systems (St Louis, MO) that have affixed to them nucleic acids that were
originally characterized by the LM.A.G.E. consortium (LLNL). This array
2 o represents 18,394 cDNA clones that have been categorized as either known
genes
or ESTs (expressed sequence tags) isolated by the consortium. This filter was
used to quantitatively determine the mRNA expression levels of all these
arrayed
cDNAs in SIHA tumor cells both under oxic conditions and hypoxic conditions
( 18 hrs, <0.2 %). Messenger RNA was isolated from control and hypoxic SIHA
cells and cDNA probe was generated using MoML reverse transcriptase. 2 ~g
mRNA was incubated with 500 ng of oligonucleotide primer (T) 1 g NM
(N=A/C/G, M=A/C/G/T) in the presence of reaction buffer, 4mM dATP, 4mM
dGTP, 4 mM dTTP and 4mM alpha ~331P dCTP and 200U reverse transcriptase.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
57
The radioactively labeled first strand cDNA that was produced from this
reaction
was then used to probe the respective filter. The filters were then exposed to
a
phosphoimager plate, the image collected and digitized for analysis, and the
relative counts on each cDNA were quantitated and compared using GDA analysis
software. The results are shown in Table 3 for the 500 genes or ESTs with the
greatest level of hypoxic induction and in Table 4 for the 500 genes or ESTs
with
the greatest level of hypoxic repression.
Table 3. Genes (identified by Genbank Accession Number) whose expression
was induced in hypoxic cells, shown with the ratio of their expression in
hypoxic cells over their expression in oxic cells.
ratio Acc. No. ratio Acc. No. ratioAcc. No. ratioAcc. No.
5.18 AA069408 7.013 AA134027 4.027AA155910 3.372AA037436
7.528 T48883 5.236 N35559. 5.343T87461 2.025H22698
9.999 N58711 5.753 ~ 863553 3:478W24548 5.454W07146
5.678 H04904 6.525 N27733 5.699T48772 5.209AA101069
6.453 H82707 3.146 N94304 4.095N94916 4.599W24109
7.825 W56465 4.218 H70730 6.52 H14897 4.996817409
9.999 N31409 1.659 AA053856 1.159834659 4.932W17090
9.999 H19264 8.836 W05763 2.492H93923 5.391824601
3.626 873213 3.394 854524 5.05428535 5.189826954
N
9.999 808251 8.246 W 15599 2.08 AA069499 2.12 AA 187216
9.999 H91612 4.216 809918 5.021W38635 3.852H86677
9.999 AA196038 9.238 AA005185 8.59 T54127 4.653H67329
9.999 W55913 4.654 T95404 4.289826319 4.993W 19173
5.143 894248 9.262 819946 6.048W07082 5.641W06851
9.999 885589 4.308 AA068998 3.12 AA194330 6.186W00378
2.9 H50204 5.203 N47831 4.594N31417 4.458AA204792
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCTNS99/06860
58
4.333 H52973 4.175 AA151009 2.424 AA069173 4.64 W48584
9.999 H60510 5.33 W46682 3.645 W52472 3.742 884764
9.915 813129 5.39 AA176700 6.109 H09049 4.049 821449
9.999 AA040826 3.612 H47207 3.843 H81775 5.074 A,A160325
2.186 N76562 7.666 W87527 5.664 H71710 2.356 897269
8.468 813125 5.965 W47502 2.494 W 17237 3.68 H86214
9.999 W52400 6.338 N44758 3.516 H06318 4.654 N40829
3.376 AA054303 2.313 AA126937 4.006 T63499 5.114 W19928
4.263 A.A042800 6.232 831353 5.829 822383 5.526 H18298
2.681 W46165 4.846 818798 3.457 T87920 2.867 N39173
8.287 W24084 4.648 T78246 5.766 W47525 3.251 876943
9.999 T77247 6.726 H29713 5.493 W32575 4.139 W07720
8.506 813073 4.524 AA026304 5.622 884635 3.604 AA085920
9.999 T95699 6.113 AA053162 5.284 818138 3.814 H18258
8.7 N30952 1.702 H65775 3.414 834648 3.192 AA035131
8.372 N29065 3.685 W32710 4.46 H80I75 2.275 AA033736
7.37 H13744 6.264 T51305 4.479 876163 3.896 871065
9.999 H46657 7.182 814403 4.381 W17182 3.534 AA074177
6.115 H83517 3.9 W38235 5.81 A,A126956 4.358 892264
6.587 AA129780 5 W47083 3.781 AA088390 3.561 N34634
6.587 N42542 5.209 W35243 4.048 852046 3.695 AA 114861
9.999 H67546 3.288 W24343 5.474 N98916 4.673 831970
6.678 AA181350 5.209 H93373 3.226 851929 3.989 H70974
7.187 T67190 6.255 826331 3.033 H69334 4.646 AA156193
6.498 AA034932 2.549 870082 5.458 W20511 3.258 AA085385
9.999 H41372 4.698 W32969 3.742 T40473 4.453 W49770
9.999 H08885 4.038 866920 5.132 882723 4.056 W37672
8.221 H90627 6.586 W25455 5.886 T50788 2.503 W38097
4.812 T91423 7.516 W38478 3.266 AA036758 5.118 N48838
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
59
5.535N49031 1.961 AA039447 3.564 W46219 5.105 H41937
7.673AA079020 3.163 H89835 3.535 N64406 1.544 AA159807
5.718H44892 3.311 AA156148 4.839 W17076 3.436 AA112478
2.058H38180 5.592 W38424 5.314 H18129 4.396 807212
3.101T48613 6.954 H75477 4.118 H93835 4.917 AA128281
3.236H12952 3.507 W31707 5.306 AA167017 4.303 N39630
2.553T87585 3.968 N40606 5.012 W24939 4.165 W39234
2.961876842 3.417 W67757 3.887 W38826 1.983 H68S87
4.505N54105 5.57 AA074760 3.791 H62991 3.358 H78277
3.424W72986 2.311 H18350 4.853 AA132801 2.968 T87597
3.738H53662 2.707 W31757 2.065 854128 3.142 H64978
1.682WOSSS 3.252 T89571 3.35 AA214334 4.376 W 16557
1
2.942H62026 3.202 W07148 3.553 W 16484 3.316 H45068
3.16 AA146611 2.404 H66389 3.902 AA122157 3.559 H08997
3.702H70850 3.621 868331 3.451 H44677 3.277 H08983
4.118W49687 4.089 AA099075 3.277 W20192 2.455 H66256
4.359AA100113 4.46 H91361 3.534 T47067 2.632 T70457
3.338AA133312 2.057 N42428 3.581 T82048 2.569 H13942
4.367T49117 3.423 W48763 3.694 AA134135 2.59 T77584
3.79 H58461 3.844 860387 2.79 825979 2.346 871543
4.806N69323 3.421 W 19744 1.586 821064 3.168 T98705
2.664W05089 2.473 859435 1.972 H29698 3.43 T78542
3.176AA070823 3.189 807238 3.507 889708 2.887 T74951
1.966W24455 2.496 AA074340 3.189 806568 3.768 W 16974
3.258863252 3.287 N78038 1.957 AA009869 3.48 888098
4.543H80571 3.935 W07144 1.627 W31940 2.021 W48791
3.593AA131550 4.133 806175 3.545 W38638 3.686 880458
4.34 N42413 3.912 N46036 1.634 860752 1.812 H29706
2.313862688 2.535 W78057 2.917 828459 3.501 AA130339
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
2.952H73881 3.614 816609 3.614 H63610 2.642H10811
5.245AA007484 3.208 H01260 3.568 866182 2.323AA074067
6.009N57562 1.818 W04913 2.77 H95908 2.419T56791
3.62 H25971 3.591 861631 2.83 H24644 2.432848720
3.583H19106 3.528 H71668 2.458 AA044130 2.881820222
3.085W46660 4.66 818905 2.039 823341 3.274H03764
3.694836401 2.311 H64449 3.785 H01679 2.71 855247
3.945809905 1.619 W07452 2.82 T91461 3.324AA005419
4.416H40081 3.462 AA203284 3.202 T74959 2.494T97640
4.498AA156956 4.843 N66473 1.924 T39976 4.157823556
3.22 AA112421 3.777 H78279 3.135 H68817 3.956N53743
2.991AA147722 3.256 AA156298 3.739 H83559 1.947W01963
4.341W2017I 3.25 N75101 1.841 T79362 3.858N94762
1.778W21312 3.961 H75277 2.551 N36269 3.655AA007521
3.42 N28517 4.366 880450 3.285 T79536 1.506800903
3.436W07026 3.532 AA057729 3.193 T87507 2.775H83982
4.393821898 2.373 T92805 3.87 T71354 2.61 H12686
3.543836586 3.167 AA005286 3.572 W 19860 2.577H78353
2.784T48691 3.33 H12796 2.896 AA039258 3.227N45476
4.06 W 16946 1.679 860831 4.245 T79534 2.799855692
3.071T85481 3.64 T85390 2.096 859467 3.196W31182
4.116T83199 2.606 N73091 3.168 H91401 2.371T91436
2.479H 19169 2.648 T56084 2.074 W24718 2.951T54610
3.768W37172 3.4 W37084 2.318 T54602 2.?35AA001324
2.146H70732 3.531 W07043 2.636 H65057 4.007H02412
4.475N75228 2.86 N44537 3.254 852482 3.547N92284
3.428W00630 3.546 T74332 2.377 H95979 2.257H78485
2.951N57398 2.2 835560 2.4 880523 3.216820594
3.62 W15521 3.945 869162 2.953 H79197 3.416H12419
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
61
3.262861036 2.337 T48694 3.711 885335 1.522 891771
2.572T68568 3.228 N31889 2.546 T56622 3.225 H09280
1.5 N40660 2.72 860420 3.107 AA205009 2.957 H63806
2.193N98743 3.234 AA004897 2.83 W00950 2.588 870441
3.464W 16685 2.893 824648 2.271 N73209 3 .093 R 19326
2.372T82120 2.187 AA063234 2.596 N31231
2.765T61346 3.766 896692 2.953 T85879
4.054AA214079 3.217 AA004891 2.655 832750
1.542AA164677 3.915 W00391 1.922 N32733
2.937A,A2117763.291 882770 3.495 W17002
3.067T87472 4.545 H 18766 2.984 W20484
3.058H61812 3.167 887818 1.377 AA136789
2.937N45602 2.214 W88806 2.364 H45241
2.927814907 3.318 T54086 1.678 W 17311
3.476H12508 2.992 H61280 3.688 887413
3.25 W19104 3.13 AA167039 3.432 814301
2.441887193 3.287 T87358 2.927 880475
2.521H92713 2.711 W03009 3.258 T79546
2.617832216 2.674 N98348 3.18 T78497
2.262AA126184 3.311 N47460 1.595 852015
3.281H09869 2.187 H73438
2.02 AA054041 2.751 W78830
2.639H66558 2.953 T49575
3.709807731 3.122 N47660
3.032T80874 3.435 H03226
3.673H51373 3.371 H42536
3.114T51726 3.049 T85153
3.137H09884 2.421 N53255
3.202T98755 2.379 870814
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
62
3.673 H51373 3.371 H42536
3.114 T51726 3.049 T85153
3.137 H09884 2.421 N53255
3.202 T98755 2.379 870814
3.201 AA194172 2.33 W47650
1.981 H13009 3.072 T78498
Table 4. Genes (identified by Genbank Accession Number) whose expression
was repressed in hypoxic cells, shown with the ratio of their expression in
oxic
cells over their expression in hypoxic cells.
ratio Acc. No. ratio Acc. No. ratio Acc. No. ratioAcc. No.
9.999 H38055 7.873 N33752 4.55 859009 9.999831317
6.948 AA057425 6.351 T54424 3.78 AA121166 6.082AA057398
9.999 AA116099 8.097 T87470 8.936 801823 6.504889643
9.999 AA057428 7.137 H06343 7.628 AA085375 7.172H70359
9.999 873197 4.887 AA058878 3.185 AA,054115 5.273N48132
9.999 848415 9.999 H46055 8.052 H96006 4.543AA054096
9.999 H 14999 6.651 T40066 5.324 T67226 5.207AA.054071
9.999 T96535 9.672 889521 5.895 862231 4.911AA078915
9.999 H27140 9.999 AA065190 4.767 AA112466 4.439H52742
9.999 T99054 7.455 H43837 4.271 N34169 3.092T67415
9:999 H46382 4.601 W81199 5.635 H51983 3.457849895
9.999 890757 9.999 H27344 6.176 N52679 4.953H16042
9.999 AA121402 9.999 H49310 6.465 H50403 6.039H45590
9.999 H38676 9.999 869813 4.812 H26200 6.767AA029349
7.52 AA057511 9.999 N32666 6.495 N30528 6.456H51981
9.999 N43944 2.046 AA053873 7.015 H47146 5.186861165
9.999 AA134018 9.999 AA125970 5.891 811999 2.541R85I83
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99148916 PCT/US99/06860
63
5.741H14566 4.712 H83338 5.442 N24303 4.764 H85692
9.999AA035019 9.999 894457 3.989 H 14332 5.288 DROS-Af
9.285N29018 4.545 N31674 6.206 864420 4.603 824904
7.813AA069149 5.6 T50828 6.203 837898 3.307 W72875
9.999876214 7.861 874161 6.251 876298 4.033 H71729
9.999823999 7.437 T99984 5.33 N98261 6.431 H 14193
9.999823880 5.5 848041 6.206 824405 8.731 N48042
9.999AA078826 7.565 H82390 4.865 H39089 4.063 H70778
7.739T92655 4.998 T54422 9.999 W72342 6.486 W24476
6.577W01565 6.317 H98046 4.976 T65484 9.999 W01642
9.59890884 7:321 N30514 3.745 H96724 7.103 T98068
5.535856663 5.137 H38881 9.999 H49809 3.784 AA10038i
8.157W00931 8.737 T99046 3.286 N57334 4.15 H60927
9.999H50385 5.465 H47440 8.214 851931 3.897 828248
8.922H28503 6.734 N42979 3.912 T79680 7.319 H56754
5.182H84008 6.802 W01319 3.824 H21568 4.617 892111
7.947W01051 7.5?2 895136 5.746 T80382 6.422 H94177
9.05866879 9.999 AA112231 9.999 887923 4.375 887352
9.999H45773 6.035 H53489 5.902 823778 6.6 831364
9.999AA126109 5.53 N94798 8.584 AA101044 3.713 AA05930:
9.999854784 6.009 N30964 3.638 AA112340 5.179 H51160
9.999N98857 9.999 N44142 8.09 890895 5.423 825798
9.999AA056159 7.759 W03129 6.3 N90458 4.448 863455
9.999877028 4.156 W03125 4.874 887886 6.596 T77139
9.999N36070 7.184 879618 4.621 N32679 4.909 863498
5.356889245 4.281 H38147 7.755 W02372 4.523 822272
9.999H51782 4.445 838004 4.624 H30637 6.954 H45355
9.999N48735 9.999 888190 8.387 801888 5.624 875964
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
64
5.379H86672 9.999 N41573 8.644 AA070426 4.742 N32681
9.999T95210 9.723 H52741 9.999 W31524 4.494 806552
7.355881942 4.401 T54426 6.574 H81786 9.097 887320
9.999N53883 4.914 AA054102 3.719 N40437 5.369 855451
7.685N24364 6.874 831243 7.48 N42402 7.075 884765
7.473H91761 7.618 AA113044 6.86 832757 4.058 H84844
x.537W 16945 5.394 896571 8.926 H21214 6.525 W21173
4.091DROS-A8 2.424 871723 4.111 H18154 1.955 AA05421
9.999AA115819 4.366 H86277 2.981 853678 3.214 N50075
x.996N36347 2.214 T53945 3.552 806539 3.359 T93912
5.587N30932 5.37 809668 4.474 H43816 4.156 T77415
2.24 880470 4.809 H40716 6.663 AA146629 2.779 AA04022'
2.746H16193 2.192 870132 4.402 801530 3.468 N26148
5.109AA004785 2.555 881899 1.884 H86896 2.744 826215
3.155N28396 4.968 N42806 4.013 868198 3.757 H58331
3.525AA047581 2.243 H66535 3.632 H16160 2.892 N34217
1.396H84204 4.049 846282 1.867 885333 2.987 AA05932~
5.795854918 5.522 849786 1.993 W 16980 3.314 823635
x.972878728 8.225 T67978 2.042 T60294 1.29 877994
1.684880601 3.362 881838 3.009 W32352 3.188 884692
7.734873050 5.364 H19572 1.589 W15194 3.837 N75231
1.832H12682 4.739 833409 2.503 H52012 4.043 AA04359~
6.87 N56601 3.959 AA069498 2.192 822397 2.645 822392
1.153880286 4.115 H44733 4.219 855158 3.933 811541
x.999N73428 5.33 AA029010 3.069 AA100975 3.244 H73503
?.754AA002135 9.999 AA113853 8.61 AA113299 2.909 AA05328~
?.948H39058 4.264 N31362 1.999 887373 3.059 H38003
1.235801799 2.793 820924 4.285 N45686 4.04 AA08838',
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/4$916 PCT/US99/06860
6.179809942 3.664 898517 3.87 W02494 4.006 H89896
1.849872766 4.656 N24477 3.84 873063 4:744 AA12688
2.963882691 4.443 AA032034 2.614 H 14441 6.651 AA08823
2.716883247 3.799 AA054203 4.806 AA058898 3.346 N59684
9.375AA070943 2.852 W95433 1.683 H89823 4.122 T92415
4.051N43796 5.221 N72527 4.392 AA037418 3.81 A.A08819i
4.754N46182 5.408 W02353 4.142 T83106 2.846 N32669
5.892832571 4.626 W01717 4.241 T81175 4.096 T5888I
3.03W72046 7.031 W79028 3.335 831471 2.097 T99924
5.486H85829 4.702 812648 3.253 886304 3.441 AA04682:
5.718875796 3.427 N32672 2.914 H86156 3.813 H85193
2.697N45640 3.989 N90836 4.004 H87311 3.134 891315
2.888H49806 3.692 N92684 2.595 T94530 3.132 T71293
5.419H52350 2.552 W06829 2.108 H27617 3.45 H79957
L515A.A0710992.537 H53375 1.565 H14143 3.198 AA05861:
3.535H51993 4.752 H67462 4.018 H49818 3.309 N34153
3.287AA069031 4.076 AA029883 1.677 897857 3.734 H61493
1.108879519 4.394 H12075 3.544 N9084I 1.687 856760
1.336N29042 3.253 T86612 3.901 AA076660 1.796 AA13275t
L075848060 9.23 T85558 4.49 N53295 2.934 H206I3
L343N77703 5.32 N29155 1.67 AA190622 6.358 AA12948t
1.491W56898 3.579 H44664 4.118 AA099ti47 3.07 H44888
?.202H28534 5.57 W 16814 4.007 AA069114 3.583 W04937
L449H62445 2.133 885191 2.606 W92014 2.521 AA04738t
1.196890798 2.161 H50471 4.047 W87655 4.349 852735
1.226835606 3.275 H30629 3.891 W01911 2.165 A,A06519:
3.224825899 4.284 880273 4.32 854194 4.009 N42453
x.999AA113119 4.063 H27411 2.085 N57249 3.688 W72149
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
66
4.2 N30471 4.231 H44693 3.472 874281 4.603 AA044942
i, T98994 2.471 W47021 4.044 AA054209 3.445 AA187560
5.466
2.577 AA075652 4.544 H69415 2.621 H62639 2.273 874076
'I2.448806926 2.425 AA181061 1.461 T78546 2.268 H65149
2.535 W45582 3.336 T97872 3.677 W40228 5.781 T48877
4.173 T820S4 3.553 H51540 6.08 AA101477 3.152 AA033945
3.359 T98110 2.63 N44493 3.907 W00916 3.032 H49111
2.703 886735 2.587 AA088784 1.846 N90969 3.741 H22503
2.77 AA045397 3.082 825304 3.006 H83363 2.252 N78086
2.893 853671 3.06 AA058600 4.484 W 17108 2.755 H71635
2.077 AA088407 2.741 H81782 1.482 N64281 2.673 N77126
2.213 T95166 3.466 H84604 2.899 AA045672 4.796 A.A083226
1.963 H27400 7.264 AA113169 2.994 H43746
5.333 T62191 3.859 N63192 2.65 891004
2.671 N32657 2.887 N29162 2.439 AA047858
3.374 T83919 1.926 828329 2.867 W21593
3.108 AA191189 3.17 AA172313 2.374 N34202
2.292 874157 2.057 834929 3.144 N80131
1.787 821095 4.695 W55902 2.458 AA043774
4.011 AA180772 2.179 813273 3.306 T56558
2.901 H82540 2.354 AA121148 3.896 AA205980
3.707 873398 5.805 AA088299 2.565 AA179841
2.658 H55982 2.233 H84617 2.973 H68783
1.826 H45128 2.011 AA059207 3.142 W30866
4.006 W25183 2.742 H39778 2.992 AA028961
4.299 T56400 3.034 H83022 2.489 826026
4.086 T95526 3.245 AA113900 5.751 AA085171
1.691 AA053901 2.716 819726 1.822 AA150817
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
67
Example 9. Analysis of Gene Expression under Hypoxia using GEMTM
microarrays
The hypoxic induction of genes in FaDu cells was analyzed by comparing
the expression of genes in FaDu cells exposed to hypoxic conditions (5% C02/S%
H2/90% N2 for 16 hours at 37 ° C) to those exposed to normal, oxic
conditions.
This differential expression was analyzed using GEMTM technology provided by
Genome Systems Inc. Messenger RNA (mRNA) was extracted from hypoxic
t o FaDu cells, and separately from oxic FaDu cells.
The total RNA was isolated from the cells essentially according to the
standard Genome Systems Inc. protocol, as follows. 500 pl Trizol was added 50-
100 mg of fresh frozen cells. The cells were then immediately homogenized. 500
~,1 Trizol was then added, and the sample was mixed well. The sample was
homogenized for five minutes at room temperature. Next, 0.2 ml chloroform was
added per 1 ml Trizol. The mixture was shaken vigorously for 15 seconds and
then allowed to incubate three minutes at room temperature. The sample was
then
centrifuged at 12,OOOX g for 15 minutes at 4°C. The aqueous phase was
transferred to a fresh centrifuge tube without disturbing the interphase. 0.5
ml of
2 o isopropanol was added and the samples were incubated for 10 minutes at
room
temperature. The RNA was pelleted by centrifuging at 12,OOOX g for 10 minutes
at 4°C. The supernatant was then removed. 1 ml of 75% ethanol was added
to the
pellet, which was then vortexed. This was followed by centrifugation at 7,SOOX
g
for S minutes at 4°C. The ethanol was removed. The pellet was dried for
10
2 5 minutes at room temperature and then dissolved in 10 p.l nuclease-free
water and
stored at -80°C.
Next, the poly A+ RNA was isolated from total RNA essentially according
to the standard Genome Systems Inc. protocol, as follows. To purify polyA RNA,
CA 02322843 2000-09-18
WO 99/48916 PCT/IJS99/06860
68
the total RNA sample was passed twice over OligoTex mRNA isolation columns
from Qiagen. After the elution of the polyA RNA, the polyA RNA was ethanol
precipitated, and the final product was brought up in DEPC H20 or TE. For 50
p,l
of elution from the OligoTex column, 40 pl of 1 X TE and 1 pl of glycogen (5
mg/ml) was added. Then 120 pl of 100% EtOH was added and the sample was
frozen at -80°C for 10 minutes. The sample was then spun at 12,000 x g
for 10
minutes at 4°C. The supernatant was removed and 250 pl of 75% EtOH was
added. The pellet was spun at 12,000 x g for 5 minutes at 4°C. The
supernatant
was again removed and the pellet dried for 10 minutes at room temperature. The
1o pellet was then dissolved in DEPC H20 to a concentration of SO ng/pl.
The purified RNA samples were sent to Genome Systems Inc. to perform a
GEM microarray analysis. From the mRNA samples, fluorescent labeled cDNA
probes were prepared by Genome Systems Inc. using standard methodologies
familiar to those skilled in the art. The cDNA probes corresponding to the
mRNA
sample from the oxic FaDu cells were labeled with a different, distinguishable
fluorescent label than the cDNA probes corresponding to the mRNA sample from
the hypoxic FaDu cells.
The two fluorescent probe samples (one from hypoxic FaDu cells, the other
from oxic FaDu cells) were then simultaneously applied by Genome Systems Inc.
2 o to their Human UniGEM V microarray for hybridization to the arrayed cDNA
molecules. The Human UniGEM V microarray contains sequence verified
Genome Systems Inc. proprietary cDNA clones representing more than 4,000
known human genes and up to 3,000 ESTs mapped to the UniGene database. (All
of the genes on the microarray were selected for criteria such as known
functions,
2 5 homologies, and presence on the human transcript map.) The genes or gene
fragments of the GEM microrarray (each 500-5000 base pairs in length) are
arrayed on glass surface to which they have been chemically bonded.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
69
Once the two fluorescent cDNA samples were sufficiently incubated with
the arrayed cDNA molecules to allow for hybridization to occur, the microarray
was washed free of probe molecules which had not hybridized. The different
gene/EST sites of the GEM microarray are then scanned for the each of the two
fluorescent labels. Presence of the fluorescent label at a particular gene
site
indicates the expression of that gene in the cell corresponding to that
fluorescent
label.
The 30 genes or ESTs which were determined on the microarray to have
the greatest level of induction in hypoxic cells (versus oxic cells) are
listed below
1 o in Table S, along with their levels of induction, functional category if
known, and
GenBank accession number.
Table 5. Genes Induced by Hypoacia in FaDu cells.
Functional Gene GenBan Induction
Category
fibroblast growth factor (FGF-3X 14445 5.6
)
Angiogenesisvascular endothelial growth XS4936 4.0
factor
(VEGF)
EPH receptor ligand MS7730 3.9
plasminogen activator inhibitorM14083 13.5
-1 (PAI-
1)
Tissue macrophage migration inhibitoryM2S639 9.3
factor
Remodeling (~F)
fibronectin receptor X062S6 6.0
lysyl hydroxylase-2 U84S73 4,g
endotheIin-2 M6S 199 4.4
B-cell translocation gene-1 (BTG-1) X61123 4.9
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
reducing agent and tunicamycin-D87953 3.8
responsive protein (RTP)
Cell Cycle CDC-like kinase (clk-1) L29219 3.0
quiescin (Q6) U97276 2.9
growth arrest DNA damage-inducibleL24498 2.9
protein 45 (GADD45)
Differentiation of Embryo AB0040 8.2
Chondrocytes-1 (DEC 1 ) 66
low density lipoprotein receptorX13916 6.0
(LDLR)
related protein
Miscellaneoushuman hairy gene homologue (HRHL 19314 4.7
adipophilin X97324 3.9
cyclooxygenase-1 (COX-1 ) U63 846 3 .0
fructose bisphosphatase AF0549 4.5
87
creatine transporter U36341, 4.1
U41163
Metabolism fatty acid binding protein M94856 3.9
glucose transporter-like proteinM20681 3.4
III
(GLUT-3 }
lactate dehyrogenase (LDH) X02152 2.9
insulin-like growth factor (IGFBP-3)M35878 11.1
Apoptosis Bcl-2-interacting killer (BIK) X89986 7.6
19 kDa-interacting protein 3 AB0047 5.3
long/Nip3-
like protein X (NipP3L/Nix) 88
Pim-1 M54915 4.4
EST AA0447 5.0
68
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
71
EST AA0545 4.7
43
KIAA0242 D87684 3.9
Unknown EST AA1562 3.5
40
EST AA1868 3.4
03
EST AA0630 3.0
84
EST H15429 2.9
EST U60873 2.9
Various modifications and variations of the present invention will be
apparent to those skilled in the art without departing from the scope and
spirit of
the invention. Although the invention has been described in connection with
s specific preferred embodiments, it should be understood that the invention
as
claimed should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the invention
which
are obvious to those skilled in the art are intended to be within the scope of
the
claims.
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
SEQUENCE LISTING
<110> Denko, Nicholas C
Giaccia, Amato J
Green. Christopher J
Laderoute, Keith R
Schindler, Cornelia
Koong. Albert C.
<120> Hypoxia-Inducible Human Genes, Proteins, and Uses
Thereof
<130> 15907-OOllP1
<140>
<14I>
<150> US 09/049,719
<151> 1998-03-27
<160> 20
<170> Patentln Ver. 2.0
<210> 1
<211> 1313
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (62)..(340)
<400> 1
cggaagccgg ttggggtgtg agaggttttc tcgctctagg gagattcttc aagcaatcac 60
t atg tca aca gac aca ggt gtt tcc ctt cct tca tat gag gaa gat cag 109
Met Ser Thr Asp Thr Gly Val Ser Leu Pro Ser Tyr Glu Glu Asp Gln
1 5 10 15
gqa tca aaa ctc att cga aaa get aaa gag gca cca ttc gta ccc gtt 157
Gly Ser Lys Leu Ile Arg Lys Ala Lys Glu Ala Pro Phe Val Pro Val
20 25 30
gga ata gcg ggt ttt gca gca att gtt gca tat gga tta tat aaa ctg 205
Gly Ile Ala Gly Phe Ala Ala Ile Val Ala Tyr Gly Leu Tyr Lys Leu
35 40 45
1
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
aag agc agg gga aat act aaa atg tcc att cat ctg atc cac atg cgt 253
Lys Ser Arg Gly Asn Thr Lys Met Ser Ile His Leu IIe His Met Arg
50 55 60
gtg gca gcc caa ggc ttt gtt gta gga gca atg act gtt ggt atg ggc 301
Val Ala Ala Gln Gly Phe Val Val Gly Ala Met Thr Val Gly Met Gly
65 70 ' 75 80
tat tcc atg tat cgg gaa ttc tgg gca aaa cct aag cct tagaagaaga 350
Tyr Ser Met Tyr Arg Glu Phe Trp Ala Lys Pro Lys Pro
85 90
gatgctgtct tggtcttgtt ggaggagctt gctttagtta gatgtcttat tattaaagtt 410
acctattatt gttggaaata aactaatttg tatgggttta gatggtaaca tggcattttg 470
aatattggct tcctttcttg caggcttgat ttgcttggtg accgaattac tagtgactag 530
tttactaact aggtcattca aggaagtcaa gttaacttaa acatgtcacc taaatgcact 590
tgatggtgtt gaaatgtcca ccttcttaaa tttttaagat gaacttagtt ctaaagaaga 650
taacaggcca atcctgaagg tactccctgt ttgctgcaga atgtcagata ttttggatgt 710
tgcataagag tcctatttgc cccagttaat tcaacttttg-tctgcctgtt ttgtggactg 770
gctggctctg ttagaactct gtccaaaaag tgcatggaat ataacttgta aagcttccca 830
caattgacaa tatatatgca tgtgtttaaa ccaaatccag aaagcttaaa caatagagct 990
gcataatagt atttattaaa gaatcacaac tgtaaacatg agaataactt aaggattcta 950
gtttagtttt ttgtaattgc aaattatatt tttgctgctg atatattaga ataattttta 1010
aatgtcatct tgaaatagaa atatgtattt taagcactca cgcaaaggta aatgaacacg 1070
ttttaaatgt gtgtgttgct aattttttcc ataagaattg taaacattga actgaacaaa 1130
ttacctataa tggatttggt taatgactta tgagcaagct ggtttggcca gacagtatac~1190
ccaaactttt atataatata cagaaggcta tcacacttgt gaaattctct tgtctaatct 1250
gaatttgcat tccatggtgt taacatggta tatgtattgt tattaaagta agtgacccat 1310
gtc 1313
<210> 2
2
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
<211> 93
<212> PRT
<213> Homo Sapiens
<400> 2
Met Ser Thr Asp Thr Gly Val Ser Leu Pro Ser Tyr Glu Glu Asp Gln
1 5 10 15
Gly Set Lys Leu Ile Arg Lys Ala Lys Glu Ala Pro Phe Val Pro Val
20 25 30
Gly ile Ala Gly Phe Ala Ala Ile Val Ala Tyr Gly Leu Tyr Lys Leu
35 40 45
Lya Ser Arg Gly Rsn Thr Lys Met Ser Ile His Leu Ile His Met Arg
50 55 60
Val Ala Ala Gln Gly Phe Val Val Gly Ala Met Thr Val Gly Met Gly
65 70 75 80
Tyr Ser Met Tyr Arg Glu Phe Trp Ala Lys Pro Lys Pro
85 90
<210>3
<211>1466
<212>DNA
<213>Homo sapiens
<220>
<221> CDS
<222> (274)..(462)
<400> 3
acaaaactgg agtccaccgc ggtggcggcc gctctagaat agtggatccc ccgggctgca 60
ggaattcggc acgagggcgc ttttgtctcc ggtgagtttt gtggcgggaa gcttctgcgc 120
tggtgcttag taaccgactt tcctccggac tcctgcacga cctgctccta cagccggcga 180
tccactcccg gctgttcccc cggagggtcc agaggccttt cagaaggaga aggcagctct 240
gtttctctgc agaggagtag ggtcctttca gcc atg aag cat gtg ttg aac ctc 294
Met Lys His Val Leu Asn Leu
1 5
tac ctg tta ggt gtg gta ctg acc cta ctc tcc atc ttc gtt aga gtg 342
Tyr Leu Leu Gly Val Val Leu Thr Leu Leu Ser Ile Phe Val Arg Val
3
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
15 20
atg gag tcc cta gaa ggc tta cta gag agc cca tcg cct ggg acc tcc 390
Met Glu Ser Leu Glu Gly Leu Leu Glu Ser Pro Ser Pro Gly Thr Ser
25 30 35
tgg acc acc aga agc caa cta gcc aac aca gag ccc acc aag ggc ctt 438
Trp Thr Thr Arg Ser Gln Leu Ala Asn Thr Glu Pro Thr Lys Gly Leu
40 45 50 55
cca gac cat cca tcc aga agc atg tgataagacc tccttccata ctggccatat 992
Pro Asp His Pro Ser Arg Ser Met
tttggaacac tgacctagac atgtccagat gggagtccca ttcctagcag acaagctgag 552
caccgttgta accagagaac tattactaqg ccttgaagaa cctgtctaac tggatgctca 612
ttgcctgggc aaggcctgtt taggccggtt gcggtggctc atgcctgtaa tcctagcact 672
ttgggaggct gaggtgggtg gatcacctga ggtcaggagt tcgagaccag cctcgccaac 732
atggcgaaac cccatctcta ctaaaaatac aaaagttagc tgggtgtggt ggcagaggcc 792
tgtaatccca gttccttggg aggctgaggc. gggagaattg-cttgaacccg gggacggagg 852
ttgcagtgaa ccgagatcgc actgctgtac ccagcctggg ccacagtgca agactccatc 9I2
tcaaaaaaaa aaagaaaaga aaaagcctgt ttaatgcaca ggtgtgagtg gattgcttat 972
ggctatgaga taggttgatc tcgcccttac cccggggtct ggtgtatgct gtgctttcct 1032
cagcagtatg gctctgacat ctcttagatg tcccaacttc agctgttggg agatggtgat 1092
attttcaacc ctacttccta aacatctgtc tggggttcct ttagtcttga atgtcttatg 1152
ctcaattatt tggtgttgag cctctcttcc acaagagctc ctccatgttt ggatagcagt 1212
tgaagaggtt gtgtgggtgg gctgttggga gtgaggatgg agtgttcagt gcccatttct 1272
cattttacat tttaaagtcg ttcctccaac atagtgtgta ttggtctgaa gggggtggtg 1332
ggatgccaaa gcctgctcaa gttatggaca ttgtggccac catgtggctt aaatgatttt 1392
ttctaactaa taaagtggaa tatatatttc aaaaaaaaaa aaaaaaaact cgaggggggc 1452
ccgtacccaa tcgc 1466
4
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
<210> 4
<211> 63
<212> ?RT
<213> !iomo Sapiens
<400> 9
Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr Leu
1 5 10 15
Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu Leu Glu
20 25 30
Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln Leu Ala Asn
35 40 45
Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro'Ser Arg Ser Met
50 55 60
<210> 5
<211> 444
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (63)..(347)
<400> 5
ggccagaaac cggcagactc ggaagggacc ccgcgtctcg gaagactctt caagaaatca 60
ca atg tca acc aac aca gac ctt tct ctc tct tca tac gat gaa ggt 107
Met Ser Thr Asn Thr Asp Leu Ser Leu Ser Ser Tyr Asp Glu Gly
1 5 10 15
cag ggg tct aag ttt att cgg aaa get aag gag aca ceg ttt gtc ccc 155
Gln Gly Ser Lys Phe Ile Arg Lys Ala Lys Glu Thr Pro Phe Val Pro
20 25. 30
att gga atg gcg ggc ttt gca gcg att gtt gcc tat ggg ttg tac aag 203
Ile Gly Met Ala Gly Phe Ala Ala Ile Val Ala Tyr Gly Leu Tyr Lys
35 90 45
ctg aag agc aga gga aat aca aag atg tcc att cac ttg atc cac atg 251
Leu Lys Ser Arg Gly Asn Thr Lys Met Ser Ile His Leu Ile His Met
50 55 60
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99106860
cgt gta gca gcc cag ggc ttt gtt gtg ggg gcc atg act ctt ggt atg 299
Arg Val Ala Ala Gln Gly Phe Val Val Gly A1a Met Thr Leu Gly Met
65 70 75
ggc tac tcc atg tat cag gaa ttc tgg gcc aac cct aag cct aag cct 34?
Gly Tyr Ser Met Tyr Gln Glu Phe Trp Ala Asn Pro Lys Pro Lys Pro
80 85 90 95
tagaagagct ggtggcatgg gaagtgcttg ctttagttag acgtctcata tt~gaggttac 407
gtgtttgtat ctacaataaa taacatgtgg gtttaga 444
<210> 6
<211> 95
<212> PAT
<213> Mus musculus
<400> 6
Met Ser Thr Aan Thr Asp Leu Ser Leu Ser Ser Tyr Asp .Glu Gly Gln
1 5 10 15
Gly Ser Lys Phe Ile Arg Lya Ala Lys Glu Thr Pro Phe Val Pro Ile
20 25 30
Gly Met Ala Gly Phe Ala Ala Ile Val Rla Tyr Gly Leu Tyr Lys Leu
35 40 45
Lys Ser Arg Gly Asn Thr Lya Met Ser Ile His Leu Ile His Met Arg
50 55 60
Val Ala Ala Gln Gly Phe Val Val Gly Ala Met Thr Leu Gly Met Gly
65 70 75 80
Tyr Ser Met Tyr Gln Glu Phe Trp Ala Asn Pro Lys Pro Lys Pro
85 90 g5
<210> 7
<211> 1521
<212> DNA
<213> Seriola quinqueradiata
<220>
<221> CDS
<222> (115)..(390)
<400> 7
6
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
cgtcaggcaa aattacttcc tccagactgt acgagggatc tgtggctcca aagactcata 60
aaataataat aattctttac agacgattca agagacactt cttaaacagt cagg atg 117
Met
1
act gcc tat gat gag aat gaa tcc aag tta atg cga aaa gta aag gag 165
Thr Ala Tyr Asp Glu Asn Glu Ser Lys Leu Met Arg Lys Val Lys Glu
10 15
aat cca ttt gtc cca gtg ggg att get gga ttc ttt gcc att gtt ggg 213
Asn Pro Phe Val Pro Val Gly Ile Ala Gly Phe Phe Ala Ile Val Gly
20 25 30
tic aga ctg atg aaa atg aaa aat cgg gga gac aca aaa atg tcg gta 261
Tyr Arg Leu Met Lys Met Lys Asn Arg Gly Asp Thr Lys Met Ser Val
35 40 45
cac ctg atc cac~atg cgt gta get gca caa ggc ttt gtg gtc gga gcc 309
His Leu Ile His Met Arg Val Ala Ala Gln Gly Phe Val Val Gly Ala
50 55 60 65
atg act gtt gga gtc ctg tat tca atg tac aga gat ttc att gta aaa 357
Met Thr Val Gly Val Leu Tyr Ser Met Tyr Arg Aap Phe Ile Val. Lys
70 75 80
ccc aga gaa gaa cag aaa tca atg caa aac aag tgaacaccac ctcttaccct 410
Pro Arg Glu Glu Gln Lys Ser Met Gln Asn Lys
85 90
ggtattttat gtcccttaat attacctcat attaaggtgt gtagagtgtt tatttttact 470
gatgggtcaa cttttatatg cacgcatcac tctagaagct cccctctact gtaaataccc 530
gtaacttatt gatcacactt gacatcttct aagtatcata ccagagggtc aagttgtcac 590
ttctgtattg agaaggagtt atattgatct cagctgtttt aacactggtt acactccttg 650
tgtttgcggt tataaacgta gctggtttga tatctgtgta gacagatgac attactgagt 710
gcagagtttt tagagcctct tattgtgatg tagtgtgtgt gaaatggcga gaagcctcga 770
atttacggca cacgtatcaa ttgtaaacac gtatgtccat ggcaaagtct ggattttaag 830
acaccccacc aattggcagg ttgcccaaca ggtttcttcc tccggccgga atttaatgtc 890
tcgtaccagc tatattgttt tatgtactaa tttaggaact ttttgccaaa taaaaaaatg 950
7
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
cttgcacctt agctcacttt tttaatgagc atcccagtgc attttgggca tcttgaggaa 1010
ggctttgaca acacttgact aacagacgaa acctaagctc ccacattgtt taaacaacta 1070
gaacacaaga ggtttttgac tcacaacagc atcattccat aaaacaacat tttaaaatca II30
tgacatgaaa ataggaaatg gaaaaaagga ctatgcatat ttcttgaccg aaactataaa 1190
gatctcttgt agaattaaaa tggattattt taatttggta cgctttccac aaaatgtctt 1250
tttttttttt tgacacaagg gggtccaata tttcaataga gcagcttcac aagccctcac 1310
gagaaatgta aaccaactga caccttcacc tgtacagact gtgcagtaat ctatagtata 1370
tcatcatata gcctaccttg tgataagctt aaatagatgc cttgttaagt tatacacaaa 1430
gttgaatttt gaatattgtg tgcaaataca gaagatgtta ttgaatgttt ttttttcctc 1490
tcgasataaa attgac~agt cttgtaattc t 1521
<210> 8
<211> 92
<212> PRT
<213> Seriola quinqueradiata
<400> 8
Met Thr Ala Tyr Asp Glu Asn Glu 5er Lys Leu Met Arg Lys Val Lys
1 5 10 15
Glu Asn Pro Phe Val Pro Val Gly Ile Ala Gly Phe Phe Ala Ile Val
20 25 30
Gly Tyr Arg Leu Met Lys Met Lys Asn Arg Gly Asp Thr Lys Met Ser
35 40 45
Val His Leu Ile His Met Arg Val Ala Ala Gln Gly Phe Val Val Gly
50 55 60
Ala Met Thr Val Gly Val Leu Tyr Ser Met Tyr Arg Asp Phe Ile Val
65' 70 75 80
Lys Pro Arg Glu Glu Gln Lys Ser Met Gln Asn Lys
85 90
<210> 9
<211> 857
8
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
<212> DNA
<213> Mus musculus
<220>
<22I> CDS
<222> (236)..(927)
<400> 9
tcggacgagg gcctcgcaga agggcgggct ttgggaggtc cgtttgtctt tggggcttat 60
ttctatccag agcagtgcct gcgtggagct tccacgttgc gactcagccg accttcttcc 120
ttactcctgc acgacctggt gtgactgtga gcagccgtct ctcaactttt ccttctgagg 180
atctagcagc agaaagcagc tctacttccc tgcaaaggag ctggqcaccg tcgcc atg 238
Met
1
aag ttc atg ctg aac ctc tat gtg ctg ggc atc atg ttg acc ctg ctt 286
Lys Phe Met Leu Asn Leu Tyr Val'Leu Gly Ile Met Leu Thr Leu Leu
10 15
tcc atc ttt gtt aga gtg atg gag tct ctg gga ggc tta.ctg gag agc 334
Ser Ile Phe Val Arg Val Met Glu Ser Leu Gly Gly Leu Leu Glu Ser
20 25 30
cca ctg ccc ggg agc tcc tgg atc acg agg ggt cag cta gcc aac aca 382
Pro Leu Pro Gly Ser Ser Trp Ile Thr Arg Gly Gln Leu Ala Asn Thr
35 40 45
cag cct cct aag ggc ctg cca gac cat cca tcc cga gga gtg cag 427
Gln Pro Pro Lys Gly Leu Pro Asp His Pro Ser Arg Gly Val Gln
50 55 60
tgaacctccc tccctgcagg catcacagct tcagcatgtc caaccacacg ttccatttct 48?
cgggaggcag catcaagtgt ctccaaagga ctcttactag gcctggaagg gctgttccct 547
taccctggaa aagagcctat ttcccctaga gctgtgagtg ggctgtctgt ggctctggga 607
tggaggtgta ccagttccag ctgtagggag aatggatttt ggtttcgttt gtttcagacc 667
tctgtcctaa aggactcttt tggacctaag tatcttctgt tggtttacca ttgagtctct 727
tccctgagag ttgtttggat ggcatcaaag gggttgtggt ttgactgtga agacagaggg 787
tggactatcc agtgtccagg tcaagttgta catttaagtt ctttctccag tgtaatgcac 847
9
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
atgtgtttgt 85?
<210> 10
<211> 64
<212> ?RT
<213> :bus musculus
<400> 10
Met Lys Phe Met Leu Asn Leu Tyr Val Leu Gly Ile Met Leu Thr Leu
1 5 10 15
Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Gly Gly Leu Leu Glu
20 25 30
Ser Pro Leu Pro Gly Ser Ser Trp Ile Thr Arg Gly Gln Leu Ala Asn
35 90 45
Thr Gln Pro Pro Lys Gly Leu Pro Asp His Pro Ser Arg Gly Val Gln
50 55 60
<210> 11
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
oligonucleotide
<400> 11
ttttaccagc ttattcaatt cggtcctctc gcacaggatq catg 44
<210> 12
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
oligonucleotide
<400> I2
catcctgtgc gagaggaccg aattgaataa gctggta 3?
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
<2I0> 13
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
oligonucleotide
<400> 13
tttttgtaga cattctagta tctcgtcaag tcggaaggat gcatg 45
<210> 14
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
oligonucleotide
<400> 14
catccttccg acttgacgag atactagaat gtctacaa 38
<210> 15
<211> 2I
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide PCR primer
<400> 15
ccagcttatt caattcggtc c 21
<210> 16
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide PCR primer
11
Substitute Sheet (Rule 26)
CA 02322843 2000-09-18
WO 99/48916 PCT/US99/06860
<400> 16
gtagacattc tagtatctcg t 21
<210> 17
<211> 32
<212> DNA
<213> Mus musculus
<400> I7
c~cgatctaga ggaagggacc ccgcgtctcg ga 32
<210> 18
<2I1> 34
<212> DNA
<213> Mus musculus
<400> 18
ggcgctcgag tctaaaccca catgttattt attg 34
<210> 19
<211> 21
<212> DNA
<213> Mus musculua
<400> 19
ccttactcct gcacgacctg g 21
<210> 20
<211> 32
<2I2> DNA
<213> Mus musculus
<400> 20
ggcgctcgag cacatgtgca ttacactgga ga 32
12
Substitute Sheet (Rule 26)
