Note: Descriptions are shown in the official language in which they were submitted.
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ESTs AND ENCODED HUMAN PROTEINS
Background of the Invention
[0001] The -estimated 50,000-100,000 genes scattered along the human
chromosomes offer
tremendous promise for the understanding, diagnosis, and treatment of human
diseases. In addition, probes
capable of specifically hybridizing to loci distributed throughout the human
genome find applications in the
construction of high resolution chromosome maps and in the identification of
individuals.
[0002] In the past, the characterization of even a single human gene was a
painstaking process,
requiring years of effort. Recent developments in the areas of cloning
vectors, DNA sequencing, and
computer technology have merged to greatly accelerate the rate at which human
genes can be isolated,
sequenced, mapped, and characterized.
[0003] Currently, two different approaches are being pursued for identifying
and characterizing
the genes distributed along the human genome. In one approach, large fragments
of genomic DNA are
isolated, cloned, and sequenced. Potential open reading frames in these
genomic sequences are identified
1 S using bioinformatics software. However, this approach entails sequencing
large stretches of human DNA,
which do not encode proteins, in order to find the protein encoding sequences
scattered throughout the
genome. In addition to requiring extensive sequencing, the bioinformatics
software may mischaracterize
the genomic sequences obtained, i.e., labeling non-coding DNA as coding DNA
and vice versa.
[0004] An alternative approach takes a more direct route to identifying and
characterizing human
genes. In this approach, complementary DNAs (cDNAs) are synthesized from
isolated messenger RNAs
(mRNAs) which encode human proteins. Using this approach, sequencing is only
performed on DNA
which is derived from protein coding portions of the genome. Often, only short
stretches of the cDNAs
are sequenced to obtain sequences called expressed sequence tags (ESTs). The
ESTs may then be used to
isolate or purify extended cDNAs which include sequences adjacent to the EST
sequences. The extended
cDNAs may contain all of the sequence of the EST which was used to obtain them
or only a portion of
the sequence of the EST which was used to obtain them. In addition, the
extended cDNAs may contain
the full coding sequence of the gene from which the EST was derived or,
alternatively, the extended
cDNAs may include portions of the coding sequence of the gene from which the
EST was derived. It will
be appreciated that there may be several extended cDNAs which include the EST
sequence as a result of
alternate splicing or the activity of alternative promoters. Alternatively,
ESTs having partially
overlapping sequences may be identified and contigs comprising the consensus
sequences of the
overlapping ESTs may be identified.
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[OOOSJ In the past, these short EST sequences were often obtained from oligo-
dT primed cDNA
libraries. Accordingly, they mainly corresponded to the 3' untranslated region
of the mRNA. In part, the
prevalence of EST sequences derived from the 3' end of the mRNA is a result of
the fact that typical
techniques for obtaining cDNAs, are not well suited for isolating cDNA
sequences derived from the 5' ends
of mRNAs (Adams et al., Nature 377:3-174, 1996, Hillier et al., Genome Res.
6:807-828, 1996).
[0006] In addition, in those reported instances where longer cDNA sequences
have been obtained,
the reported sequences typically correspond to coding sequences and do not
include the full 5' untranslated
region (5'UTR;) of the mRNA from which the cDNA is derived. Indeed, 5'UTRs
have been shown to affect
either the stability or translation of mRNAs. Thus, regulation of gene
expression may be achieved through
the use of alternative 5'UTRs as shown, for instance, for the translation of
the tissue inhibitor of
metalloprotease mRNA in mitogenically activated cells (Waterhouse et al, J
Biol Chem. 265:5585-9.
1990). Furthermore, modification of 5'UTR through mutation, insertion or
translocation events may even
be implied in pathogenesis. For instance, the fragile X syndrome, the most
common cause of inherited
mental retardation, is partly due to an insertion of multiple CGG
trinucleotides in the 5'UTR of the fragile
X mRNA resulting in the inhibition of protein synthesis via ribosome stalling
(Feng et al, Science
268:731-4, 1995). An aberrant mutation in regions of the 5'UTR known to
inhibit translation of the
proto-oncogene c-myc was shown to result in upregulation of C-myc protein
levels in cells derived from
patients with multiple myelomas (Willis et al, Curr Top Microbiol Immunol
224:269-76, 1997). In
addition, the use of oligo-dT primed cDNA libraries does not allow the
isolation of complete 5'UTRs since
such incomplete sequences obtained by this process may not include the first
exon of the mRNA, particularly
in situations where the first exon is short. Furthermore, they may not include
some exons, often short ones,
which are located upstream of splicing sites. Thus, there is a need to obtain
sequences derived from the 5'
ends of mRNAs.
[0007] While many sequences derived from human chromosomes have practical
applications,
approaches based on the identification and characterization of those
chromosomal sequences which encode a
protein product are particularly relevant to diagnostic and therapeutic uses.
In some instances, the sequences
used in such therapeutic or diagnostic techniques may be sequences which
encode proteins which are
secreted from the cell in which they are synthesized. Those sequences encoding
secreted proteins as well as
the secreted proteins themselves, are particularly valuable as potential
therapeutic agents. Such proteins are
often involved in cell to cell communication and may be responsible for
producing a clinically relevant
response in their target cells. In fact, several secretory proteins, including
tissue plasminogen activator, G-
CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon-,
interferon-, interferon-, and
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interleukin-2, are currently in clinical use. These proteins are used to treat
a wide range of conditions,
including acute myocardial infarction, acute ischemic stroke, anemia,
diabetes, growth hormone deficiency,
hepatitis, kidney carcinoma, chemotherapy-induced neutropenia and multiple
sclerosis. For these reasons,
extended cDNAs encoding secreted proteins or portions thereof represent a
valuable source of therapeutic
agents. Thus, there is a need for the identification and characterization of
secreted proteins and the nucleic
acids encoding them.
[0008] In addition to being therapeutically useful themselves, secretory
proteins include short
peptides, called signal peptides, at their amino termini which direct their
secretion. These signal peptides are
encoded by the signal sequences located at the 5' ends of the coding sequences
of genes encoding secreted
proteins. These signal peptides can be used to direct the extracellular
secretion of any protein to which they
are operably linked. In addition, portions of the signal peptides called
membrane-translocating sequences
may also be used to direct the intracellular import of a peptide or protein of
interest. This may prove
beneficial in gene therapy strategies in which it is desired to deliver a
particular gene product to cells other
than the cells in which it is produced. Signal sequences encoding signal
peptides also find application in
simplifying protein purification techniques. In such applications, the
extracellular secretion of the desired
protein greatly facilitates purification by reducing the number of undesired
proteins from which the desired
protein must be selected. Thus, there exists a need to identify and
characterize the S' portions of the genes for
secretory proteins which encode signal peptides.
[0009] Sequences coding for non-secreted proteins may also find application as
therapeutics or
diagnostics. In particular, such sequences may be used to determine whether an
individual is likely to express
a detectable phenotype, such as a disease, as a consequence of a mutation in
the coding sequence of a protein.
In instances where the individual is at risk of suffering from a disease or
other undesirable phenotype as a
result of a mutation in such a coding sequence, the undesirable phenotype may
be corrected by introducing a
normal coding sequence using gene therapy. Alternatively, if the undesirable
phenotype results from
overexpression of the protein encoded by the coding sequence, expression of
the protein may be reduced
using antisense or triple helix based strategies.
[0010] The secreted or non-secreted human polypeptides encoded by the coding
sequences may
also be used as therapeutics by administering them directly to an individual
having a condition, such as a
disease, resulting from a mutation in the sequence encoding the polypeptide.
In such an instance, the
condition can be cured or ameliorated by administering the polypeptide to the
individual.
[0011] In addition, the secreted or non-secreted human polypeptides or
portions thereof may be
used to generate antibodies useful in determining the tissue type or species
of origin of a biological sample.
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For example, to distinguish between human and non-human cells and tissues or
to distinguish between
human tissues that do and do not express the polypeptides. The antibodies may
also be used to determine the
cellular localization of the secreted or non-secreted human polypeptides or
the cellular localization of
polypeptides which have been fused to the human polypeptides. In addition, the
antibodies may also be used
in immunoaffinity chromatography techniques to isolate, purify, or enrich the
human polypeptide or a target
polypeptide which has been fused to the human polypeptide.
[0012] Public information on the number of human genes for which the promoters
and upstream
regulatory regions have been identified and characterized is quite limited. In
part, this may be due to the
difficulty of isolating such regulatory sequences. Upstream regulatory
sequences such as transcription factor
binding sites are typically too short to be utilized as probes for isolating
promoters from human genomic
libraries. Recently, some approaches have been developed to isolate human
promoters. One of them consists
of making a CpG island library (Cross et al., Nature Genetics 6: 236-244,
1994). The second consists of
isolating human genomic DNA sequences containing SpeI binding sites by the use
of SpeI binding protein
(Mortlock et al., Genome Res. 6:327-335, 1996). Both of these approaches have
their limits due to a lack of
1 S specificity and of comprehensiveness. Thus, there exists a need to
identify and systematically characterize
the 5' portions of the genes.
[0013] The present 5' ESTs may be used to efficiently identify and isolate
5'UTRs and upstream
regulatory regions which control the location, developmental stage, rate, and
quantity of protein synthesis, as
well as the stability of the mRNA. Once identified and characterized, these
regulatory regions may be
utilized in gene therapy or protein purification schemes to obtain the desired
amount and locations of protein
synthesis or to inhibit, reduce, or prevent the synthesis of undesirable gene
products. The regulatory regions
may also be used for expressing polypeptides in cell types from which the 5'
EST of the present invention
were isolated.
[0014] In addition, ESTs containing the S' ends of protein genes may include
sequences useful as
probes for chromosome mapping and the identification of individuals. Thus,
there is a need to identify and
characterize the sequences upstream of the 5' coding sequences of genes.
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Summary of the Invention
[0014] The present invention relates to purified, isolated, or enriched 5'
ESTs which include
sequences derived from the authentic 5' ends of their corresponding mRNAs. The
terns "corresponding
mRNA" refers to the.mRNA which was the template for the cDNA synthesis which
produced the S' EST.
These sequences will be referred to hereinafter as "5' ESTs". The present
invention also includes purified,
isolated or enriched nucleic acids comprising contigs assembled by determining
a consensus sequences from
a plurality of ESTs containing overlapping sequences. These contigs will be
referred to herein as "consensus
contigated ESTs."
[0015] As used herein, the term "purified" does not require absolute purity;
rather, it is intended as a
relative definition. Individual 5' EST clones isolated from a cDNA library
have been conventionally purified
to electrophoretic homogeneity. The sequences obtained from these clones could
not be obtained directly
either from the library or from total human DNA. The cDNA clones are not
naturally occurring as such, but
rather are obtained via manipulation of a partially purified naturally
occurring substance (messenger RNA).
The conversion of mRNA into a cDNA library involves the creation of a
synthetic substance (cDNA) and
pure individual cDNA clones can be isolated from the synthetic library by
clonal selection. Thus, creating a
cDNA library from messenger RNA and subsequently isolating individual clones
from that library results in
an approximately 104-106 fold purification of the native message. Purification
of starting material or natural
material to at least one order of magnitude, preferably two or three orders,
and more preferably four or five
orders of magnitude is expressly contemplated. Alternatively, purification may
be expressed as "at least" a
percent purity relative to heterologous polynucleotides (DNA, RNA or both). As
a preferred embodiment,
the polynucleotides of the present invention are at least; 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%,
95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologous
polynucleotides. As a further
preferred embodiment the polynucleotides have an "at least" purity ranging
from any number, to the
thousandth position, between 90% and 100% (e.g., S' EST at least 99.995% pure)
relative to heterologous
polynucleotides. Additionally, purity of the polynucleotides may be expressed
as a percentage (as described
above) relative to all materials and compounds other than the carrier
solution. Each number, to the
thousandth position, may be claimed as individual species of purity.
[0016] As used herein, the term "isolated" requires that the material be
removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a naturally-occurring
polynucleotide present in a living animal is not isolated, but the same
polynucleotide, separated from some or
all of the coexisting materials in the natural system, is isolated.
Specifically excluded from the definition of
"isolated" are: naturally occurring chromosomes (e.g., chromosome spreads)
artificial chromosome libraries,
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genomic libraries, and cDNA libraries that exist either as an in vitro nucleic
acid preparation or as a
transfected/transformed host cell preparation, wherein the host cells are
either an in vitro heterogeneous
preparation or plated as a heterogeneous population of single colonies. Also
specifically excluded are the
above libraries wherein a specified 5' EST makes up less than 5% of the number
of nucleic acid inserts in the
S vector molecules. Further specifically excluded are whole cell genomic DNA
or whole cell RNA
preparations (including said whole cell preparations which are mechanically
sheared or enzymaticly
digested). Further specifically excluded are the above whole cell preparations
as either an in vitro preparation
or as a heterogeneous mixture separated by electrophoresis (including blot
transfers of the same) wherein the
polynucleotide of the invention has not further been separated from the
heterologous polynucleotides in the
electrophoresis medium (e.g., further separating by excising a single band
from a heterogeneous band
population in an agarose gel or nylon blot).
[0017] As used herein, the term "recombinant" means that the 5' EST is
adjacent to "backbone"
nucleic acid to which it is not adjacent in its natural environment.
Additionally, to be "enriched" the 5' ESTs
will represent S% or more of the number of nucleic acid inserts in a
population of nucleic acid backbone
molecules. Backbone molecules according to the present invention include
nucleic acids such as expression
vectors, self replicating nucleic acids, viruses, integrating nucleic acids,
and other vectors or nucleic acids
used to maintain or manipulate a nucleic acid insert of interest. Preferably,
the enriched 5' ESTs represent
15% or more of the number of nucleic acid inserts in the population of
recombinant backbone molecules.
More preferably, the enriched 5' ESTs represent 50% or more of the number of
nucleic acid inserts in the
population of recombinant backbone molecules. In a highly preferred
embodiment, the enriched 5' ESTs
represent 90% or more (including any integer between 90 and 100%, to the
thousandth position, e.g., 99.5%)
of the number of nucleic acid inserts in the population of recombinant
backbone molecules.
[0018] "Stringent", "moderate," and "low" hybridization conditions are as
defined below.
[0019] The term "polypeptide" refers to a polymer of amino acids without
regard to the length of
the polymer; thus, peptides, oligopeptides, and proteins are included within
the definition of polypeptide.
This term also does not specify or exclude chemical or post-expression
modifications of the polypeptides
of the invention, although chemical or post-expression modifications of these
polypeptides may be
included excluded as specific embodiments. Therefore, for example,
modifications to polypeptides which
include the covalent attachment of glycosyl groups, acetyl groups, phosphate
groups, lipid groups and the
like are expressly encompassed by the term polypeptide. Further, polyeptides
with these modifications,
described herein, may be specified as individual species to be included or
excluded from the present
invention. The natural or other chemical modifications, such as those listed
in example above, can occur
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anywhere in a polypeptide, including the peptide backbone, the amino acid side-
chains and the amino or
carboxyl termini. It will be appreciated that the same type of modification
may be present in the same or
varying degrees at several sites in a given polypeptide. Also, a given
polypeptide may contain many
types of modifications. Polypeptides may be branched, for example, as a result
of ubiquitination, and
they may be cyclic, with or without branching. Modifications include
acetylation, acylation, ADP-
ribosylation, arnidation, covalent attachment of flavin, covalent attachment
of a heme moiety, covalent
attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation,
demethylation, formation of covalent cross-links, formation of cysteine,
formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination,
methylation, myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids to proteins such as
arginylation, and ubiquitination. (See, for instance, PROTEINS - S'TRUCTURE
AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York
(1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,
Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol
182:626-646 (1990); Rattan et
al., Ann NY Acad Sci 663:48-62 (1992).). Also included within the definition
are polypeptides which
contain one or more analogs of an amino acid (including, for example, non-
naturally occurring amino
acids, amino acids which only occur naturally in an unrelated biological
system, modified amino acids
from mammalian systems etc.), polypeptides with substituted linkages, as well
as other modifications
known in the art, both naturally occurring and non-naturally occurring.
[0020] As used interchangeably herein, the terms "nucleic acids",
"oligonucleotides", and
"polynucleotides" include RNA or DNA (either double or single stranded (coding
or antisense), or
RNA/DNA hybrid sequences of more than one nucleotide in either single chain or
duplex form (although
each of the above species may be particularly specified). The term
"nucleotide" as used herein as an
adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid
sequences of any length in
single-stranded or duplex form. The term "nucleotide" is also used herein as a
noun to refer to individual
nucleotides or varieties of nucleotides, meaning a molecule, or individual
unit in a larger nucleic acid
molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar
moiety, and a phosphate
group, or phosphodiester linkage in the case of nucleotides within an
oligonucleotide or polynucleotide.
Although the term "nucleotide" is also used herein to encompass "modified
nucleotides" which comprise
at least one modifications (a) an alternative linking group, (b) an analogous
form of purine, (c) an
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analogous form of pyrimidine, or (d) an analogous sugar, for examples of
analogous linking groups,
purine, pyrimidines, and sugars see for example PCT publication No. WO
95/04064. Preferred
modifications of the present invention include, but are not limited to, 5-
fluorouracil, 5-bromouracil, 5-
chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenme, t-methylguanme, t-
metnymosme,
dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-
mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-
isopentenyladenine, uracil-5-oxyacetic acid (v) ybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
oxyacetic acid methylester, uracil-
S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)
uracil, and 2,6-diaminopurine.
Methylenemethylimino linked oligonucleosides as well as mixed backbone
compounds having, may be
prepared as described in U.S. Pat. Nos. 5,378,825; 5,386,023; 5,489,677;
5,602,240; and 5,610,289.
1 S Formacetal and thioformacetal linked oligonucleosides may be prepared as
described in U.S. Pat. Nos.
5,264,562 and 5,264,564. Ethylene oxide linked oligonucleosides may be
prepared as described in U.S.
Pat. No. 5,223,618. Phosphinate oligonucleotides may be prepared as described
in U.S. Pat. No.
5,508,270. Alkyl phosphonate oligonucleotides may be prepared as described in
U.S. Pat. No. 4,469,863.
3'-Deoxy-3'-methylene phosphonate oligonucleotides may be prepared as
described in U.S. Pat. Nos.
5,610,289 or 5,625,050. Phosphoramidite oligonucleotides may be prepared as
described in U.S. Pat. No.
5,256,775 or U.S. Pat. No. 5,366,878. Alkylphosphonothioate oligonucleotides
may be prepared as
described in published PCT applications WO 94/17093 and WO 94/02499. 3'-Deoxy-
3'-amino
phosphoramidate oligonucleotides may be prepared as described in U.S. Pat. No.
5,476,925.
Phosphotriester oligonucleotides may be prepared as described in U.S. Pat. No.
5,023,243. Borano
phosphate oligonucleotides may be prepared as described in U.S. Pat. Nos.
5,130,302 and 5,177,198.
[0021] The polynucleotide sequences of the invention may be prepared by any
known method,
including synthetic, recombinant, ex vivo generation, or a combination
thereof, as well as utilizing any
purification methods known in the art.
(0022] In specific embodiments, the polynucleotides of the invention are at
least 15, at least 30,
at least 50, at least 100, at least 125, at least 500, or at least 1000
continuous nucleotides but are less than
or equal to 300kb, 200kb, 100kb, SOkb, lOkb, 7.Skb, Skb, 2.Skb, 2kb, I.Skb, or
lkb in length. In a further
embodiment, polynucleotides of the invention comprise a portion of the coding
sequences, as disclosed
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herein, but do not comprise all or a portion of any intron. In another
embodiment, the polynucleotides
comprising coding sequences do not contain coding sequences of a genomic
flanking gene (i.e., 5' or 3' to
the gene of interest in the genome). In other embodiments, the polynucleotides
of the invention do not
contain the coding sequence of more than 1000, 500, 250, 100, 75, 50, 25, 20,
15, 10, 5, 4, 3, 2, or 1
genomic flanking gene(s).
[0023] The terms "base paired" and "Watson & Crick base paired" are used
interchangeably
herein to refer to nucleotides which can be hydrogen bonded to one another be
virtue of their sequence
identities in a manner like that found in double-helical DNA with thymine or
uracil residues linked to
adenine residues by two hydrogen bonds and cytosine and guanine residues
linked by three hydrogen
bonds (See Stryer, L., Biochemistry, 4"' edition, 1995).
[0024] The terms "complementary" or "complement thereo:P' are used herein to
refer to the
sequences of polynucleotides which is capable of forming Watson & Crick base
pairing with another
specified polynucleotide throughout the entirety of the complementary region.
For the purpose of the
present invention, a first polynucleotide is deemed to be complementary to a
second polynucleotide when
each base in the first polynucleotide is paired with its complementary base.
Complementary bases are,
generally, A and T (or A and U), or C and G. "Complement" is used herein as a
synonym from
"complementary polynucleotide", "complementary nucleic acid" and
"complementary nucleotide
sequence". These terms are applied to pairs of polynucleotides based solely
upon their sequences and not
any particular set of conditions under which the two polynucleotides would
actually bind. Preferably, a
"complementary" sequence is a sequence which an A at each position where there
is a T on the opposite
strand, a T at each position where there is an A on the opposite strand, a G
at each position where there is a C
on the opposite strand and a C at each position where there is a G on the
opposite strand.
[0025] The terms "vertebrate nucleic acid" and "vertebrate polypeptide" are
used herein to refer to
any nucleic acid or polypeptide respectively which are derived from a
vertebrate species including birds and
more usually mammals, preferably primates such as humans, farm animals such as
swine, goats, sheep,
donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used
herein, the term
"vertebrate" is used to refer to any vertebrate, preferably a mammal. The term
"vertebrate" expressly
embraces human subjects unless preceded with the term "non-human".
[0026] Thus, S' ESTs in cDNA libraries in which one or more 5' ESTs make up 5%
or more of the
number of nucleic acid inserts in the backbone molecules are "enriched
recombinant 5' ESTs" as defined
herein. Likewise, 5' ESTs in a population of plasmids in which one or more 5'
ESTs of the present invention
have been inserted such that they represent 5% or more of the number of
inserts in the plasmid backbone are
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"enriched recombinant 5' ESTs" as defined herein. However, 5' ESTs in cDNA
libraries in which S' ESTs
constitute less than 5% of the number of nucleic acid inserts in the
population of backbone molecules, such as
libraries in which backbone molecules having a S' EST insert are extremely
rare, are not "enriched
recombinant 5' ESTs."
[0027] The term "capable of hybridizing to the polyA tail of said mRNA" refers
to and embraces all
primers containing stretches of thymidine residues, so-called oligo(dT)
primers, that hybridize to the 3' end of
eukaryotic poly(A)+ mRNAs to prime the synthesis of a first cDNA strand.
Techniques for generating said
oligo(dT) primers and hybridizing them to mRNA to subsequently prime the
reverse transcription of said
hybridized mRNA to generate a first cDNA strand are well known to those
skilled in the art and are described
in Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1997 and
Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989, the entire
disclosures of which are incorporated herein by reference. Preferably, said
oligo(dT) primers are present in a
large excess in order to allow the hybridization of all mRNA 3'ends to at
least one oligo(dT) molecule. The
priming and reverse transcription steps are preferably performed between
37°C and 55°C depending on the
type of reverse transcriptase used.
[0028] Preferred oligo(dT) primers for priming reverse transcription of mRNAs
are
oligonucleotides containing a stretch of thymidine residues of sufficient
length to hybridize specifically to the
polyA tail of mRNAs, preferably of 12 to 18 thymidine residues in length. More
preferably, such oligo(T)
primers comprise an additional sequence upstream of the poly(dT) stretch in
order to allow the addition of a
given sequence to the 5'end of all first cDNA strands which may then be used
to facilitate subsequent
manipulation of the cDNA. Preferably, this added sequence is 8 to 60 residues
in length. For instance, the
addition of a restriction site in 5' of cDNAs facilitates subcloning of the
obtained eDNA. Alternatively, such
an added 5'end may also be used to design primers of PCR to specifically
amplify cDNA clones of interest.
[0029] In some embodiments, the present invention relates to 5' ESTs which are
derived from
genes encoding secreted proteins. As used herein, a "secreted" protein is one
which, when expressed in a
suitable host cell, is transported across or through a membrane, including
transport as a result of signal
peptides in its amino acid sequence. "Secreted" proteins include without
limitation proteins secreted wholly
(e.g. soluble proteins), or partially (e.g. receptors) from the cell in which
they are expressed. "Secreted"
proteins also include without limitation proteins which are transported across
the membrane of the
endoplasmic reticulum.
[0030] Such 5' ESTs include nucleic acid sequences, called signal sequences,
which encode signal
peptides which direct the extracellular secretion of the proteins encoded by
the genes from which the 5' ESTs
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are derived. Generally, the signal peptides are located at the amino termini
of secreted proteins. Polypeptides
comprising these signal peptides (as delineated in the sequence listing), and
polynucleotides encoding the
same, are preferred embodiments of the present invention.
[0031] Secreted proteins are translated by ribosomes associated with the
"rough" endoplasmic
reticulum. Generally, secreted proteins are co-translationally transferred to
the membrane of the endoplasmic
reticulum. Association of the ribosome with the endoplasmic reticulum during
translation of secreted
proteins is mediated by the signal peptide. The signal peptide is typically
cleaved following its co
translational entry into the endoplasmic reticulum. After delivery to the
endoplasmic reticulum, secreted
proteins may proceed through the Golgi apparatus. In the Golgi apparatus, the
proteins may undergo post
translational modification before entering secretory vesicles which transport
them across the cell membrane.
[0032] The 5' ESTs of the present invention have several important
applications. For example, the
5'EST sequences of the sequence listing, and fragments thereof, may be used to
distinguish human tissues or
cells from non-human tissues or cells and to distinguish between human tissues
or cells that do and do not
express polynucleotides comprising the 5' EST sequences of the present
invention. By knowing the tissue
expression pattern of the 5' EST sequences, either through routine
experimentation or by using the Tables
herein, the polynucleotides of the present invention may be used in methods of
determining the identity of an
unknown tissue or cell sample. For example, if a 5' EST is expressed in a
particular tissue or cell type, as
shown in the Tables below, and the unknown tissue or cell sample does not
express the 5' EST, it may be
inferred that the unknown tissue or cells are either not human or not the same
human tissue or cell type as that
which expresses the 5' EST. Conversely, if a 5' EST is not expressed in a
particular tissue or cell type, as
shown in the Tables below, and the unknown tissue or cell sample does express
the 5' EST, it may be inferred
that the unknown tissue or cells are either not human or not the same human
tissue or cell type as that which
does not express the 5' EST. The above procedure may be used for either
homogeneous tissue or cell
samples or heterogeneous tissue or cell samples since one may only want to
narrow the identity to human or
non-human or to a tissue type. Further assays may be used in conjunction with
the above methods to narrow
or confirm the identification process. These methods of determining tissue or
cell identity are based on
methods which detect the presence or absence of the 5' EST sequences in a
tissue or cell sample using
methods well know in the art (e.g., hybridisation or PCR methods).
[0033] In other useful applications, fragments of the 5' EST sequences
encoding signal peptides as
well as degenerate polynucleotides encoding the same, may be ligated to
sequences encoding either the
polypeptide from the same gene or to sequences encoding a heterologous
polypeptide to facilitate secretion.
The 5'EST sequences, and fragments thereof, may also be used to obtain and
express cDNA clones which
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include the full protein coding sequences of the corresponding gene products,
including the authentic
translation start sites derived from the 5' ends of the coding sequences of
the mRNAs from which the 5'
ESTs are derived. These cDNAs will be referred to hereinafter as "full-length
cDNAs." These cDNAs may
also include DNA derived from mRNA sequences upstream of the translation start
site. The full-length
cDNA sequences may be used to express the proteins corresponding to the 5'
ESTs. As discussed above,
secreted proteins and non-secreted proteins may be therapeutically important.
Thus, the proteins expressed
from the cDNAs may be useful in treating or controlling a variety of human
conditions. The 5' ESTs may
also be used to obtain the corresponding genomic DNA. The term "corresponding
genomic DNA" refers to
the genomic DNA which encodes the mRNA from which the 5' EST was derived.
[0034] Another use of the polynucleotides of the present invention is to map
and clone promoter
regions and open reading frames from a genomic sequence. For example, the 5'
ESTs can be used in
combination with the sequence information from genome sequencing projects,
such as the U.S. Human
Genome Project or other public and private genome sequencing projects, to map
and clone regions of the
genome that comprise promoters and expressed open reading frames. The
polynucleotides of the present
invention are particularly useful for mapping and identifying coding regions
(regions containing expressed
open reading frames) from a genomic sequence since the vast majority of the
human genome does not encode
expressed genes and because of the difficulty in identifying authentic open
reading frames (open reading
frames that encode expressed genes). The 5' EST sequences of the present
invention can be used in
conjunction with various algorithms to identify promoter or entire ORF
sequences.
[0035] Alternatively, the 5' ESTs may be used to obtain and express extended
cDNAs encoding
portions of the protein. In the case of secreted proteins, the portions may
comprise the signal peptides of the
secreted proteins or the mature proteins generated when the signal peptide is
cleaved off.
[0036] T'he present invention includes isolated, purified, or enriched "EST-
related nucleic acids."
The terms "isolated", "purified" or "enriched" have the meanings provided
above. As used herein, the term
"EST-related nucleic acids" means the nucleic acids of SEQ )D NOs. 24-13309
and 26596-52153, extended
cDNAs obtainable using the nucleic acids of SEQ >D NOs. 24-13309 and 26596-
52153, full-length cDNAs
obtainable using the nucleic acids of SEQ )D NOs. 24-13309 and 26596-52153 or
genomic DNAs obtainable
using the nucleic acids of SEQ )D NOs. 24-13309 and 26596-52153. The present
invention also includes the
sequences complementary to, or allelic variants of (including single
nucleotide polymorphisms or SNPs), the
EST-related nucleic acids.
[0037] The present invention also includes isolated, purified, or enriched
"fragments of EST-related
nucleic acids." The terms "isolated", "purified" and "eruiched" have the
meanings described above. As used
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herein the term "fragments of EST-related nucleic acids" means fragments
comprising at least 8, 10, 12, 15,
18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300, 500, or 1000
consecutive nucleotides of the EST-related
nucleic acids to the extent that fragments of these lengths are consistent
with the lengths of the particular
EST-related nucleic acids being referred to. The present invention also
includes the sequences
complementary to the fragments of the EST-related nucleic acids. In a
preferred embodiment, fragments of
EST-related nucleic acids refer to the polynucleotides described in Tables IVa
and IVb, and polynucleotides
described in Tables IVa and IVb updated as defined below.
[0038] The present invention also includes isolated, purified, or enriched
"positional segments of
EST-related nucleic acids." The terns "isolated", "purified", or "enriched"
have the meanings provided
above. As used herein, the term "positional segments of EST-related nucleic
acids" includes segments
comprising nucleotides 1-25, 26-50, 51-75, 76-100, 101-125, 126-150, 151-175,
176-200, 201-225, 226-250,
251-300, 301-325, 326-350, 351-375, 376-400, 401-425, 426-450, 451-475, 476-
500, 501-525, 526-550,
551-575, 576-600 and 601-the terminal nucleotide of the EST-related nucleic
acids to the extent that such
nucleotide positions are consistent with the lengths of the particular EST-
related nucleic acids being referred
to, and wherein position "1" is defined as the 5' most position defined in the
sequence listing or Tables
below. The term "positional segments of EST-related nucleic acids also
includes segments comprising
nucleotides 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-
400, 401-450, 450-500, 501-
550, 551-600 or 601-the terminal nucleotide of the EST-related nucleic acids
to the extent that such
nucleotide positions are consistent with the lengths of the particular EST-
related nucleic acids being referred
to. The term "positional segments of EST-related nucleic acids" also includes
segments comprising
nucleotides 1-100, 101-200, 201-300, 301-400, 501-500, 500-600, or 601-the
terminal nucleotide of the EST-
related nucleic acids to the extent that such nucleotide positions are
consistent with the lengths of the
particular EST-related nucleic acids being referred to. In addition, the term
"positional segments of EST-
related nucleic acids" includes segments comprising nucleotides 1-200, 201-
400, 400-600, or 601-the
terminal nucleotide of the EST-related nucleic acids to the extent that such
nucleotide positions are consistent
with the lengths of the particular EST related nucleic acids being referred
to. The present invention also
includes the sequences complementary to the positional segments of EST-related
nucleic acids.
[0039] The present invention also includes isolated, purified, or enriched
"fragments of positional
segments of EST-related nucleic acids." The terms "isolated", "purified", or
"enriched" have the meanings
provided above. As used herein, the terns "fragments of positional segments of
EST-related nucleic acids"
refers to fragments comprising at least 8, 10, 15, 18, 20, 23, 25, 28, 30, 35,
40, 50, 75, 100, 150, or 200
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consecutive nucleotides of the positional segments of EST-related nucleic
acids. The present invention also
includes the sequences complementary to the fragments of positional segments
of EST-related nucleic acids.
[0040] In addition to the above "positional segments of EST-related nucleic
acids" and "fragments
of positional segments of EST-related nucleic acids", for the nucleic acids of
SEQ >D NOs. 24-13309 and
26596-52153, further preferred nucleic acids comprise at least 8 nucleotides,
wherein "at least 8" is defined
as any integer between 8 and the integer representing the 3' most nucleotide
position in the sequence listing
or Tables below. Further included are nucleic acid fragments at least 8
nucleotides in length, as described
above, that are further specified in terms of their 5' and 3' position. The 5'
and 3' positions are represented
by the positional numbering set forth in the sequence listing below.
Therefore, every combination of a 5' and
3' nucleotide position that a fragment at least 8 contiguous nucleotides in
length could occupy is included in
the invention as an individual species. The polynucleotide fragments specified
by 5' and 3' positions can be
immediately envisaged and are therefore not individually listed solely for the
purpose of not unnecessarily
lengthening the specifications. It is noted that the above species of
polynucleotide fragments of the present
invention may alternatively be described by the formula "a to b"; where "a"
equals the 5" nucleotide position
and "b" equals the 3 " nucleotide position of the polynucleotide fragment; and
further where "a" equals an
integer between 1 and the number of nucleotides of the polynucleotide sequence
of the present invention
minus 8, and where "b" equals an integer between 9 and the number of
nucleotides of the polynucleotide
sequence of the present invention; and where "a" is an integer smaller then
"b" by at least 8.
[0041] The present invention also provides for the exclusion of any
polynucleotide fragments
specified by 5' and 3' positions or by size in nucleotides as described above.
Any number of fragment
species specified by 5' and 3' positions or sub-genus specified by size in
nucleotides, as described above,
may be excluded from the present invention.
[0042] The present invention also includes isolated or purified "EST-related
polypeptides." The
terms "isolated" or "purified" have the meanings provided above. As used
herein, the term "EST-related
polypeptides" means the polypeptides encoded by the EST-related nucleic acids,
including the polypeptides
of SEQ ID NOs. 13310-26595.
[0043] The present invention also includes isolated or purified "fragments of
EST-related
polypeptides." The terms "isolated" or "purified" have the meanings provided
above. As used herein, the
term "fragments of EST-related polypeptides" means fragments comprising at
least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids of an EST-related polypeptide
to the extent that fragments of
these lengths are consistent with the lengths of the particular EST-related
polypeptides being referred to. In
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particular, fragments of EST-related polypeptides refer to polypeptides
encoded by polynucleotides described
in the Tables herein.
[0044] The present invention also includes isolated or purified "positional
segments of EST-related
polypeptides." As used herein, the term "positional segments of EST-related
polypeptides" includes
polypeptides comprising amino acid residues 1-25, 26-50, 51-75, 76-100, 101-
125, 126-150, 151-175, 176-
200, or 201-the C-terminal amino acid of the EST-related polypeptides to the
extent that such amino acid
residues are consistent with the lengths of the particular EST-related
polypeptides being referred to. The term
"positional segments of EST-related polypeptides also includes segments
comprising amino acid residues 1-
50, 51-100, 101-150, 151-200 or 201-the C-terminal amino acid of the EST-
related polypeptides to the extent
that such amino acid residues are consistent with the lengths of the
particular EST-related polypeptides being
referred to. The term "positional segments of EST-related polypeptides" also
includes segments comprising
amino acids 1-100 or 101-200 of the EST-related polypeptides to the extent
that such amino acid residues are
consistent with the lengths of particular EST-related polypeptides being
referred to. In addition, the term
"positional segments of EST-related polypeptides" includes segments comprising
amino acid residues 1-200
or 201-the C-terminal amino acid of the EST-related polypeptides to the extent
that amino acid residues are
consistent with the lengths of the particular EST related polypeptides being
referred to.
[0045] The present invention also includes isolated or purified "fragments of
positional segments of
EST-related polypeptides." The terms "isolated" or "purified" have the
meanings provided above. As used
herein, the term "fragments of positional segments of EST-related
polypeptides" means fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids of positional
segments of EST-related polypeptides to the extent that fragments of these
lengths are consistent with the
lengths of the particular EST-related polypeptides being referred to.
[0046] In addition to the above "positional segments of EST-related
polypeptides" and
"fragments of positional segments of EST-related polypeptides", for the
polypeptides of the present
invention, further preferred polypeptides comprise at least 8 amino acids,
wherein "at least 8" is defined
as any integer between 8 and the integer representing the C-terminal amino
acid of the polypeptide of the
present invention including the polypeptide sequences of the sequence listing
below. Further included are
polypeptide fragments at least 8 amino acids in length, as described above,
that are further specified in
terms of their N-terminal and C-terminal positions. Preferred polypeptide
fragment species specified by
their N-terminal and C-terminal positions include the signal peptides
delineated in the sequence listing
below. However, included in the present invention as individual species are
all polypeptide fragments, at
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least 5 amino acids in length, as described above, and may be particularly
specified by a N-terminal and
C-terminal position.
[0047) The present invention also provides for the exclusion of any fragments
specified by
N-terminal and C-terminal positions or by size in amino acid residues as
described above. Any number of
fragment species specified by N-terminal and C-terminal positions or sub-genus
of fragments specified by
size in amino acid residues as described above may be excluded from the
present invention.
[0048] The polypeptide fragments of the present invention can be immediately
envisaged using
the above description and are therefore not individually listed solely for the
purpose of not unnecessarily
lengthening the specification. The above fragments need not be active since
they would be useful, for
example, in immunoassays, in epitope mapping, epitope tagging, as vaccines, to
raise antibodies,
stimulate an immune response in a heterologous species, and as molecular
weight markers. The above
fragments may also be used to generate antibodies to a particular portion of
the polypeptide. These
antibodies can then be used in immunoassays well known in the art to
distinguish between human and
non-human cells and tissues or to determine whether cells or tissues in a
biological sample are or are not
of the same type which express the polypeptide of the present invention.
Further preferred polypeptide
fragments of the present invention comprise the signal peptides as delineated
in the sequence listing.
These signal peptides may be used to facilitate secretion of either the
polypeptide of the same gene or a
heterologous polypeptide.
[0049] The present invention also includes antibodies which specifically
recognize the EST-related
polypeptides, fragments of EST-related polypeptides, positional segments of
EST-related polypeptides, or
fragments of positional segments of EST-related polypeptides. In the case of
secreted proteins, such as those
of SEQ )D NOs. 24-1027, 4597-6443, 13310-14313, 17883-19729, antibodies which
specifically recognize
the mature protein generated when the signal peptide is cleaved may also be
obtained as described below.
Similarly, antibodies which specifically recognize the signal peptides these
sequences, for example, may also
be obtained.
[0050] In some embodiments and in the case of secreted proteins, the EST-
related nucleic acids,
fragments of EST-related nucleic acids, positional segments of EST-related
nucleic acids, or fragments of
positional segments of nucleic acids include a signal sequence. In other
embodiments, the EST-related
nucleic acids, fragments of EST-related nucleic acids, positional segments of
EST-related nucleic acids, or
fragments of positional segments of nucleic acids may include the full coding
sequence for the protein or, in
the case of secreted proteins, the full coding sequence of the mature protein
(i.e. the protein generated when
the signal polypeptide is cleaved off). In addition, the EST-related nucleic
acids, fragments of EST-related
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nucleic acids, positional segments of EST-related nucleic acids, or fragments
of positional segments of
nucleic acids may include regulatory regions upstream of the translation start
site or downstream of the stop
codon which control the amount, location, or developmental stage of gene
expression.
[0051] As discussed above, both secreted and non-secreted human proteins may
be therapeutically
important. Thus, the proteins expressed from the EST-related nucleic acids,
fragments of EST-related nucleic
acids, positional segments of EST-related nucleic acids, or fragments of
positional segments of nucleic acids
may be useful in treating or controlling a variety of human conditions.
[0052] The EST-related nucleic acids, fragments of EST-related nucleic acids,
positional segments
of EST-related nucleic acids, or fragments of positional segments of nucleic
acids may be used in forensic
procedures to identify individuals or in diagnostic procedures to identify
individuals having genetic diseases
resulting from abnormal gene expression. In addition, the EST-related nucleic
acids, fragments of EST-
related nucleic acids, positional segments of EST-related nucleic acids, or
fragments of positional segments
of nucleic acids are useful for constructing a high resolution map of the
human chromosomes.
[0053] The present invention also relates to secretion vectors capable of
directing the secretion of a
protein of interest, whether a protein of the present invention or a
heterologous protein. The vectors may also
be used gene therapy strategies in which it is desired to produce a gene
product in one cell which is to be
delivered to another location in the body. Secretion vectors may also
facilitate the purification of desired
proteins. The secretion vectors may also be used to express a desired protein,
such as a heterologous protein,
such that the protein is secreted into the culture medium, thereby
facilitating purification. The cells
expressing the desired protein may either be transiently or stably transfected
with the vectors. The cells may
be, for example, human, other animal, bacterial, yeast, or insect cells.
[0054] The present invention also relates to expression vectors capable of
directing the expression
of an inserted gene in a desired spatial or temporal manner or at a desired
level. Such vectors may include
sequences upstream of the EST-related nucleic acids, fragments of EST-related
nucleic acids, positional
segments of EST-related nucleic acids, or fragments of positional segments of
nucleic acids, such as
promoters or upstream regulatory sequences. Preferred chimeric polypeptides,
and vectors encoding the
same, comprise a signal peptide set forth in the sequence listing below.
[0055] The present invention also comprises fusion vectors for making chimeric
polypeptides
comprising a first polypeptide and a second polypeptide. Such vectors are
useful for determining the cellular
localization of the chimeric polypeptides or for isolating, purifying or
enriching the chimeric polypeptides.
[0056] The EST-related nucleic acids, fragments of EST-related nucleic acids,
positional segments
of EST-related nucleic acids, or fragments of positional segments of nucleic
acids may also be used for gene
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therapy to contt-ol or treat genetic diseases. In the case of secreted
proteins, signal peptides may be fused to
heterologous proteins to direct their extracellular secretion.
[0057] Bacterial clones containing Bluescript plasmids having inserts
containing the sequence of
the non-aligned 5'ESTs, also referred to as singletons, and sequences of the
5'ESTs which were aligned to
yield consensus contigated 5' ESTs are presently stored at -80°C in 4%
(v/v) glycerol in the inventor's
laboratories under internal designations. The non-aligned 5'ESTs of the
invention are those sequences which
are present in the sequence listing but which identification number either
corresponds to a single EST from a
single tissue in the second column of Table V or is absent from the first
column of Table V. The inserts may
be recovered from the stored materials by growing the appropriate clones on a
suitable medium. The
Bluescript DNA can then be isolated using plasmid isolation procedures
familiar to those skilled in the art
such as alkaline lysis minipreps or large scale alkaline lysis plasmid
isolation procedures. If desired the
plasmid DNA may be further enriched by centrifugation on a cesium chloride
gradient, size exclusion
chromatography, or anion exchange chromatography. The plasmid DNA obtained
using these procedures
may then be manipulated using standard cloning techniques familiar to those
skilled in the art. Alternatively,
a PCR can be performed with primers designed at both ends of the inserted EST-
related nucleic acids,
fragments of EST-related nucleic acids, positional segments of EST-related
nucleic acids, or fragments of
positional segments of nucleic acids. The PCR product which corresponds to the
EST-related nucleic acids,
fragments of EST-related nucleic acids, positional segments of EST-related
nucleic acids, or fragments of
positional segments of nucleic acids can then be manipulated using standard
cloning techniques familiar to
those skilled in the art.
[0058] One embodiment of the present invention is a purified nucleic acid
comprising a
sequence selected from the group consisting of SEQ ID NOs. 24-13309 and SEQ ID
NOs. 26596-52153
and sequences complementary to the sequences of SEQ ID NOs. 24-13309 and SEQ
ID NOs. 26596-
52153.
[0059] Another embodiment of the present invention is a purified nucleic acid
comprising at
least 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300,
500, or 1000 consecutive
nucleotides, to the extent that fragments of these lengths are consistent with
the specific sequence, of a
sequence selected from the group consisting of SEQ ID NOs. 24-13309 and SEQ ID
NOs. 26596-52153
and sequences complementary to the sequences of SEQ ID NOs. 24-13309 and SEQ
ID NOs. 26596-
52153.
[0060 A further aspect of this embodiment is a purified vertebrate nucleic
acid comprising at
least 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300,
500, or 1000 consecutive
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nucleotides, to the extent that fragments of these lengths are consistent with
the specific sequence, of a
sequence selected from the group consisting of SEQ ID NOs. 24-13309 and SEQ >D
NOs. 26596-52153
and sequences complementary to the sequences of SEQ ID NOs. 24-13309 and SEQ
)D NOs. 26596-
52153.
[0061] A further aspect of this embodiment is a purified human nucleic acid
comprising at least
8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300, 500, or
1000 consecutive nucleotides, to
the extent that fragments of these lengths are consistent with the specific
sequence, of a sequence selected
from the group consisting of SEQ ID NOs. 24-13309 and SEQ 1D NOs. 26596-52153
and sequences
complementary to the sequences of SEQ ID NOs. 24-13309 and SEQ ID NOs. 26596-
52153.
[0062] Another embodiment of the present invention is a purified nucleic acid
comprising at
least 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300,
500, or 1000 consecutive
nucleotides, to the extent that fragments of these lengths are consistent with
the specific sequence, of a
sequence selected from the group consisting of the preferred polynucleotides
described in Tables IVa and
IVb and sequences complementary to the sequences the preferred polynucleotides
described in Tables
IVa and IVb.
[0063] Another embodiment of the present invention is a purified nucleic acid
comprising at
least 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300,
500, or 1000 consecutive
nucleotides, to the extent that fragments of these lengths are consistent with
the specific sequence, of a
sequence selected from the group consisting of the preferred polynucleotides
described in Tables IVa and
IVb updated and sequences complementary to the sequences the preferred
polynucleotides described in
Tables IVa and IVb updated.
[0064] Another embodiment of the present invention is a purified nucleic acid
comprising at
least 15 consecutive nucleotides of a sequence selected from the group
consisting of SEQ >D NOs. 24-
13309 and SEQ ID NOs. 26596-52153 and sequences complementary to the sequences
of SEQ 117 NOs.
24-13309 and SEQ )D NOs. 26596-52153.
[0065] A further aspect of this embodiment is a purified vertebrate nucleic
acid comprising at
least 15 consecutive nucleotides of a sequence selected from the group
consisting of SEQ >D NOs. 24-
13309 and SEQ ID NOs. 26596-52153 and sequences complementary to the sequences
of SEQ ID NOs.
24-13309 and SEQ >D NOs. 26596-52153.
[0066] A further aspect of this embodiment is a purified human nucleic acid
comprising at least
15 consecutive nucleotides of a sequence selected from the group consisting of
SEQ ID NOs. 24-13309
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and SEQ ID NOs. 26596-52153 and sequences complementary to the sequences of
SEQ ID NOs. 24-
13309 and SEQ ID NOs. 26596-52153.
[0067] Another embodiment of the present invention is a purified nucleic acid
comprising at
least 15 consecutive_ nucleotides of a sequence selected from the group
consisting of the preferred
polynucleotides described in Tables IVa and IVb and sequences complementary to
the sequences of the
preferred polynucleotides described in Tables IVa and IVb.
[0068] A further embodiment of the present invention is a purified nucleic
acid comprising the
coding sequence of a sequence selected from the group consisting of 24-13309.
[0069] Yet another embodiment of the present invention is a purified nucleic
acid comprising the
full coding sequences of a sequence selected from the group consisting of SEQ
ID NOs. 4597-6443
wherein the full coding sequence comprises the sequence encoding the signal
peptide and the sequence
encoding the mature protein.
[0070] Still another embodiment of the present invention is a purified nucleic
acid comprising a
contiguous span of a sequence selected from the group consisting of SEQ ID
NOs. 4597-6443 which
encodes the mature protein.
[0071] Another embodiment of the present invention is a purified nucleic acid
comprising a
contiguous span of a sequence selected from the group consisting of SEQ >D
NOs. 24-1027 and 4597-
6443 which encodes the signal peptide.
[0072] Another embodiment of the present invention is a purified nucleic acid
encoding a
polypeptide comprising a sequence selected from the group consisting of the
sequences of SEQ ID NOs.
13310-26595.
[0073] Another embodiment of the present invention is a purified nucleic acid
encoding a
polypeptide comprising a sequence selected from the group consisting of the
sequences of SEQ ID NOs.
17883-19729.
[0074] Another embodiment of the present invention is a purified nucleic acid
encoding a
polypeptide comprising a mature protein included in a sequence selected from
the group consisting of the
sequences of SEQ ID NOs. 17883-19729.
[0075] Another embodiment of the present invention is a purified nucleic acid
encoding a
polypeptide comprising a signal peptide included in a sequence selected from
the group consisting of the
sequences of SEQ ID NOs. 13310-14313 and 17883-19729.
[0076] Another embodiment of the present invention is a purified nucleic acid
of at least 15,18,
20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300, 500 or 1000 nucleotides in
length which hybridizes
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under stringent conditions to a sequence selected from the group consisting of
SEQ ID NOs. 24-13309
and SEQ ID NOs. 26596-52153 and sequences complementary to the sequences of
SEQ ID NOs. 24-
13309 and SEQ ID NOs. 26596-52153.
[0077] Another embodiment of the present invention is a vertebrate purified
nucleic acid of at
least 15,18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300, 500 or 1000
nucleotides in length which
hybridizes under stringent conditions to a sequence selected from the group
consisting of SEQ ID NOs.
24-13309 and SEQ ID NOs. 26596-52153 and sequences complementary to the
sequences of SEQ )D
NOs. 24-13309 and SEQ ID NOs. 26596-52153.
(0078] Another embodiment of the present invention is a human purified nucleic
acid of at least
15,18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, 300, 500 or 1000
nucleotides in length which
hybridizes under stringent conditions to a sequence selected from the group
consisting of SEQ ID NOs.
24-13309 and SEQ ID NOs. 26596-52153 and sequences complementary to the
sequences of SEQ ID
NOs. 24-13309 and SEQ ID NOs. 26596-52153.
[0079] Another embodiment of the present invention is a purified or isolated
polypeptide
comprising a sequence selected from the group consisting of the sequences of
SEQ ID NOs. 13310-
26595.
[0080] Another embodiment of the present invention is a purified or isolated
polypeptide
comprising a sequence selected li-om the group consisting of SEQ ID NOs. 17883-
19729.
[0081] Another embodiment of the present invention is a purified or isolated
polypeptide
comprising a mature protein of a polypeptide selected from the group
consisting of SEQ ID NOs. 17883-
19729.
[0082] Another embodiment of the present invention is a purified or isolated
polypeptide
comprising a signal peptide of a sequence selected from the group consisting
of the polypeptides of SEQ
1D NOs. 13310-14313 and 17883-19729.
[0083] Another embodiment of the present invention is a purified or isolated
polypeptide
comprising at least 5, 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75,
100, 200, 300, 500, or 1000
consecutive amino acids, to the extent that fragments of these lengths are
consistent with the specific
sequence, of a sequence selected from the group consisting of the sequences of
SEQ ID NOs. 13310-
26595.
[0084] Another embodiment of the present invention is a method of making a
cDNA comprising
the steps of contacting a collection of mRNA molecules from human cells with a
primer comprising at
least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or 50 consecutive nucleotides of
a sequence selected from the
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group consisting of the sequences complementary to SEQ >D NOs. 24-13309 and
SEQ ID NOs. 26596-
52153, hybridizing said primer to an mRNA in said collection that encodes said
protein, reverse
transcribing said hybridized primer to make a first cDNA strand from said
mRNA, making a second
cDNA strand complementary to said first cDNA strand and isolating the
resulting cDNA encoding said
protein comprising said first cDNA strand and said second cDNA strand.
[0085] Another embodiment of the present invention is a purified cDNA
obtainable by the
method of the preceding paragraph. In one aspect of this embodiment, the cDNA
encodes at least a
portion of a human polypeptide.
[0086] Another embodiment of the present invention is a purified cDNA obtained
by a method
of making a cDNA of the invention. In one aspect of this embodiment, the cDNA
encodes at least a
portion of a human polypeptide.
(0087] Another embodiment of the present invention is a method of making a
cDNA comprising
the steps of contacting a cDNA collection with a detectable probe comprising
at least 12, 15, 18, 20, 23,
25, 28, 30, 35, 40, or 50 consecutive nucleotides of a sequence selected from
the group consisting of SEQ
ID NOs. 24-13309 and SEQ ID NOs. 26596-52153 and the sequences complementary
to SEQ )D NOs.
24-13309 and SEQ ID NOs. 26596-52153 under conditions which permit said probe
to hybridize to
cDNA, identifying a cDNA which hybridizes to said detectable probe, and
isolating said cDNA which
hybridizes to said probe.
[0088] Another embodiment of the present invention is a purified cDNA
obtainable by the
method of the preceding paragraph. In one aspect of this embodiment, the cDNA
encodes at least a
portion of a human polypeptide.
[0089] Another embodiment of the present invention is a method of making a
cDNA comprising
the steps of contacting a collection of mRNA molecules from human cells with a
first primer capable of
hybridizing to the polyA tail of said mRNA, hybridizing said first primer to
said polyA tail, reverse
transcribing said mRNA to make a first cDNA strand, making a second cDNA
strand complementary to
said first cDNA strand using at least one primer comprising at least 12, 15,
18, 20, 23, 25, 28, 30, 35, 40, or
50 consecutive nucleotides of a sequence selected from the group consisting of
SEQ >D NOs. 24-13309
and SEQ )D NOs. 26596-52153, and isolating the resulting cDNA comprising said
first cDNA strand and
said second cDNA strand. In another aspect of this method the second cDNA
strand is made by
contacting said first cDNA strand with a second primer comprising at least 12,
15, 18, 20, 23, 25, 28, 30,
35, 40, or 50 consecutive nucleotides of a sequence selected from the group
consisting of SEQ ID NOs.
24-13309 and SEQ ID NOs. 26596-52153 and a third primer which sequence is
fully included within the
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sequence of said first primer, performing a first polymerase chain reaction
with said second and third
primers to generate a first PCR product, contacting said first PCR product
with a fourth primer,
comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or 50 consecutive
nucleotides of said sequence
selected from the group consisting of SEQ ID NOs. 24-13309 and SEQ )D NOs.
26596-52153, and a fifth
primer, which sequence is fully included within the sequence of said third
primer, wherein said fourth and
fifth hybridize to sequences within said first PCR product, and performing a
second polymerase chain
reaction, thereby generating a second PCR product. Alternatively, the second
cDNA strand may be made
by contacting said first cDNA strand with a second primer comprising at least
12, 15, 18, 20, 23, 25, 28,
30, 35, 40, or 50 consecutive nucleotides of a sequence selected from the
group consisting of SEQ ID
NOs. 24-13309 and SEQ ID NOs. 26596-52153 and a third primer which sequence is
fully included
within the sequence of said first primer, performing a polymerase chain
reaction with said second and
third primers to generate said second cDNA strand. Alternatively, the second
cDNA strand may be made
by contacting said first cDNA strand with a second primer comprising at least
12, 15, 18, 20, 23, 25, 28,
30, 35, 40, or 50 consecutive nucleotides of a sequence selected from the
group consisting of SEQ ID
NOs. 24-13309 and SEQ ID NOs. 26596-52153, hybridizing said second primer to
said first strand
cDNA, and extending said hybridized second primer to generate said second cDNA
strand.
[0090] Another embodiment of the present invention is a purified cDNA
obtainable by a method
of making a cDNA of the invention. In one aspect of this embodiment, said cDNA
encodes at least a
portion of a human polypeptide.
[0091] Another embodiment of the present invention is a method of making a
polypeptide
comprising the steps of obtaining a cDNA which encodes a polypeptide encoded
by a nucleic acid
comprising a sequence selected from the group consisting of SEQ ID NOs. 24-
13309 or a cDNA which
encodes a polypeptide comprising at least 6, 8, 10, 12, 15, 18, 20, 23, 25,
28, 30, 35, 40, or 50 consecutive
amino acids of a polypeptide encoded by a sequence selected from the group
consisting of SEQ ID NOs.
24-13309, inserting said cDNA in an expression vector such that said cDNA is
operably linked to a
promoter, introducing said expression vector into a host cell whereby said
host cell produces the protein
encoded by said cDNA, and isolating said protein.
[0092] Another embodiment of the present invention is a method of obtaining a
promoter DNA
comprising the steps of obtaining genomic DNA located upstream of a nucleic
acid comprising a
sequence selected from the group consisting of SEQ ID NOs. 24-13309 and SEQ )D
NOs. 26596-52153
and the sequences complementary to the sequences of SEQ ID NOs. 24-13309 and
SEQ 117 NOs. 26596-
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52153, screening said genomic DNA to identify a promoter capable of directing
transcription initiation,
and isolating said DNA comprising said identified promoter.
[0093] In one aspect of this embodiment, said obtaining step comprises walking
from genomic
DNA comprising a sequence selected from the group consisting of SEQ ID NOs. 24-
13309 and SEQ ID
NOs. 26596-52153 and the sequences complementary to SEQ ID NOs. 24-13309 and
SEQ ID NOs.
26596-52153. In another aspect of this embodiment, said screening step
comprises inserting genomic
DNA located upstream of a sequence selected from the group consisting of SEQ
ID NOs. 24-13309 and
SEQ 1D NOs. 26596-52153 and the sequences complementary to SEQ ID NOs. 24-
13309 and SEQ ID
NOs. 26596-52153 into a promoter reporter vector. For example, said screening
step may comprise
identifying motifs in genomic DNA located upstream of a sequence selected from
the group consisting of
SEQ >D NOs. 24-13309 and SEQ >D NOs. 26596-52153 and the sequences
complementary to SEQ 117
NOs. 24-13309 and SEQ 117 NOs. 26596-52153 which are transcription factor
binding sites or
transcription start sites.
[0094] Another embodiment of the present invention is a isolated promoter
obtainable by the
methods of the above paragraphs.
[0095] Another embodiment of the present invention is a isolated promoter
obtained by the
methods described in the above paragraphs.
[0096] Another embodiment of the present invention is the inclusion of at
least one sequence
selected from the group consisting of SEQ m NOs. 24-13309 and SEQ ID NOs.
26596-52153, the
sequences complementary to the sequences of SEQ ID NOs. 24-13309 and SEQ ID
NOs. 26596-52153
and fragments comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, 50,
or 100 consecutive nucleotides
of said sequence in an array of discrete ESTs or fragments thereof of at least
12, 15, 18, 20, 23, 25, 28, 30,
35, 40, 50, or 100 nucleotides in length. In some aspects of this embodiment,
the array includes at least
two sequences selected from the group consisting of SEQ ID NOs. 24-13309 and
SEQ ID NOs. 26596-
52153, the sequences complementary to the sequences of SEQ >D NOs. 24-13309
and SEQ ID NOs.
26596-52153, and fragments comprising at least 12, 15, 18, 20, 23, 25, 28, 30,
35, 40, 50, or 100
consecutive nucleotides of said sequences. In another aspect of this
embodiment, the array includes at
least one, three, five, ten, fifteen, or twenty sequences selected from the
group consisting of SEQ ID NOs.
24-13309 and SEQ B7 NOs. 26596-52153, the sequences complementary to the
sequences of SEQ ID
NOs. 24-13309 and SEQ ff~ NOs. 26596-52153 and fragments comprising at least
12, 15, 18, 20, 23, 25,
28, 30, 35, 40, 50, or 100 consecutive nucleotides of said sequences.
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[0097] Another embodiment of the present invention is an enriched population
of recombinant
nucleic acids, said recombinant nucleic acids comprising an insert nucleic
acid and a backbone nucleic
acid, wherein at least 0.01%, 0.05°/o, 0.1%, 0.5%, 1%, 2%, 5%, 10%, or
20% of said insert nucleic acids
in said population comprise a sequence selected from the group consisting of
SEQ 117 NOs. 24-13309 and
SEQ ID NOs. 26596-52153 and the sequences complementary to SEQ ID NOs. 24-
13309 and SEQ ID
NOs. 26596-52153.
[0098] Another embodiment of the present invention is a purified or isolated
antibody capable of
specifically binding to a polypeptide comprising a sequence selected from the
group consisting of SEQ )D
NOs. 13310-26595.
[0099] Another embodiment of the present invention is a purified or isolated
antibody capable of
specifically binding to a polypeptide comprising at least 6, 8, 10, 12, 15,
18, 20, 23, 25, 28, 30, 35, 40, or
50 consecutive amino acids of a sequence selected from the group consisting of
SEQ ID NOs. 13310-
26595.
[0100] Yet, another embodiment of the present invention is an antibody
composition capable of
selectively binding to an epitope-containing fragment of a polypeptide
comprising a contiguous span of at
least 8, 10, 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or 50 amino acids of any
of SEQ ID NOs. 13310-26595,
wherein said antibody is polyclonal or monoclonal.
[0101] Another embodiment of the present invention is a computer readable
medium having
stored thereon a sequence selected from the group consisting of a nucleic acid
code of SEQ ID NOs. 24-
13309 and 26596-52153 and a polypeptide code of SEQ ID NOs. 13310-26595.
[0102] Another embodiment of the present invention is a computer system
comprising a
processor and a data storage device wherein said data storage device has
stored thereon a sequence
selected from the group consisting of a nucleic acid code of SEQ ID NOs. 24-
13309 and 26596-52153
and a polypeptide code of SEQ lD NOs. 13310-26595. In one aspect of this
embodiment the computer
system further comprises a sequence comparer and a data storage device having
reference sequences
stored thereon. For example, the sequence comparer may comprise a computer
program which indicates
polymorphisms.
[0103] In another aspect of this embodiment, the computer system further
comprises an
identifier which identifies features in said sequence.
[0104] Another embodiment of the present invention is a method for comparing a
first sequence
to a reference sequence wherein said first sequence is selected from the group
consisting of a nucleic acid
code of SEQII) NOs. 24-13309 and 26596-52153 and a polypeptide code of SEQ )D
NOs. 13310-26595
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comprising the steps of reading said first sequence and said reference
sequence through use of a computer
program which compares sequences and determining differences between said
first sequence and said
reference sequence with said computer program. In some aspects of this
embodiment, saW step of
determining differences between the first sequence and the reference sequence
comprises identifying
polymorphisms.
[0105] Another embodiment of the present invention is a method for identifying
a feature in a
sequence selected from the group consisting of a nucleic acid code of SEQ >D
NOs. 24-13309 and 26596-
52153 and a polypeptide code of SEQ )D NOs. 13310-26595 comprising the steps
of reading said
sequence through the use of a computer program which identifies features in
sequences and identifying
features in said sequence with said computer program.
[0106] Another embodiment of the present invention is a vector comprising a
nucleic acid
according to any one of the nucleic acids described above.
[0107] Another embodiment of the present invention is a host cell containing
the above vector.
(0108] Another embodiment of the present invention is a method of making any
of the nucleic
acids described above comprising the steps of introducing said nucleic acid
into a host cell such that said
nucleic acid is present in multiple copies in each host cell and isolating
said nucleic acid from said host cell.
[0109] Another embodiment of the present invention is a method of making a
nucleic acid of any
of the nucleic acids described above comprising the step of sequentially
linking together the nucleotides
in said nucleic acids.
[0110] Another embodiment of the present invention is a method of making any
of the
polypeptides described above wherein said polypeptides is 150 amino acids in
length or less comprising
the step of sequentially linking together the amino acids in said polypeptide.
[0111] Another embodiment of the present invention is a method of making any
of the
polypeptides described above wherein said polypeptides is 120 amino acids in
length or less comprising
the step of sequentially linking together the amino acids in said
polypeptides.
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Brief Description of the Sequence Listing
[0112) SEQ 1D NOs. l, 3, 5, 7, 9, 11, and 13 are full-length cDNAs prepared
using the methods
described herein.
[0113] SEQ_>D NOs. 2, 4 and 6 are the signal peptides encoded by the nucleic
acids of SEQ 1D
NOs. 1, 3 and 5 respectively.
[0114] SEQ )D NOs. 8, 10, 12, and 14 are the polypeptides encoded by the
nucleic acids of SEQ ID
NOs. 7, 9, 1 I , and 13 respectively.
[0115] SEQ 1D NOs. 15, 16, 18, 19, 21 and 22 are primers whose use is
described in the
specification.
[0116] SEQ ID NOs. 17, 20, and 23 are the sequences of nucleic acids
containing transcription
factor binding sites which were obtained as described below.
[0117] SEQ ID NOs. 24-1027 are nucleic acids having an incomplete OltF which
encodes a signal
peptide. As used herein, an "incomplete ORF"' is an open reading frame in
which a start codon has been
identified but no stop codon has been identified. The locations of the
incomplete ORFs and sequences
encoding signal peptides are listed in the accompanying Sequence Listing. In
addition, the von Heijne score
of the signal peptide computed as described below is listed as the "score" in
the accompanying Sequence
Listing. The sequence of the signal-peptide is listed as "seq" in the
accompanying Sequence Listing. The "/"
in the signal peptide sequence indicates the location where proteolytic
cleavage of the signal peptide occurs to
generate a mature protein.
[0118] SEQ 1D NOs. 1028-4596 are nucleic acids having an incomplete ORF' in
which no sequence
encoding a signal peptide has been identified to date. However, it remains
possible that subsequent analysis
will identify a sequence encoding a signal peptide in these nucleic acids. The
locations of the incomplete
ORFs are listed in the accompanying Sequence Listing.
[0119) SEQ )D NOs. 4597-6443 are nucleic acids having a complete OItF which
encodes a signal
peptide. As used herein, a "complete ORF" is an open reading frame in which a
start codon and a stop codon
have been identified. The locations of the complete ORFs and sequences
encoding signal peptides are listed
in the accompanying Sequence Listing. In addition, the von Heijne score of the
signal peptide computed as
described below is listed as the "score" in the accompanying Sequence Listing.
The sequence of the signal-
peptide is listed as "seq" in the accompanying Sequence Listing. The "/" in
the signal peptide sequence
indicates the location where proteolytic cleavage of the signal peptide occurs
to generate a mature protein.
[0120] SEQ 1D NOs. 6444-13309 are nucleic acids having a complete 012F in
which no sequence
encoding a signal peptide has been identified to date. Flowever, it remains
possible that subsequent analysis
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will identify a sequence encoding a signal peptide in these nucleic acids. The
locations of the complete ORFs
are listed in the accompanying Sequence Listing.
[0121] SEQ )D NOs. 13310-14313 are "incomplete polypeptide sequences" which
include a signal
peptide. Incomplete polypeptide sequences" are polypeptide sequences encoded
by nucleic acids in which a
start codon has been identified but no stop codon has been identified. These
polypeptides are encoded by the
nucleic acids of SEQ )D NOs. 24-1027. The location of the signal peptide is
listed in the accompanying
Sequence Listing.
[0122] SEQ )D NOs. 14314-17882 are incomplete polypeptide sequences in which
no signal
peptide has been identified to date. However, it remains possible that
subsequent analysis will identify a
signal peptide in these polypeptides. These polypeptides are encoded by the
nucleic acids of SEQ >D NOs.
1028-4596.
[0123] SEQ )D NOs. 17883-19729 are "complete polypeptide sequences" which
include a signal
peptide. "Complete polypeptide sequences" are polypeptide sequences encoded by
nucleic acids in which a
start codon and a stop codon have been identified. These polypeptides are
encoded by the nucleic acids of
SEQ )D NOs. 4597-6443. The location of the signal peptide is listed in the
accompanying Sequence Listing.
(0124] SEQ )D NOs. 19730-26595 are complete polypeptide sequences in which no
signal peptide
has been identified to date. However, it remains possible that subsequent
analysis will identify a signal
peptide in these polypeptides. These polypeptides are encoded by the nucleic
acids of SEQ 1D NOs.6444-
13309.
[0125] SEQ ID NOs. 26596-52153 are nucleic acid sequences in which no open
reading frame of at
least 150 nucleotides has been conclusively identified to date. However, it
remains possible subsequent
analysis will identify an open reading frame in these nucleic acids.
[0126] In the accompanying Sequence Listing, all instances of the symbol "n"
in the nucleic acid
sequences mean that the nucleotide can be adenine, guanine, cytosine or
thymine. In some instances the
polypeptide sequences in the Sequence Listing contain the symbol "Xaa." These
"Xaa" symbols indicate
either (1) a residue which cannot be identified because of nucleotide sequence
ambiguity or (2) a stop codon
in the determined sequence where applicants believe one should not exist (if
the sequence were determined
more accurately). In some instances, several possible identities of the
unlrnown amino acids may be
suggested by the genetic code.
[0127] In the case of secreted proteins, it should be noted that, in
accordance with the regulations
governing Sequence Listings, in the appended Sequence Listing, the encoded
protein (i.e. the protein
containing the signal peptide and the mature protein or part thereof) extends
from an amino acid residue
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having a negative number through a positively numbered amino acid residue.
Thus, the first amino acid
of the mature protein resulting from cleavage of the sigmal peptide is
designated as amino acid number l,
and the first amino acid of the signal peptide is designated with the
appropriate negative number.
[0127.1] The Sequence Listing, in computer readable format on CD ROM, is
incorporated herein by reference. The Sequence Listing is identified as
SEQLST.txt which
contains 48,524 IB and was recorded on March 6, 2001 at 3:21 P.M.
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Brief Description of the Drawings
[0128] Figure 1 summarizes the computer analysis procedure for obtaining
consensus contigated
ESTs.
[0129] Figure 2 is an analysis of the 43 amino terminal amino acids of all
human SwissProt proteins
to determine the frequency of false positives and false negatives using the
techniques for signal peptide
identification described herein.
[0130] Figure 3 summarizes a general RT-PCR-based-method used to clone and
sequence extended
cDNAs containing sequences adjacent to 5'ESTs.
[0131] Figure 4 provides a schematic description of the promoters isolated and
the way they are
assembled with the corresponding S'ESTs.
[0132] Figure 5 describes the transcription factor binding sites present in
each of the promoters of
Figure 4.
[0133] Figure 6 is a block diagram of an exemplary computer system.
[0134] Figure 7 is a flow diagram illustrating one embodiment of a process 200
for comparing a
new nucleotide or protein sequence with a database of sequences in order to
determine the homology levels
between the new sequence and the sequences in the database.
[0135] Figure 8 is a flow diagram illustrating one embodiment of a process 250
in a computer
for determining whether two sequences are homologous.
[0136] Figure 9 is a flow diagram illustrating one embodiment of an identifier
process 300 for
detecting the presence of a feature in a sequence.
[0137] Figure 10 is a table describing algorithms, parameters and criteria
that can be used for each
step of extended cDNA analysis.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
I. General Methods for Obtaining 5' ESTs derived from mRNAs with intact 5'
ends
[0138] The 5'ESTs of the present invention were obtained from cDNA libraries
derived from
mRNAs having intact 5' ends as described in Examples 1 to 5 using either a
chemical or enzymatic approach.
EXAMPLE 1
Preparation of mRNA
[0139] Total human RNAs or polyA+ RNAs derived from different tissues were
respectively
purchased from LABIMO and CI,ONTECH and used to generate cDNA libraries as
described below. The
purchased RNA had been isolated from cells or tissues using acid guanidium
thiocyanate-phenol-chloroform
extraction (Chamczyniski and Sacchi, Analytical Biochemistry 162:156-159,
1987). PolyA+ RNA was
isolated from total RNA (LABIMO) by two passes of oligo dT chromatography, as
described by Aviv and
Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972) in order to eliminate
ribosomal RNA.
[0140] The quality and the integrity of the polyA+ RNAs were checked. Northern
blots hybridized
with a probe corresponding to an ubiquitous mRNA, such as elongation factor 1
or elongation factor 2, were
used to confirm that the mRNAs were not degraded. Contamination of the polyA+
mRNAs by ribosomal
sequences was checked using Northern blots and a probe derived from the
sequence of the 28S rRNA.
Preparations of mRNAs with less than 5% of rRNAs were used in library
construction. To avoid constructing
libraries with RNAs contaminated by exogenous sequences (prokaryotic or
fungal), the presence of bacterial
16S ribosomal sequences and of two highly expressed fungal mRNAs was examined
using PCR.
EXAMPLE 2
Methods for Obtaining mRNAs having Intact 5' Ends
[0141] Following preparation of the mRNAs from various tissues as described
above, selection of
mRNA with intact 5' ends and specific attachment of an oligonucleotide tag to
the 5' end of said mRNA is
performed using either a chemical or enzymatic approach. Both techniques take
advantage of the presence of
the "cap" structure, which characterizes the 5'end of intact mRNAs and which
comprises a guanosine
generally methylated once, at the 7 position.
[0142] The chemical modification approach involves the optional elimination of
the 2', 3'-cis diol
of the 3' terminal ribose, the oxidation of the 2', 3', -cis diol of the
ribose linked to the cap of the S' ends of
the mRNAs into a dialdehyde, and the coupling of the said obtained dialdehyde
to a derivatized
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oligonucleotide tag. Further detail regarding the chemical approaches for
obtaining mRNAs having intact 5'
ends are disclosed in International Application No. W096/34981, published
November 7, 1996, the
disclosure of which is incorporated herein by reference in its entirety.
[0143] The enzymatic approach for ligating the oligonucleotide tag to the 5'
ends of mRNAs with
S intact 5' ends involves the removal of the phosphate groups present on the
5' ends of uncapped incomplete
mRNAs, the subsequent decapping of mRNAs with intact 5' ends and the ligation
of the phosphate present at
the 5' end of the decapped mRNA to an oligonucleotide tag. Further detail
regarding the enzymatic
approaches for obtaining mRNAs having intact 5' ends are disclosed in Dumas
Milne Edwards J.B. (Doctoral
Thesis of Paris VI University, Le clonage des ADNc complets: difficultes et
perspectives nouvelles. Apports
pour (etude de la regulation de 1'expression de la tryptophane hydroxylase de
rat, 20 Dec. 1993), EPO 625572
and Kato et al., Gene 150:243-250 (1994), the disclosures of which are
incorporated herein by reference in
their entireties.
[0144] In either the chemical or the enzymatic approach, the oligonucleotide
tag has a restriction
enzyme site (e.g. Eco RI sites) therein to facilitate later cloning
procedures. Following attachment of the
1 S oligonucleotide tag to the mRNA, the integrity of the mRNA was then
examined by performing a Northern
blot using a probe complementary to the oligonucleotide tag.
EXAMPLE 3
cDNA Synthesis Using mRNA Templates Having Intact 5' Ends
[0145] For the mRNAs joined to oligonucleotide tags using either the chemical
or the enzymatic
method, first strand cDNA synthesis was performed using a thermostable reverse
transcriptase with an oligo-
dT primer. In some instances, this oligo-dT primer contained an internal tag
of at least 4 nucleotides which is
different from one tissue to the other. In order to protect internal EcoRI
sites in the cDNA from digestion at
later steps in the procedure, methylated dCTP was used for first strand
synthesis. After removal of RNA by
an alkaline hydrolysis, the first strand of cDNA was precipitated using
isopropanol in order to eliminate
residual primers.
[0146] The second strand of the cDNA was then synthesized with a Klenow
fragment using a
primer corresponding to the 5'end of the ligated oligonucleotide. Preferably,
the primer is 20-25 bases in
length. Methylated dCTP was also used for second strand synthesis in order to
protect internal EcoRI sites in
the cDNA from digestion during the cloning process.
EXAMPLE 4
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Cloning of cDNAs derived from mRNA with intact 5' ends
[0147] Following second strand synthesis, the cDNAs were cloned into the
phagemid pBIueScript
II SK- vector (Stratagene) or one of its derivative. The ends of the cDNAs
were blunted with T4 DNA
polymerase (Biolabs) _and the cDNA was digested with EcoRI. Since rnethylated
dCTP was used during
cDNA synthesis, the EcoRI site present in the tag was the only hemi-methylated
site, hence the only site
susceptible to EcoRI digestion. In some instances, to facilitate subcloning,
an Hind III adaptor was added to
the 3' end of cDNAs.
[0148] The cDNAs were then size fractionated using either exclusion
chromatography (AcA,
Biosepra) or electrophoretic separation which yields 3 or 6 different
fractions. The cDNAs were then
directionally cloned either into pBlueScript using either the EcoRI and SmaI
restriction sites or the EcoRI and
Hind III restriction sites when the Hind III adaptator was present in the
cDNAs. The ligation mixture was
electroporated into bacteria and propagated under appropriate antibiotic
selection.
EXAMPLE 5
1 S Selection of Clones Having the Oli~onucleotide Tag Attached Thereto
[0149] Clones containing the oligonucleotide tag attached to cDNAs were then
selected as follows.
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[0150] The plasmid DNAs containing cDNA libraries made as described above were
purified
(Qiagen). A positive selection of the tagged clones was performed as follows.
Briefly, in this selection
procedure, the plasmid DNA was converted to single stranded DNA using gene II
endonuclease of the phage
F1 in combination with an exonuclease (Chang et al., Gene 127:95-8, 1993) such
as exonuclease III or T7
gene 6 exonuclease. The resulting single stranded DNA was then purified using
paramagnetic beads as
described by Fry et al., Biotechnigues, 13: 124-131, 1992. In this procedure,
the single stranded DNA was
hybridized with a biotinylated oligonucleotide having a sequence corresponding
to the 3' end of the
oligonucleotide tag described in Example 2. Preferably, the primer has a
length of 20-25 bases. Clones
including a sequence complementary to the biotinylated oligonucleotide were
captured by incubation with
streptavidin coated magnetic beads followed by magnetic selection. After
capture of the positive clones, the
plasmid DNA was released from the magnetic beads and converted into double
stranded DNA using a DNA
polymerase such as the ThermoSequenase obtained from Amersham Pharmacia
Biotech. Alternatively,
protocols such as the Gene Trapper kit (Gibco BRL) may be used. The double
stranded DNA was then
electroporated into bacteria. The percentage of positive clones having the 5'
tag oligonucleotide was
I 5 estimated to typically rank between 90 and 98% using dot blot analysis.
[0151] Following electroporation, the libraries were ordered in 384-microtiter
plates (MTP). A
copy of the MTP was stored for future needs. Then the libraries were
transferred into 96 MTP and
sequenced.
EXAMPLE 6
Sequencing of Inserts in Selected Clones
[0152] Plasmid inserts were first amplified by PCR on PE-9600/ PE-9700
thermocyclers (PE
Biosystems, Applied Biosystems Division, Foster City, CA) or tetrades
thermocyclers (MJ Research), using
L7AF3 and SETA primers (Genset SA), ExTaq polymerase (Takara), dNTPs
(Boehringer), buffer and
cycling conditions as recommended by the PE Biosystems Corporation. PCR
products were then sequenced
using MegaBace Capillary sequencers (Molecular Dynamics). Sequencing reactions
were performed using
PE 9600 / PE-9700 thermocyclers with ET primer (Energy Transfer) chemistry and
ThermoSequenase
(Amersham Pharmacia Biotech). The primer used was Reverse Primer (RP)
(Amersham Pharmacia Biotech)
as appropriate. The dNTPs and ddNTPs used in the sequencing reactions were
purchased from Boehringer.
Sequencing buffer, reagent concentrations and cycling conditions were as
recommended by Amersham.
Following the sequencing reaction, the samples were purified with Sephadex
(G50) and injected in the
capillaries of the MegaBace. Injection was performed for 12 seconds at 10000 V
and electrophoresis for 100
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minutes at 10000V. The sequence data were collected and analyzed using the
Instrument Control Manager
analysis software of the MegaBace prior to the Genset's proprietary sequence
verification software.
[0153] Alternatively, plasmid inserts were first amplified by PC:R on PE-9600
thermocyclers (PE
Biosystems, Applied Biosystems Division, Foster City, CA), using standard SETA-
A and SETA-B primers
(Genset SA), AmpliTaqGold (PE Biosystems), dNTPs (Boehringer), buffer and
cycling conditions as
recommended by the PE Biosystems Corporation. PCR products were then sequenced
using automatic ABI
Prism 377 sequencers (PE Biosystems). Sequencing reactions were performed
using PE 9600 thermocyclers
with standard dye-primer chemistry and ThermoSequenase (Amersham Pharmacia
Biotech). The primers
used were either T7 or 21M13 (available from Genset SA) as appropriate. The
primers were labeled with the
JOE, FAM, ROX and TAMRA dyes. The dNTPs and ddNTPs used in the sequencing
reactions were
purchased from Boehringer. Sequencing buffer, reagent concentrations and
cycling conditions were as
recommended by Amersham. Following the sequencing reaction, the samples were
precipitated with ethanol,
resuspended in formamide loading buffer, and loaded on a standard 4%
acrylamide gel. Electrophoresis was
performed for 2.5 hours at 3000V on an ABI 377 sequencer, and the sequence
data were collected and
analyzed using the ABI Prism DNA Sequencing Analysis Software, version 2.1.2.
II. Computer Analysis of the Isolated 5' ESTs
[0154] The sequence data from the different cDNA libraries made as described
above were
transferred to a database, where quality control and validation steps were
performed. A base-caller, working
using a Unix system, automatically flagged suspect peaks, taking into account
the shape of the peaks, the
inter-peak resolution, and the noise level. The proprietary base-caller also
performed an automatic trimming.
Any stretch of 25 or fewer bases having more than 4 suspect peaks was
considered unreliable and was
discarded. Sequences corresponding to cloning vector or ligation
oligonucleotides were automatically
removed from the 5'EST sequences. However, the resulting 5'EST sequences may
contain 1 to 5 bases
belonging to the above mentioned sequences at their 5' end. If needed, these
can easily be removed on a case
to case basis.
[0155] Following sequencing as described above, the sequences of the 5' ESTs
were entered in a
database for storage and manipulation as described below and as depicted in
Figure 1. Before searching the
5'ESTs in the database for sequences of interest, 5'ESTs derived from mRNAs
which were not of interest
were identified. Briefly, such undesired sequences may be of three types.
First, contaminants of either
endogenous (ribosomal RNAs, transfert RNAs, mitochondria) RNAs) or exogenous
(prokaryotic RNAs and
fungal RNAs) origins were identified. Second, uninformative sequences, namely
redundant sequences, small
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sequences and highly degenerate sequences were identified. Third, repeated
sequences (Alu, L1, THE and
MER repeats, SSTR sequences or satellite, micro-satellite, or telomeric
repeats) were identified and masked
in further processing.
[0156] Then, in order to determine the accuracy of the sequencing procedure as
well as the
efficiency of the 5' selection described above, the analyses described in
Examples 7 and 8 respectively were
performed on 5'ESTs.
EXAMPLE 7
Measurement of Sequencing Accuracy by Comparison to Known Sequences
[0157] To further determine the accuracy of the sequencing procedure described
in Example 6, the
sequences of 5' ESTs derived from known sequences were identified and compared
to the original known
sequences. First, a FASTA analysis with overhangs shorter than 5 by on both
ends was conducted on the 5'
ESTs to identify those matching an entry in the public human mRNA database
available at the time of filing
the present documents. The 5' ESTs which matched a known human mRNA were then
realigned with their
cognate mRNA and dynamic programming was used to include substitutions,
insertions, and deletions in the
list of "errors" which would be recognized. Errors occurring in the last 10
bases of the 5' EST sequences
were ignored to avoid the inclusion of spurious cloning sites in the analysis
of sequencing accuracy. This
analysis revealed that the sequences incorporated in the database had an
accuracy of more than 99.3% using
Megabaces Capillary sequencers and more than 99.5% using ABI 377 sequencers.
EXAMPLE 8
Determination of Efficiency of 5' EST Selection
[0158] To determine the efficiency at which the above selection procedures
isolated cDNAs which
include the 5' ends of their corresponding mRNAs, the sequences of 5'ESTs were
aligned with a reference
pool of complete mRNA/cDNA extracted from the EMBL release 57 using the FASTA
algorithm. The
reference mRNA/eDNA starting at the most 5' transcription start site was
obtained, and then compared to the
5' transcription start site position of the 5'EST. More than 75% of 5'ESTs had
their 5' ends close to the S'
ends of the known sequence. As some of the mRNA sequences available in the
EMBL database are deduced
from genomic sequences, a 5' end matching with these sequences will be counted
as an internal match. Thus,
the method used here underestimates the yield of 5'ESTs including the
authentic 5' ends of their
corresponding mRNAs.
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EXAMPLE 9
Generation of (:onsensus Conti~ated 5' ESTs
[0159] Since the cDNA libraries made above include multiple 5' ESTs derived
from the same
mRNA, overlapping 5'ESTs may be assembled into continuous sequences. The
following method describes
how to efficiently align multiple 5'ESTs in order to yield not only consensus
contigated 5'EST sequences for
mRNAs derived from different genes but also consensus contigated 5'EST
sequences for different mRNAs,
so called variants, transcribed from the same gene such as alternatively
spliced mRNAs.
[0160] A subset of 5'ESTs free from endogenous contaminants and uninformative
sequences, and
following the masking of repeats, was first selected.
[0161 ] This whole set of sequences was first partitioned into small clusters
containing sequences
which exhibited perfect matches with each other on a given length and which
derived from a small
number of different genes. Some 5'EST sequences, so called singletons, were
not aligned using this
approach because they were not homologous to any other sequence.
[0162] Thereafter, all variants of a given gene were identified in each
cluster using a proprietary
software. 5'EST sequences belonging to the same variant were then contigated
and consensus contigated
5'EST sequences generated for each variant. All consensus contigated 5' EST
sequences were
subsequently compared to the whole set of individual 5'EST sequences used to
obtained them.
[0163] If desired, the consensus contigated 5'EST sequences may be verified by
identifying
clones in nucleic acid samples derived from biological tissues, such as cDNA
libraries, which hybridize to
the probes based on the sequences of the consensus contigated S'ESTs using any
methods described
herein and sequencing those clones.
[0164] To assess the yield of new sequences, the 5'ESTs obtained and consensus
contigated S'ESTs
were compared to all known complete human mRNAs extracted from the EMBL
release 58 using BLASTN
with the following parameters S=1000, S2=1000, V=5 and B=5. All sequences with
high scoring pairs
whose significance was above e-100 were kept. Then, the obtained 5'ESTs and
consensus contigated 5'ESTs
were compared to all the human proteins extracted from SwissProt release 37,
TrEMBL release 58 and
Genseqp (Derwent's database of patented amino acid sequences) release 35.3 on
both strands using blastx
with the following parameters: S=450, S2=450, V=5 and B=5. All sequences with
high scoring pairs whose
significance was above e-50 were kept. Using this process, about 86% of 5'ESTs
or consensus assembled
5'ESTs were considered unidentified.
EXAMPLE 10
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Identification of the Most Probable Open Readies, Frame of 5' ESTs
[0165] Subsequently, consensus contigated 5'ESTs and 5'ESTs were screened to
identify those
having an open reading frame (ORF).
[0166] The nucleic acid sequence was first divided into several subsequences
which coding
propensity was evaluated separately using one or several different methods
lrnown to those skilled in the
art such as the evaluation of N-mer frequency and its variants (Fickett and
Tung, Nucleic Acids
Res;20:6441-50 (1992)) or the Average Mutual Information method (Grosse et al,
International
Conference on Intelligent Systems for Molecular Biology, Montreal, Canada.
June 28-July 1, 1998).
Each of the scores obtained by the techniques described above were then
normalized by their distribution
extremities and then fused using a neural network into a unique score that
represents the coding
probability of a given subsequence. The coding probability scores obtained for
each subsequence, thus
the probability score profiles obtained for each reading frame, was then
linked to the initiation codons
present on the sequence. For each open reading frame, defined as a nucleic
acid sequence beginning with
an ATG codon, an ORF score was determined. Preferably, this score is the sum
of the probability scores
computed for each subsequence corresponding to the considered ORF in the
correct reading frame
corrected by a function that negatively accounts for locally high score values
and positively accounts for
sustained high score values. The most probable ORF with the highest score was
selected.
[0167] Alternatively, open reading frames were simply defined as uninterrupted
nucleic acid
sequences longer than 150 nucleotides and beginning with an ATG codon.
[016$] In some embodiments, nucleic acid sequences encoding an "incomplete
ORF", as described
herein, namely an open reading frame in which a start codon has been
identified but no stop codon has been
identified, were obtained.
[0169] In other embodiments, nucleic acid sequences encoding a "complete ORF",
as used herein,
namely an open reading frame in which a start codon and a stop codon have been
identified, are obtained.
[0170] In a preferred embodiment, open reading frames encoding polypeptides of
at least 50 amino
acids were obtained.
[0171 ] To confirm that the chosen ORF actually encodes a polypeptide, the
consensus contigated
5'EST or 5'EST may be used to obtain an extended cDNA using any of the
techniques described herein, and
especially those described in Examples 17 and 18. Then, such obtained extended
cDNAs may be screened
for the most probable open reading frame using any of the techniques described
herein. The amino acid
sequence of the ORF encoded by the consensus contigated 5'EST or 5'EST may
then be compared to the
amino acid sequence of the ORF encoded by the extended cDNA using any of the
algorithms and parameters
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described herein in order to determine whether the ORF encoded by the extended
cDNA is basically the same
as the one encoded by the consensus contigated 5'EST or 5'EST.
[0172] Alternatively, to confirm that the chosen ORF actually encodes a
polypeptide, the consensus
contigated 5'ES'T or 5'EST may be used to obtain an extended cDNA using any of
the techniques described
herein, and especially those described in Examples 17 and 18. Such an extended
cDNA may then be inserted
into an appropriate expression vector and used to express the polypeptide
encoded by the extended cDNA as
described herein. The expressed polypeptide may be isolated, purified, or
enriched as described herein.
Several methods known to those skilled in the art may then be used, including
in combination, to determine
whether the expressed polypeptide is the one actually encoded by the chosen
ORF, herein referred to as the
expected polypeptide. Such methods are based on the determination of
predictable features of the expressed
polypeptide, including but not limited to its amino acid sequence, its size or
its charge, and the comparison of
these features to those predicted for the expected polypeptide. The following
paragraphs present examples of
such methods.
[0173] One of these methods involves the determination of at least a portion
of the amino acid
sequence of the expressed polypeptide using any technique known to those
skilled in the art. For example,
the amino-terminal residues may be determined using automated techniques based
on Edman's degradation
of polypeptides in which N-terminal residues are sequentially labeled and
cleaved from the polypeptide of
interest (see Stryer, Exploring proteins in Biochemistry, Freeman and Company,
New York, (1995)). The
amino acid sequence of the expressed polypeptide may then be compared to the
one predicted for the
expected polypeptide using any algorithm and parameters described therein.
[0174] Alternatively, the size of the expressed polypeptides may be determined
using techniques
familiar to those skilled in the art such as Coomassie blue or silver staining
and subsequently compared to the
size predicted for the expected polypeptide. Generally, the band corresponding
to the expressed polypeptide
will have a mobility near that expected based on the number of amino acids in
the open reading frame of the
extended cDNA. However, the band may have a mobility different than that
expected as a result of
modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
[0175] Alternatively, specific antibodies or antipeptides may be generated
against the expected
polypeptide as described in Example 33 and used to perform immunoblotting or
immunoprecipitation studies
against the expressed polypeptide. The presence of a band in samples from
cells containing the expression
vector with the extended cDNA which is absent in samples from cells containing
the expression vector
encoding an irrelevant polypeptide indicates that the expected polypeptide or
portion thereof is being
expressed. Generally, the band corresponding to the expressed polypeptide will
have a mobility near that
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expected based on the number of amino acids in the open reading frame of the
extended cDNA. However,
the band may have a mobility different than that expected as a result of
modifications such as glycosylation,
ubiquitination, or enzymatic cleavage.
S EXAMPLE 11
Identification of Potential Signal Sequences in 5' ESTs
[0176] The 5'ESTs or consensus contigated 5'ESTs found to include an ORF were
then searched to
identify potential signal motifs using slight modifications of the procedures
disclosed in Von Heijne, Nucleic
Acids Res. 14:4683-4690, 1986. 'those sequences encoding a peptide with a
score of at least 3.5 in the Von
Heijne signal peptide identification matrix were considered to possess a
signal sequence.
EXAMPLE 12
Confirmation of Accurate of Identification of Potential Signal Sequences in 5'
ESTs
[0177] The accuracy of the above procedure for identifying signal sequences
encoding signal
peptides was evaluated by applying the method to the 43 amino acids located at
the N terminus of all human
SwissProt proteins. The computed Von Heijne score for each protein was
compared with the known
characterization of the protein as being a secreted protein or a non- secreted
protein. In this manner, the
number of non-secreted proteins having a score higher than 3.5 (false
positives) and the number of secreted
proteins having a score lower than 3.5 (false negatives) could be calculated.
[0178] Using the results of the above analysis, the probability that a peptide
encoded by the 5'
region of the mRNA is in fact a genuine signal peptide based on its Von
Heijne's score was calculated based
on either the assumption that 10% of human proteins are secreted or the
assumption that 20% of human
proteins are secreted. The results of this analysis are shown in Figure 2.
[0179] Using the above method of identification of secretory proteins, 5' ESTs
of the following
polypeptides Imown to be secreted were obtained: human glucagon, gamma
interferon induced monokine
precursor, secreted cyclophilin-like protein, human pleiotropin, and human
biotinidase precursor. Thus, the
above method successfully identified those S' ESTs which encode a signal
peptide.
[0180] To confirm that the signal peptide encoded by the 5' ESTs or consensus
contigated 5' ESTs
actually functions as a signal peptide, the signal sequences from the 5' ESTs
or consensus contigated 5' ESTs
may be cloned into a vector designed for the identification of signal
peptides. Such vectors are designed to
confer the ability to grow in selective medium only to host cells containing a
vector with an operably linked
signal sequence. For example, to confirm that a 5' EST or consensus contigated
5' EST encodes a genuine
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signal peptide, the signal sequence of the 5' EST or consensus contigated 5'
EST may be inserted upstream
and in frame with a non-secreted form of the yeast invertase gene in signal
peptide selection vectors such as
those described in U.S. Patent No. 5,536,637. Growth of host cells containing
signal sequence selection
vectors with the correctly inserted 5' EST or consensus contigated 5' EST
signal sequence confirms that the
5' EST or consensus contigated 5' ESTs encodes a genuine signal peptide.
[0181] Alternatively, the presence of a signal peptide may be confirmed by
cloning the extended
cDNAs obtained using the ESTs or consensus contigated 5' ESTs into expression
vectors such as pXTl as
described below, or by constructing promoter-signal sequence-reporter gene
vectors which encode fusion
proteins between the signal peptide and an assayable reporter protein. After
introduction of these vectors into
a suitable host cell, such as COS cells or NIH 3T3 cells, the growth medium
may be harvested and analyzed
for the presence of the secreted protein. The medium from these cells is
compared to the medium from
control cells containing vectors lacking the signal sequence or extended cDNA
insert to identify vectors
which encode a functional signal peptide or an authentic secreted protein.
EXAMPLE 13
Analysis of the Sequences of the Invention
[0181] The set of the nucleic acid sequences of the invention (SEQ ID NOs. 24-
13309 and
26596-52153) was obtained as described in Example 9. Subsequently, the most
probable open reading
frame was determined and signal sequences were searched, as described in
Examples 10 and 11, for all
sequences of the invention.
[0182] The nucleotide sequences of the SEQ >D NOs. 24-13309 and 26596-52153
and the
preferred polypeptides sequences encoded by SEQ ID NOs. 24-13309 (i.e.
polypeptide sequences of SEQ
ID NOs. 1331(1-26595) are provided in the appended sequence listing. In
addition, for each of the nucleic
acid sequences of the invention as referred to by its sequence identification
number in the first column,
Table I provides the nucleotide positions of the first and last codons for
each of the corresponding open
reading frames in the second column.
[0183] For each of the consensus contigated 5'ESTs of the invention as
referred to by its
sequence identification number in the first column of Table II, the second
column gives a list of the
internal clone :IDs, ending in a letter, followed by the nucleotide positions
of the 5'EST clones that were
used to obtain this consensus contigated 5'EST. The internal clone IDs and the
nucleotide positions are
separated by a colon and each clone ID with its nucleotide positions are
separated by a semi-colon. For
example, where the first column indicates 40 and the second column indicates
105-045-3-0-G6-F:1-
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364;107-003-1-0-B3-F:11-245;117-005-1-0-DS-F:12-354, this means that the
consensus contigated
5'ESTs of SEQ ID N0:40 was computed from 3 different 5'EST clones, the first
one having the clone ID
105-045-3-0-G6-F matching from nucleotide positions 1 to 364 of the consensus
contigated 5'EST, the
second clone, 107-003-1-0-B3-F, from nucleotide positions 11 to 245 of the
consensus contigated 5'EST,
and the third clone, 117-005-1-0-DS-F, from nucleotide positions 12 to 354 of
the consensus contigated
5'EST.
[0184] If one of the nucleic acid sequences of SEQ ID NOs. 24-13309 and 26596-
52153 is
suspected of containing one or more incorrect or ambiguous nucleotides, the
ambiguities can readily be
resolved by resequencing a fragment containing the nucleotides to be
evaluated. If one or more incorrect
or ambiguous nucleotides are detected, the corrected sequences should be
included in the clusters from
which the sequences were isolated, and used to compute other consensus
contigated sequences on which
other ORFs would be identified. Nucleic acid fragments for resolving
sequencing errors or ambiguities
may be obtained from deposited clones or can be isolated using the techniques
described herein.
Resolution of any such ambiguities or errors may be facilitated by using
primers which hybridize to
1 S sequences located close to the ambiguous or erroneous sequences. For
example, the primers may
hybridize to sequences within 50-75 bases of the ambiguity or error. Upon
resolution of an error or
ambiguity, the corresponding corrections can be made in the protein sequences
encoded by the DNA
containing the error or ambiguity. The amino acid sequence of the protein
encoded by a particular clone
can also be determined by expression of the clone in a suitable host cell,
collecting the protein, and
determining its sequence.
[0185] In addition, if one of the sequences of SEQ >D NOs. 13310-26595 is
suspected of containing
a truncated ORF as the result of a frameshift in the sequence, such
frameshifting errors may be corrected by
combining the following two approaches. The first one involves thorough
examination of all double
predictions, i.e. all cases where the probability scores as defined in Example
10 for two ORFs located on
2S different reading frames are high and close, preferably different by less
than 0.4. The fine examination of
the region where the two possible ORFs overlap may help to detect the
frameshift. In the second
approach, homologies with known proteins are used to correct suspected
frameshifts.
[0186] Of the identified clusters, some were shown to be multivariant, i.e. to
contain several
variants of the same gene. Table III lists the internal reference ID for each
of the multivariant clusters (first
column) and the corresponding list of the SEQ )D Nos. for all variant
consensus contigated 5'ESTs (second
column).
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EXAMPLE 14
Categorization of 5' ESTs and Consensus Conti~ated 5'ESTs
[0187] The nucleic acid sequences of the present invention (SEQ ID NOs. 24-
13309 and 26596-
52153) were grouped based on their homology to known sequences as follows. All
sequences were
compared to the nucleic acid sequences of all vertebrates present in the EMBL
release 58 and Genseqn
(Derwent 's database of patented nucleic acids) releases 35.3 or release 36.
It should be noted that, because
of the large number of sequences of the invention, the comparison of the
polynucleotides of the invention to
public sequences was done in a time frame of 15 days, meaning that the first
sequences were compared to
Geneseqn release 35.3 and the last ones with Geneseqn release 36. The
comparison to EMBL vertebrate
sequences was performed after masking of repeated sequences and using blastn
with the parameters S=108
and X=16 followed by fasta. 7'he comparison to Geneseqn was performed after
masking of repeated
sequences and using blastn with the parameters S=108 and X=16.
(0188] All matches with a minimum of 30 nucleotides with 95% identity or 100%
identity were
retrieved and used to compute Tables IVa and IVb respectively. Tables IVa and
Nb give for each sequence
of the invention referred to by its sequence identification number in the
first column, the positions of their
preferred fragments in the second column entitled "Positions of preferred
fragments." These preferred
fragments are novel fragments which do not match any publicly available
vertebrate sequence according to
the algorithm, parameters and criteria defined above. As used herein the term
"polynucleotides described in
Tables IVa and IVb" refers to all of the preferred polynucleotide fragments
defined in Tables IVa and IVb in
this manner.
[0189] The term "polynucleotides described in Tables IVa and IVb updated"
refers to all of the
preferred polynucleotide fragments defined in manner described above except
that the most recent updates of
the EMBL and Derwent databases are used to define the preferred fragments as
of the filing date of the
instant application.
[0190] The present invention encompasses isolated, purified, or recombinant
nucleic acids which
consist of, consist essentially of, or comprise a contiguous span of at least
8, 10, 12, 15, 18, 20, 25, 35, 40,
50, 70, 80, 100, 250, or 500 nucleotides in length, to the extent that a
contiguous span of these lengths is
consistent with the lengths of the particular polynucleotide, of a
polynucleotide described in Tables IVa
and IVb, or a sequence complementary thereto, wherein said polynucleotide
described in Tables IVa and IVb
is selected individually or in any combination from the polynucleotides
described in Tables IVa and IVb. In
particular, the present invention encompasses isolated, purified, or
recombinant vertebrate nucleic acids
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which consist of; consist essentially of, or comprise a contiguous span of at
least 8, 10, 12, 15, 18, 20, 25, 35,
40, 50, 70, 80, 100, 250, or 500 nucleotides in length, to the extent that a
contiguous span of these lengths
is consistent with the lengths of the particular polynucleotide, of a
polynucleotide described in Tables IVa
and Nb, or a sequence complementary thereto, wherein said polynucleotide
described in Tables IVa and IVb
is selected individually or in any combination from the polynucleotides
described in Tables IVa and IVb. In
particular, the present invention encompasses isolated, purified, or
recombinant human nucleic acids which
consist of, consist essentially of, or comprise a contiguous span of at least
8, 10, 12, 15, 18, 20, 25, 35, 40,
50, 70, 80, 100, 250, or 500 nucleotides in length, to the extent that a
contiguous span of these lengths is
consistent with the lengths of the particular polynucleotide, of a
polynucleotide described in Tables IVa
and IVb, or a sequence complementary thereto, wherein said polynucleotide
described in Tables IVa and IVb
is selected individually or in any combination from the polynucleotides
described in Tables Na and IVb.
[0191 ] The present invention also encompasses isolated, purified, or
recombinant nucleic acids
which comprise, consist of, or consist essentially of a polynucleotide
described in Tables IVa and IVb, or a
sequence complementary thereto, wherein said polynucleotide is selected
individually or in any combination
from the polynucleotides described in Tables IVa and IVb. In particular, the
present invention encompasses
isolated, purified, or recombinant vertebrate nucleic acids which comprise,
consist of, or consist essentially of
a polynucleotide described in Tables IVa and IVb, or a sequence complementary
thereto, wherein said
polynucleotide is selected individually or in any combination from the
polynucleotides described in Tables
Na and IVb. In particular, the present invention encompasses isolated,
purified, or recombinant human
nucleic acids which comprise, consist of, or consist essentially of a
polynucleotide described in Tables IVa
and IVb, or a sequence complementary thereto, wherein said polynucleotide is
selected individually or in any
combination from the polynucleotides described in Tables IVa and IVb.
[0192] The present invention encompasses isolated, purified, or recombinant
nucleic acids which
consist of, consist essentially of, or comprise a contiguous span of at least
8, 10, 12, 15, 18, 20, 25, 35, 40,
50, 70, 80, 10(1, 250, or 500 nucleotides in length, to the extent that a
contiguous span of these lengths is
consistent with the lengths of the particular polynucleotide, of a
polynucleotide described in Tables IVa
and IVb, or a sequence complementary thereto, wherein said polynucleotide
described in Tables IVa and IVb
is selected individually or in any combination from the polynucleotides
described in Tables Na and IVb
updated. In particular, the present invention encompasses isolated, purified,
or recombinant vertebrate
nucleic acids which consist of, consist essentially of, or comprise a
contiguous span of at least 8, 10, 12, 15,
18, 20, 25, 35, 40, 50, 70, 80, 100, 250, or 500 nucleotides in length, to the
extent that a contiguous span
of these lengths is consistent with the lengths of the particular
polynucleotide, of a polynucleotide
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described in Tables IVa and IVb, or a sequence complementary thereto, wherein
said polynucleotide
described in Tables IVa and IVb is selected individually or in any combination
from the polynucleotides
described in Tables IVa and IVb updated. In particular, the present invention
encompasses isolated, purified,
or recombinant human nucleic acids which consist of, consist essentially of,
or comprise a contiguous span of
at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, or 500
nucleotides in length, to the extent
that a contiguous span of these lengths is consistent with the lengths of the
particular polynucleotide, of a
polynucleotide described in Tables IVa and IVb, or a sequence complementary
thereto, wherein said
polynucleotide described in Tables IVa and IVb is selected individually or in
any combination from the
polynucleotides described in Tables TVa and IVb updated.
[0193] The present invention also encompasses isolated, purified, or
recombinant nucleic acids
which comprise, consist of, or consist essentially of a polynucleotide
described in Tables 1Va and IVb, or a
sequence complementary thereto, wherein said polynucleotide is selected
individually or in any combination
from the polynucleotides described in Tables Na and Nb updated. In particular,
the present invention
encompasses isolated, purified, or recombinant vertebrate nucleic acids which
consist of or consist essentially
of a polynucleotide described in Tables IVa and IVb, or a sequence
complementary thereto, wherein said
polynucleotide is selected individually or in any combination from the
polynucleotides described in Tables
IVa and IVb updated. In particular, the present invention encompasses
isolated, purified, or recombinant
human nucleic acids which consist of or consist essentially of a
polynucleotide described in Tables 1Va and
IVb, or a sequence complementary thereto, wherein said polynucleotide is
selected individually or in any
combination from the polynucleotides described in Tables IVa and IVb updated.
III Evaluation of Spatial and Temporal Expression of mRNAs Corresponding to
the 5'ESTs or
Extended cDNAs
[0194] Each of the SEQ )D NOs. 24-13309 and 26596-52153 was also categorized
based on the
tissue from which its corresponding mRNA was obtained, as described below in
Example 15.
EXAMPLE 15
Expression Patterns of mRNAs From Which the 5'ESTs were obtained
[0195] Table V shows the spatial distribution of each nucleic acid sequence of
the invention (SEQ
1D NOs. 24-13309 and 26596-52153) referred to by its internal designation in
the first column and the
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number of individual 5'ESTs used to assemble the consensus contigated 5'ESTs
per tissue in the second
column. A singleton is thus represented by a single 5'EST from a single
tissue. Each type of tissue listed in
Table V is encoded by a letter. T'he correspondence between the letter code
and the tissue type is given in
Table VI. For example, if the first column contains 47 and the second column
contains the following list: A:1
C:4 F:3, this means that the consensus contigated 5'EST of SEQ ID NO. 24 was
obtained from 17 5'EST
from brain, and thirty 5'ESTs from placenta.
[0196] The bias in spatial distribution of the polynucleotide sequences of the
sequence listing were
examined by comparing the relative proportions of the biological 5'ESTs of a
given tissue in each cluster
using the following statistical analysis. The under- or over-representation of
5'ESTs of a given cluster in a
given tissue was performed using the normal approximation of the binomial
distribution. When the observed
proportion of 5'ESTs of a given tissue in a given consensus had less than 1%
chance to occur randomly
according to the chit test, the frequency bias was reported as "low" or
"high". The results are given in Table
VII as follows. For each consensus contigated 5'ESTs showing a bias in tissue
distribution as referred to by
its sequence identification number in the first column, the list of tissues
where some 5'ESTs were under-
represented is given in the second column entitled "low frequency" and the
list of tissues where some 5'ESTs
are over-represented is given in the third column entitled "high frequency".
[0197] The bias in spatial distribution of the polynucleotide sequences of the
sequence listing were
also examined by comparing them to the polynucleotide sequences in pubic
databases. Table VIII lists
tissues and cell types which express the polynucleotides of the sequence
listing.. Column one lists the SEQ
ID NO and column two lists the corresponding tissues and cell types that were
found to express the
polynucleotide sequences using information from public databases. The number
in parentheses to the right of
the tissue or cell type in column two represents the number of files in the
databases listing that tissue or cell
type as expressing the sequence of column 1.
[0198] The chromosomal location of the 5' ESTs were also determined using
sequence information
from public databases. Table IX lists the SEQ ID NO in column one and the
corresponding chromosomal
location in column two.
[0199] In addition to categorizing the 5' ESTs and consensus contigated 5'
ESTs with respect to
their tissue of origin, the spatial and temporal expression patterns of the
mRNAs corresponding to the 5'
ESTs and consensus contigated 5' ESTs, as well as their expression levels, may
be determined as described in
Example 16 below.
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[0200] Characterization of the spatial and temporal expression patterns and
expression levels of
these mRNAs is useful for constructing expression vectors capable of producing
a desired level of gene
product in a desired spatial or temporal manner, as will be discussed in more
detail below.
[0201 ] Furthermore, 5' E STs and consensus contigated 5' ESTs whose
corresponding mRNAs are
associated with disease states may also be identified. For example, a
particular disease may result from the
lack of expression, over expression, or under expression of a mRNA
corresponding to a 5' EST or consensus
contigated 5' EST. By comparing mRNA expression patterns and quantities in
samples taken from healthy
individuals with those from individuals suffering from a particular disease,
S' ESTs or consensus contigated
5' ESTs responsible for the disease may be identified.
[0202] It will be appreciated that the results of the above characterization
procedures for 5' ESTs
and consensus contigated 5' ESTs also apply to extended cDNAs (obtainable as
described below) which
contain sequences adjacent to the 5' ESTs and consensus contigated 5' ESTs. It
will also be appreciated that
if desired, characterization may be delayed until extended cDNAs have been
obtained rather than
characterizing the 5' ESTs or consensus contigated 5' ESTs themselves.
EXAMPLE 16
Evaluation of Expression Levels and Patterns of mRNAs Corresponding to EST-
Related Nucleic Acids
[0203] Expression levels and patterns of mRNAs corresponding to EST-related
nucleic acids may
be analyzed by solution hybridization with long probes as described in
International Patent Application No.
WO 97/05277, the entire contents of which are hereby incorporated by
reference. Briefly, an EST-related
nucleic acid, fragment of an EST related nucleic acid, positional segment of
an EST-related nucleic acid, or
fragment of a positional segment of an EST-related nucleic acid corresponding
to the gene encoding the
mRNA to be characterized is inserted at a cloning site immediately downstream
of a bacteriophage (T3, T7 or
SP6) RNA polymerase promoter to produce antisense RNA. Preferably, the EST-
related nucleic acid,
fragment of an EST related nucleic acid, positional segment of an EST-related
nucleic acid, or fragment of a
positional segment of an EST-related nucleic acid is 100 or more nucleotides
in length. The plasmid is
linearized and transcribed in the presence of ribonucleotides comprising
modified ribonucleotides (i.e. biotin-
UTP and DIG-UTP). An excess of this doubly labeled RNA is hybridized in
solution with mRNA isolated
from cells or tissues of interest. The hybridizations are performed under
standard stringent conditions (40-
50°C for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The
unhybridized probe is removed
by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases
CL3, TI, Phy M, U2 or A).
The presence of the biotin-UTP modification enables capture of the hybrid on a
microtitration plate coated
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with streptavidin. The presence of the DIG modification enables the hybrid to
be detected and quantified by
ELISA using an anti-DIG antibody coupled to alkaline phosphatase.
[0204] The EST-related nucleic acid, fragment of an EST related nucleic acid,
positional segment of
an EST-related nucleic acid, or fragment of a positional segment of an EST-
related nucleic acid may also be
tagged with nucleotide sequences for the serial analysis of gene expression
(SAGE) as disclosed in LTK Patent
Application No. 2 305 241 A, the entire contents of which are incorporated by
reference. In this method,
cDNAs are prepared from a cell, tissue, organism or other source of nucleic
acid for which gene expression
patterns must be determined. The resulting cDNAs are separated into two pools.
The cDNAs in each pool
are cleaved with a first restriction endonuclease, called an anchoring enzyme,
having a recognition site which
is likely to be present at least once in most cDNAs. The fragments which
contain the 5' or 3' most region of
the cleaved eDNA are isolated by binding to a capture medium such as
streptavidin coated beads. A first
oligonucleotide linker having a first sequence for hybridization of an
amplification primer and an internal
restriction site for a so called tagging endonuclease is ligated to the
digested cDNAs in the first pool.
Digestion with the second endonuclease produces short tag fragments from the
cDNAs.
[0205] A second oligonucleotide having a second sequence for hybridization of
an amplification
primer and an internal restriction site is ligated to the digested cDNAs in
the second pool. The cDNA
fragments in the second pool are also digested with the tagging endonuclease
to generate short tag fragments
derived from the cDNAs in the second pool. The tags resulting from digestion
of the first and second pools
with the anchoring enzyme and the tagging endonuclease are ligated to one
another to produce so called
ditags. In some embodiments, the ditags are concatamerized to produce ligation
products containing from 2
to 200 ditags. The tag sequences are then determined and compared to the
sequences of the EST-related
nucleic acid, fragment of an EST related nucleic acid, positional segment of
an EST-related nucleic acid, or
fragment of a positional segment of an EST-related nucleic acid to determine
which 5' ESTs, consensus
contigated 5' ESTs, or extended cDNAs are expressed in the cell, tissue,
organism, or other source of nucleic
acids from which the tags were derived. In this way, the expression pattern of
the S' ESTs, consensus
contigated 5' ESTs, or extended cDNAs in the cell, tissue, organism, or other
source of nucleic acids is
obtained.
[0206] Quantitative analysis of gene expression may also be performed using
arrays. As used
herein, the term array means a one-dimensional, two-dimensional, or
multidimensional arrangement of EST-
related nucleic acids, fragments of EST related nucleic acids, positional
segments EST-related nucleic acids,
or fragments of positional segments of EST-related nucleic acids. Preferably,
the EST-related nucleic acids,
fragments of EST related nucleic acids, positional segments EST-related
nucleic acids, or fragments of
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positional segments of EST-related nucleic acids are at least 15 nucleotides
in length. More preferably, the
EST-related nucleic acids, fragments of EST related nucleic acids, positional
segments EST-related nucleic
acids, or fragments of positional segments of EST-related nucleic acids are at
least 100 nucleotide long.
More preferably, the fragments are more than 100 nucleotides in length. In
some embodiments, the EST-
related nucleic acids, fragments of EST related nucleic acids, positional
segments EST-related nucleic acids,
or fragments of positional segments of EST-related nucleic acids may be more
than 500 nucleotides long.
[0207] For example, quantitative analysis of gene expression may be performed
with EST-related
nucleic acids, fragments of EST related nucleic acids, positional segments EST-
related nucleic acids, or
fragments of positional segments of EST-related nucleic acids in a
complementary DNA microarray as
described by Schena et al. (Science 270:467-470, 1995; Proc. Natl. Acad. Sci.
U.S.A. 93:10614-10619, 1996).
EST-related nucleic acids, fragments of EST related nucleic acids, positional
segments EST-related nucleic
acids, or fragments of positional segments of EST-related nucleic acids are
amplified by PCR and arrayed
from 96-well microtiter plates onto silylated microscope slides using high-
speed robotics. Printed arrays are
incubated in a humid chamber to allow rehydration of the array elements and
rinsed, once in 0.2% SDS for 1
min, twice in water for 1 min and once for 5 min in sodium borohydride
solution. The arrays are submerged
in water for 2 min at 95°C, transferred into 0.2% SDS for 1 min, rinsed
twice with water, air dried and stored
in the dark at 25°C.
[0208] Cell or tissue mRNA is isolated or commercially obtained and probes are
prepared by a
single round of reverse transcription. Probes are hybridized to 1 cmZ
microarrays under a 14 x 14 mm glass
coverslip for 6-12 hours at 60°C. Arrays are washed for 5 min at
25°C in low stringency wash buffer (1 x
SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash
buffer (0.1 x SSC/0.2% SDS).
Arrays are scanned in 0.1 x SSC using a fluorescence laser scanning device
fitted with a custom filter set.
Accurate differential expression measurements are obtained by taking the
average of the ratios of two
independent hybridizations.
[0209] Quantitative analysis of the expression of genes may also be performed
with EST-related
nucleic acids, fragments of EST related nucleic acids, positional segments EST-
related nucleic acids, or
fragments of positional segments of EST-related nucleic acids in complementary
DNA arrays as described by
Pietu et al. (Genome Research 6:492-503, 1996). The EST-related nucleic acids,
fragments of EST related
nucleic acids, positional segments EST-related nucleic acids, or fragments of
positional segments of EST-
related nucleic acids thereof are PCR amplified and spotted on membranes.
Then, mRNAs originating from
various tissues or cells are labeled with radioactive nucleotides. After
hybridization and washing in
controlled conditions, the hybridized mRNAs are detected by phospho-imaging or
autoradiography.
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Duplicate experiments are performed and a quantitative analysis of
differentially expressed mRNAs is then
performed.
[0210] Alternatively, expression analysis of the EST-related nucleic acids,
fragments of EST related
nucleic acids, positional segments EST-related nucleic acids, or fragments of
positional segments of EST-
related nucleic acids can be done through high density nucleotide arrays as
described by Lockhart et al.
(Nature Biotechnology 14: 1675-1680, 1996) and Sosnowsky et al. (Proc. Natl.
Acad. Sci. 94:1119-1123,
1997). Oligonucleotides of 15-50 nucleotides corresponding to sequences of EST-
related nucleic acids,
fragments of EST related nucleic acids, positional segments EST-related
nucleic acids, or fragments of
positional segments of EST-related nucleic acids are synthesized directly on
the chip (Lockhart et al., supra)
or synthesized and then addressed to the chip (Sosnowsky et al., supra).
Preferably, the oligonucleotides are
about 20 nucleotides in length.
[0211] cDNA probes labeled with an appropriate compound, such as biotin,
digoxigenin or
fluorescent dye, are synthesized from the appropriate mRNA population and then
randomly fragmented to an
average size of 50 to 100 nucleotides. The said probes are then hybridized to
the chip. After washing as
described in Lockhart et al, supra and application of different electric
fields (Sonowsky et al, supra.), the
dyes or labeling compounds are detected and quantified. Duplicate
hybridizations are performed.
Comparative analysis of the intensity of the signal originating from cDNA
probes on the same target
oligonucleotide in different cDNA samples indicates a differential expression
of the mRNA corresponding to
the 5' EST, consensus contigated 5' EST or extended cDNA from which the
oligonucleotide sequence has
been designed.
IV. Use of 5' ESTs or Consensus Conti~ated 5'ESTs to Clone Extended cDNAs and
to Clone the
Correspondins~ Genomic DNAs
[0212] Once 5' ESTs or consensus contigated 5' ESTs which include the 5' end
of the
corresponding mRNAs have been selected using the procedures described above,
they can be utilized to
isolate extended cDNAs which contain sequences adjacent to the 5' ESTs or
consensus contigated 5' ESTs.
The extended cDNAs may include the entire coding sequence of the protein
encoded by the corresponding
mRNA, including the authentic translation start site. If the extended cDNA
encodes a secreted protein, it may
contain the signal sequence, and the sequence encoding the mature protein
remaining after cleavage of the
signal peptide. Extended cDNAs which include the entire coding sequence of the
protein encoded by the
corresponding mRNA are referred to herein as "full-length cDNAs."
Alternatively, the extended cDNAs
may not include the entire coding sequence of the protein encoded by the
corresponding mRNA, although
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they do include sequences adjacent to the 5'ESTs or consensus contigated 5'
ESTs. In some embodiments in
which the extended cDNAs are derived from an mRNA encoding a secreted protein,
the extended cDNAs
may include only the sequence encoding the mature protein remaining after
cleavage of the signal peptide, or
only the sequence encoding the signal peptide.
[0213] Example 17 below describes a general PCR based method for obtaining
extended cDNAs
using 5' ESTs or consensus contigated 5' ESTs. Example 18 describes
hybridization based methods to
obtain genomic DNAs which encode the mRNAs from which the 5' ESTs or consensus
contigated 5' ESTS
were derived, mRNAs from which the 5' ESTs or consensus contigated 5' ESTS
were derived, or nucleic
acids which are homologous to 5' ESTs-related nucleic acids. Example 19 below
describes the cloning and
sequencing of several extended eDNAs, including extended cDNAs which include
the entire coding sequence
and authentic 5' end of the corresponding mRNA for several secreted proteins.
[0214] The methods of Examples 17 and 18 can also be used to obtain extended
cDNAs which
encode less than the entire coding sequence of proteins encoded by the genes
corresponding to the 5' ESTs or
consensus contigated ESTs. In some embodiments, the extended cDNAs isolated
using these methods
encode at least 5,10, 15, 20, 25, 3U, 35, 40, 50, 75, 100, or 150 consecutive
amino acids of one of the proteins
encoded by the sequences of SEQ 1D NOs. 24-13309 and 26596-52153. In some
embodiments, the extended
cDNAs isolated using these methods encode at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150
consecutive amino acids of one of the proteins encoded by the sequences of SEQ
>D NOs. 24-13309.
EXAMPLE 17
General Method for Using 5' ESTs or C',onsensus Conti~ated 5'ESTs to Clone and
Sequence Extended
cDNAs which Include the Entire Coding Region and the Authentic 5'End of the
Corres~ondin~ mRNA
[0215] The following general method may be used to quickly and efficiently
isolate extended
cDNAs including sequence adjacent to the sequences of the 5' ESTs or consensus
contigated 5'ESTs used to
obtain them. This method may be applied to obtain extended cDNAs for any 5'
EST or consensus contigated
5' EST of the invention, including those 5' ESTs and consensus contigated 5'
ESTs encoding secreted
proteins. This method is illustrated in Figure 3.
I . Obtaining Extended cDNAs
[0216] The method takes advantage of the known 5' sequence of the mRNA. A
reverse
transcription reaction is conducted on purified mRNA with a poly dT primer
containing a nucleotide
sequence at its 5' end allowing the addition of a known sequence at the end of
the cDNA which corresponds
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to the 3' end of the mRNA. Such a primer and a commercially-available reverse
transcriptase enzyme are
added to a buffered mRNA sample yielding a reverse transcript anchored at the
3' polyA site of the RNAs.
Preferably, a thermostable enzyme is used. Nucleotide monomers are then added
to complete the first strand
synthesis.
[0217] After removal of the mRNA hybridized to the first cDNA strand by
alkaline hydrolysis, the
products of the alkaline hydrolysis and the residual poly dT primer can be
eliminated with an exclusion
column.
[0218] Subsequently, a pair of nested primers on each end of the cDNA to be
amplified is designed
based on the known 5' sequence from the 5' EST or consensus contigated 5' EST
and the known 3' end
added by the poly dT primer used in the first strand synthesis. Software used
to design primers are either
based on GC content and melting temperatures of oligonucleotides, such as OSP
(Illier and Green, PCR
Meth. Appl. 1:124-128, 1991), or based on the octamer frequency disparity
method (Griffais et al., Nucleic
Acids Res. 19: 3887-3891, 1991 ) such as PC-Rare (http://
bioinformatics.weizmann.ac.il/software/PC-
Rare/doc/manuel.html). Preferably, the nested primers at the 5' end and the
nested primers at the 3' end are
1 S separated from one another by four to nine bases. These primer sequences
may be selected to have melting
temperatures and specificities suitable for use in PCR.
[0219] A first PCR run is performed using the outer primer from each of the
nested pairs. A second
PCR run using the inner primer from each of the nested pairs is then performed
on a small sample of the first
PCR product. Thereafter, the primers and remaining nucleotide monomers are
removed.
[0220] It will be appreciated that a simple PCR reaction using a primer on
each end of the cDNA to
be amplified may also be performed rather than using a couple of primers for
in a nested PCR procedure.
However because of the possibility of PCR artifacts in this method, a nested
PCR protocol is preferred.
2. Seguencing Extended cDNAs or Fragments Thereof
[0221] Due to the lack of position constraints on the design of 5' nested
primers compatible for
PCR use using the OSP software, amplicons of two types are obtained.
Preferably, the second S' primer is
located upstream of the translation initiation codon thus yielding a nested
PCR product containing the entire
coding sequence. Such an extended cDNA may be used in a direct cloning
procedure as described in section
a below. However, in some cases, the second 5' primer is located downstream of
the translation initiation
codon, thereby yielding a PCR product containing only part of the ORF. Such
incomplete PCR products are
submitted to a modified procedure described in section b below.
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a) Nested PCR products containing complete ORFs
[0222] When the resulting nested PCR product contains the complete coding
sequence, as predicted
from the 5'EST or consensus contigated 5' EST sequence, it is directly cloned
in an appropriate vector as
described in section 3..
b) Nested PCR products containing incomplete ORFs
[0223] When the amplicon does not contain the complete coding sequence,
intermediate steps
are necessary to obtain both the complete coding sequence and a PCR product
containing the full coding
sequence. The complete coding sequence can be assembled from several partial
sequences determined
directly from different PCR products.
[0224] Once the full coding sequence has been completely determined, new
primers compatible
for PCR use are then designed to obtain amplicons containing the whole coding
region. However, in such
cases, 3' primers compatible for PCR use are located inside the 3' UTR of the
corresponding mRNA, thus
yielding amplicons which lack part of this region, i.e. the polyA tract and
sometimes the polyadenylation
signal, as illustrated in Figure 3. Such extended cDNAs are then cloned into
an appropriate vector as
described in section 3.
c) Sequencing extended cDNAs
[0225] Sequencing of extended cDNAs can be performed using a Dye Terminator
approach with
the AmpliTaq DNA polymerase FS kit available from Perkin Elmer.
[0226] In order to sequence long PCR fragments, primer walking is performed
using software such
as OSP to choose primers and automated computer software such as ASMG (Sutton
et al., Genome Science
Technol. 1: 9-19, 1995) to construct contigs of walking sequences including
the initial 5' tag. Preferably,
primer walking is performed until the sequences of full length cDNAs are
obtained.
[0227] Completion of the sequencing of a given extended cDNA fragment may be
assessed by
comparing the sequence length to the size of the corresponding nested PCR
product. When Northern blot
data are available, the size of the mRNA detected for a given PCR product may
also be used to finally assess
that the sequence is complete. Sequences which do not fulfill these criteria
are discarded and will undergo a
new isolation procedure.
3. Cloning~Extended cDNAs
[0228] The PCR product containing the full coding sequence is then cloned in
an appropriate
vector. For example, the extended cDNAs can be cloned into any expression
vector known in the art, such as
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pED6dpc2 for extended cDNA encoding potentially secreted proteins
(DiscoverEase, Genetics Institute,
Cambridge, MA).
[0229] Cloned PCR products are then entirely sequenced in order to obtain at
least two
sequences per clone.. Preferably, the sequences are obtained from both sense
and antisense strands
according to the aforementioned procedure with the following modifications.
First, both 5' and 3' ends of
cloned PCR products are sequenced in order to confirm the identity of the
clone. Second, primer walking
is performed if the full coding region has not been obtained yet. Contigation
is then performed using
primer walking sequences for cloned products as well as walking sequences that
have already contigated
for uncloned PC'.R products. The sequence is considered complete when the
resulting contigs include the
whole coding region as well as overlapping sequences with vector DNA on both
ends. All the contigated
sequences for each cloned amplicon are then used to obtain a consensus
sequence.
4. Selection of Cloned Full length Sequences
a) Computer analysis of extended cDNAs
[0230] Following identification of contaminants and masking of repeats,
structural features, e.g.
polyA tail and polyadenylation signal, of the sequences of extended cDNAs are
subsequently determined
using methods known to those skilled in the art. For example, the algorithm,
parameters and criteria
defined in Figure 10 may be used. Briefly, a polyA tail is defined as a
homopolymeric stretch of at least
I1 A with at most one alternative base within it. The polyA tail search is
restricted to the last 20
nucleotides of the sequence and limited to stretches of 11 consecutive A's
because sequencing reactions
are often not readable after such a polyA stretch. To search for a
polyadenylation signal, the polyA tail is
clipped from the full-length sequence. The 50 nucleotides preceding the polyA
tail are searched for the
canonic polyadenylation AAUAAA signal allowing one mismatch to account for
possible sequencing
errors as well as known variation in the canonical sequence of the
polyadenylation signal.
[0231] Functional features, e.g. ORFs and signal sequences, of the sequences
of extended cDNAs
are subsequently determined as follows. The 3 upper strand frames of extended
cDNAs are searched for
ORFs defined as the maximum length fragments beginning with a translation
initiation codon and ending
with a stop codon. ORFs encoding at least 80 amino acids are preferred. If
extended cDNAs encoding
secreted proteins are desired, each identified ORF is then scanned for the
presence of a signal peptide using
the matrix method described in Example 1 1.
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[0232] Sequences of extended cDNAs are then compared, on a nucleotidic or
proteic basis, to
public sequences available at the time of filing.
b) Selection of full-length cDNAs of interest
[0233] A negative selection may then be performed in order to eliminate
unwanted cloned
sequences resulting from either contaminants or PCR artifacts as follows.
Sequences matching contaminant
sequences such as vector DNA, tRNA, mtRNA, rRNA sequences are discarded as
well as those encoding
ORF sequences exhibiting extensive homology to repeats. Sequences obtained by
direct cloning (section la)
but lacking polyA tail may be discarded. Only ORFs ending either before the
polyA tail (section la) or
before the end of the cloned 3'LTTR (section lb) may be selected. If extended
cDNAs encoding secreted
proteins are desired, ORFs containing a signal peptide are considered. In
addition, ORFs containing unlikely
mature proteins such as mature proteins which size is less than 20 amino acids
or less than 25% of the
immature protein size may be eliminated if necessary.
[0234] Then, for each remaining full length cDNA containing several ORFs, a
preselection of ORFs
may be performed using the following criteria. The longest ORF is preferred.
If extended cDNAs encoding
secreted proteins are desired and if the ORF sizes are similar, the chosen ORF
is the one which signal peptide
has the highest score according to Von Heijne method.
[0235] Sequences of full length cDNA clones may then be compared pair-wise
after masking of the
repeat sequences. Full-length cDNA sequences exhibiting extensive homology may
be clustered in the same
class. Each cluster may then be subjected to a cluster analysis that detects
sequences resulting from internal
priming or from alternative splicing, identical sequences or sequences with
several frameshifts. A selection
may be operated between clones belonging to the same class in order to detect
clones encoding homologous
but distinct ORFs which may be both selected if they both contain sequences of
interest.
[0236] Selection of full-length cDNA clones encoding sequences of interest may
subsequently be
performed using the following criteria. Structural parameters (initial tag,
polyadenylation site and signal) are
first checked. 'IMen, homologies with known nucleic acids and proteins are
examined in order to determine
whether the clone sequence match a known nucleotide/protein sequence and, in
the latter case, its covering
rate and the date at which the sequence became public. If there is no
extensive match with sequences other
than ESTs or genomic DNA, or if the clone sequence provides substantial new
information, such as encoding
a protein resulting from alternative splicing of an mRNA coding for an already
known protein, the sequence
is kept. Examples of such cloned full-length cDNAs containing sequences of
interest are described in
Example 19. Sequences resulting from chimera or double inserts or located on
chromosome breaking points
as assessed by homology to other sequences may be discarded during this
procedure.
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[0237] Extended cDNAs prepared as described above may be subsequently
engineered to obtain
nucleic acids which include desired portions of the extended eDNA using
conventional techniques such as
subcloning, PCR, or in vitro oligonucleotide synthesis. For example, nucleic
acids which include only the
full coding sequences may be obtained using techniques known to those skilled
in the art. Alternatively,
conventional techniques may be applied to obtain nucleic acids which contain
only part of the coding
sequences. In the case of nucleic acids encoding secreted proteins, nucleic
acids containing only the coding
sequence for the mature protein remaining after the signal peptide is cleaved
off or nucleic acids which
contain only the coding sequences for the signal peptides may be obtained.
[0238] Similarly, nucleic acids containing any other desired portion of the
coding sequences for the
encoded protein may be obtained. For example, the nucleic acid may contain at
least 10, 15, 18, 20, 25, 28,
30, 35, 40, 50, ?5, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive
bases of an extended cDNA.
[0239] Once an extended cDNA has been obtained, it can be sequenced to
determine the amino acid
sequence it encodes. Once the encoded amino acid sequence has been determined,
one can create and
identify any of the many conceivable cDNAs that will encode that protein by
simply using the degeneracy of
the genetic code. For example, allelic variants or other homologous nucleic
acids can be identified as
described below. Alternatively, nucleic acids encoding the desired amino acid
sequence can be synthesized
in vitro.
[0240] In a preferred embodiment, the coding sequence may be selected using
the known codon or
codon pair preferences for the host organism in which the cDNA is to be
expressed.
[0241] In addition to PC'R based methods for obtaining cDNAs which include the
authentic 5'end
of the corresponding mRNA as well as the complete protein coding sequence of
the corresponding mRNA,
traditional hybridization based methods may also be employed. These methods
may also be used to obtain
the genomic DNAs which encode the mRNAs from which the 5' ESTs or consensus
contigated 5' ESTS
were derived, mRNAs from which the 5' ESTs or consensus contigated 5' ESTS
were derived, or nucleic
acids which are homologous to EST-related nucleic acids. In particular, such
methods may be used to obtain
extended cDNAs which include the entire coding region of the mRNAs from which
the 5'EST or consensus
contigated 5'ESTs was derived. Example 18 below provides examples of such
methods.
EXAMPLE 18
Methods for Obtaining Extended cDNAs which Include the Entire Codin~~ion and
the Authentic
5'End of the Corresponding mRNA or Nucleic Acids Homologous to Extended eDNAs,
5' ESTs or
Consensus Conti~ated 5' ESTs
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[0242] A full-length cDNA library can be made using the strategies described
in Examples 1-5.
Alternatively, a cDNA library or genomic DNA library may be obtained from a
commercial source or made
using techniques familiar to those skilled in the art.
[0243] Such cDNA or genomic DNA libraries may be used to isolate extended
cDNAs obtained
from 5' ESTs or consensus contigated 5' ESTs or nucleic acids homologous to
extended cDNAs, 5' ESTs, or
consensus contigated 5' ESTs as follows. The cDNA library or genomic DNA
library is hybridized to a
detectable probe. The detectable probe may comprise at least 10, 1 S, 18, 20,
25, 28, 30, 35, 40, 50, 75, 100,
150, 200, 300, 400 or 500 consecutive nucleotides of the 5' EST, consensus
contigated 5' EST, or extended
cDNA. Techniques for identifying cDNA clones in a cDNA library which hybridize
to a given probe
sequence are disclosed in Sambrook et al., Molecular C,'loning: A Laboratory
Manual 2d Ed., Cold Spring
Harbor Laboratory Press, 1989, the disclosure of which is incorporated herein
by reference. The same
techniques may be used to isolate genomic DNAs.
[0244] Briefly, cDNA or genomic DNA clones which hybridize to the detectable
probe are
identified and isolated for further manipulation as follows. The detectable
probe described in the preceding
paragraph is labeled with a detectable label such as a radioisotope or a
fluorescent molecule. Techniques for
labeling the probe are well lrnown and include phosphorylation with
polynucleotide kinase, nick translation,
in vitro transcription, and non radioactive techniques. The cDNAs or genomic
DNAs in the library are
transferred to a nitrocellulose or nylon filter and denatured. After blocking
of non specific sites, the filter is
incubated with the labeled probe for an amount of time sufficient to allow
binding of the probe to cDNAs or
genomic DNAs containing a sequence capable of hybridizing thereto.
[0245] By varying the stringency of the hybridization conditions used to
identify cDNAs or
genomic DNAs which hybridize to the detectable probe, cDNAs or genomic DNAs
having different levels of
homology to the probe can be identified and isolated as described below.
1. Identification of cDNA or Genomic DNA Sequences Havin;~a High Degree of
Homology to the
Labeled Probe
(0246] To identify cDNAs or genomic DNAs having a high degree of homology to
the probe
sequence, the melting temperature of the probe may be calculated using the
following formulas:
[0247] For probes between 14 and 70 nucleotides in length the melting
temperature (Tm) is
calculated using the formula: Tm=81.5+16.6(log (Na+))+0.41(fraction G+C)-
(600/N) where N is the length
of the probe.
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[0248] If the hybridization is carried out in a solution containing formamide,
the melting
temperature may be calculated using the equation Tm=81.5+16.6(log
(Na+))+0.41(fraction G+C)-(0.63%
formamide)-(600/I~ where N is the length of the probe.
[0249] Prehybridization may be earned out in 6X SSC, 5X Denhardt's reagent,
0.5% SDS, 100 ~g
denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5%
SDS, 100 ~g denatured
fragmented salmon sperm DNA, 50% formamide. The formulas for SSC and
Denhardt's solutions are listed
in Sambrook et al., supra.
[0250] Hybridization is conducted by adding the detectable probe to the
prehybridization solutions
listed above. Where the probe comprises double stranded DNA, it is denatured
before addition to the
hybridization solution. The filter is contacted with the hybridization
solution for a sufficient period of time to
allow the probe to hybridize to extended cDNAs or genomic DNAs containing
sequences complementary
thereto or homologous thereto. For probes over 200 nucleotides in length, the
hybridization may be carried
out at 15-25°C below the Tm. For shorter probes, such as
oligonucleotide probes, the hybridization may be
conducted at 15-25°C below the Tm. Preferably, for hybridizations in 6X
SSC, the hybridization is
I 5 conducted at approximately 68°C. Preferably, for hybridizations in
50% formamide containing solutions, the
hybridization is conducted at approximately 42°C.
[0251] All of the foregoing hybridizations would be considered to be under
"stringent" conditions.
[0252] Following hybridization, the filter is washed in 2X SSC, 0.1% SDS at
room temperature for
minutes. The filter is then washed with O.1X SSC, 0.5% SDS at room temperature
for 30 minutes to 1
hour. Thereafter, the solution is washed at the hybridization temperature in
O.1X SSC, 0.5% SDS. A final
wash is conducted in O.1X SSC at room temperature.
[0253] cDNAs or genomic DNAs which have hybridized to the probe are identified
by
autoradiography or other conventional techniques.
2 Obtaining-cDNA or Genomic DNA Sequences Having Lower Degrees of Homology to
the Labeled
Probe
[0254] The above procedure may be modified to identify cDNAs or genomic DNAs
having
decreasing levels of homology to the probe sequence. For example, to obtain
cDNAs or genomic DNAs of
decreasing homology to the detectable probe, less stringent conditions may be
used. For example, the
hybridization temperature may be decreased in increments of 5°C from
68°C to 42°C in a hybridization
buffer having a sodium concentration of approximately 1 M. Following
hybridization, the filter may be
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washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These
conditions are considered to be
"moderate" conditions above 50°C and "low" conditions below
50°C.
(0255) Alternatively, the hybridization may be carried out in buffers, such as
6X SSC, containing
formamide at a temperature of 42°C. In this case, the concentration of
formamide in the hybridization buffer
may be reduced in 5% increments from 50% to 0% to identify clones having
decreasing levels of homology
to the probe. Following hybridization, the filter may be washed with bx ~~L,
u.~ ~° ~t~~ at ~u-~. i nese
conditions are considered to be "moderate" conditions above 25% formamide and
"low" conditions below
25% formamide.
[0256] cDNAs or genomic DNAs which have hybridized to the probe are identified
by
autoradiography.
3 Determination of the Degree of Homology between the Obtained cDNAs or
Genomic DNAs and
5'ESTs, Consensus Conti~ated 5'ESTs or Extended cDNAs or Between the
Polypeptides Encoded by the
Obtained cDNAs or Genomic DNAs and the Polypeptides Encoded by the 5'ESTs,
Consensus Conti aeYted
5'ESTs, or Extended cDNAs
[0257] To determine the level of homology between the hybridized cDNA or
genomic DNA and
the 5'EST, consensus contigated 5'EST or extended cDNA from which the probe
was derived, the nucleotide
sequences of the hybridized nucleic acid and the 5'EST, consensus contigated
5'EST or extended cDNA
from which the probe was derived are compared. The sequences of the 5'EST,
consensus contigated 5'EST
or extended cDNA from which the probe was derived and the sequences of the
cDNA or genomic DNA
which hybridized to the detectable probe may be stored on a computer readable
medium as described below
and compared to one another using any of a variety of algorithms familiar to
those skilled in the art, those
described below.
[0258] To determine the level of homology between the polypeptide encoded by
the hybridizing
cDNA or genomic DNA and the polypeptide encoded by the 5'EST, consensus
contigated 5'EST or extended
cDNA from which the probe was derived, the polypeptide sequence encoded by the
hybridized nucleic acid
and the polypeptide sequence encoded by the 5'EST, consensus contigated 5'EST
or extended cDNA from
which the probe was derived are compared. The sequences of the polypeptide
encoded by the 5'EST,
consensus contigated 5'EST or extended cDNA from which the probe was derived
and the polypeptide
sequence encoded by the cDNA or genomic DNA which hybridized to the detectable
probe may be stored on
a computer readable medium as described below and compared to one another
using any of a variety of
algorithms familiar to those skilled in the art, those described below.
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[0259] Protein and/or nucleic acid sequence homologies may be evaluated using
any of the
variety of sequence comparison algorithms and programs known in the art. Such
algorithms and
programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA,
TFASTA, and
CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-
2448; Altschul et al.,
1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res.
22(2):4673-4680; Higgins
et al., 1996, Methods Enrymol. 266:383-402; Altschul et al., 1990, J. Mol.
Biol. 215(3):403-410; Altschul
et al., 1993, Nature Genetics 3:266-272).
[0260] In a particularly preferred embodiment, protein and nucleic acid
sequence homologies are
evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well
known in the art (see,
e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268;
Altschul et al., 1990, J. Mol.
Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul
et al., 1997, Nuc. Acids
Res. 25:3389-3402). In particular, five specific BLAST programs are used to
perform the following task:
(1) BLASTP and BLAST3 compare an amino acid query sequence against a
protein sequence database;
(2) BLASTN compares a nucleotide query sequence against a nucleotide
sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query
nucleotide sequence (both strands) against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide
sequence database translated in all six reading frames (both strands); and
(5) TBLASTX compares the six-frame translations of a nucleotide query
sequence against the six-frame translations of a nucleotide sequence
database.
[0261] The BLAST programs identify homologous sequences by identifying similar
segments,
which are referred to herein as "high-scoring segment pairs," between a query
amino or nucleic acid
sequence and a test sequence which is preferably obtained from a protein or
nucleic acid sequence
database. High-scoring segment pairs are preferably identified (i.e., aligned)
by means of a scoring
matrix, many of which are known in the art. Preferably, the scoring matrix
used is the BLOSUM62
matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff,
1993, Proteins 17:49-61).
Less preferably, the PAM or PAM250 matrices may also be used (see, e.g.,
Schwartz and Dayhoff, eds.,
1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence
and Structure,
Washington: National Biomedical Research Foundation)
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[0262) The BLAST programs evaluate the statistical significance of all high-
scoring segment
pairs identified, and preferably selects those segments which satisfy a user-
specified threshold of
significance, such as a user-specified percent homology. Preferably, the
statistical significance of a high-
scoring segment pair is evaluated using the statistical significance formula
of Karlin (see, e.g., Karlin and
S Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).
[0263] The parameters used with the above algorithms may be adapted depending
on the sequence
length and degree of homology studied. In some embodiments, the parameters may
be the default parameters
used by the algorithms in the absence of instructions from the user.
[0264] In some embodiments, the level of homology between the hybridized
nucleic acid and the
extended cDNA, 5'EST, or 5' consensus contigated EST from which the probe was
derived may be
determined using the FASTDB algorithm described in Brutlag et al. Comp. App.
Biosci. 6:237-245, 1990. In
such analyses the parameters may be selected as follows: Matrix=Unitary, k-
tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the sequence which hybridizes
to the probe, whichever is
shorter. Because the FASTDB program does not consider 5'or 3,'truncations when
calculating homology
levels, if the sequence which hybridizes to the probe is truncated relative to
the sequence of the extended
cDNA, 5'EST, or consensus contigated 5'EST from which the probe was derived
the homology level is
manually adjusted by calculating the number of nucleotides of the extended
cDNA, 5'EST, or consensus
contigated 5' EST which are not matched or aligned with the hybridizing
sequence, determining the
percentage of total nucleotides of the hybridizing sequence which the non-
matched or non-aligned
nucleotides represent, and subtracting this percentage from the homology
level. For example, if the
hybridizing sequence is 700 nucleotides in length and the extended cDNA,
5'EST, or consensus contigated 5'
EST sequence is 1000 nucleotides in length wherein the first 300 bases at the
5'end of the extended cDNA,
5'EST, or consensus contigated 5' EST are absent from the hybridizing
sequence, and wherein the
overlapping 700 nucleotides are identical, the homology level would be
adjusted as follows. The non-
matched, non-aligned 300 bases represent 30% of the length of the extended
cDNA, 5'EST, or consensus
contigated 5' EST. If the overlapping 700 nucleotides are 100% identical, the
adjusted homology level
would be 100-:30=70% homology. It should be noted that the preceding
adjustments are only made when the
non-matched or non-aligned nucleotides are at the 5,'or 3'ends. No adjustments
are made if the non-matched
or non-aligned sequences are internal or under any other conditions.
[0265] For example, using the above methods, nucleic acids having at least 95%
nucleic acid
homology, at least 96% nucleic acid homology, at least 97% nucleic acid
homology, at least 98% nucleic acid
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homology, at least 99% nucleic acid homology, or more than 99% nucleic acid
homology to the extended
cDNA, 5'EST, or consensus contigated 5' EST from which the probe was derived
may be obtained and
identified. Such nucleic acids may be allelic variants or related nucleic
acids from other species. Similarly,
by using progressively less stringent hybridization conditions one can obtain
and identify nucleic acids
having at least 90%, at least 85%, at least 80% or at least 75% homology to
the extended cDNA, 5'EST, or
consensus contigated 5' EST from which the probe was derived.
[0266] Using the above methods and algorithms such as FASTA with parameters
depending on the
sequence length and degree of homology studied, for example the default
parameters used by the algorithms
in the absence of instructions from the user, one can obtain nucleic acids
encoding proteins having at least
99%, at least 98%, at least 97%, at least 96'%, at least 95%, at least 90%, at
least 85%, at least 80% or at least
75% homology to the protein encoded by the extended cDNA, 5'EST, or consensus
contigated 5' EST from
which the probe was derived. In some embodiments, the homology levels can be
determined using the
"default" opening penalty and the "default" gap penalty, and a scoring matrix
such as PAM 250 (a
standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and
Structure, Vol. 5, Supp. 3
(1978)).
[0267] Alternatively, the level of polypeptide homology may be determined
using the FASTDB
algorithm described by Brutlag et al. Comp. App. Biosci. 6:237-245, 1990. In
such analyses the parameters
may be selected as follows: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,
Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=l, Window Size=Sequence Length, Gap
Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the homologous sequence,
whichever is shorter. If the
homologous amino acid sequence is shorter than the amino acid sequence encoded
by the extended cDNA,
5'EST, or consensus contigated 5' EST as a result of an N terminal and/or C
terminal deletion the results may
be manually corrected as follows. First, the number of amino acid residues of
the amino acid sequence
encoded by the extended cDNA, 5'EST, or consensus contigated 5' EST which are
not matched or aligned
with the homologous sequence is determined. Then, the percentage of the length
of the sequence encoded by
the extended cDNA, 5'EST, or consensus contigated 5' EST which the non-matched
or non-aligned amino
acids represent is calculated. This percentage is subtracted from the homology
level. For example wherein
the amino acid sequence encoded by the extended cDNA, 5'EST, or consensus
contigated 5' EST is 100
amino acids in length and the length of the homologous sequence is 80 amino
acids and wherein the amino
acid sequence encoded by the extended cDNA or 5'EST is truncated at the N
terminal end with respect to the
homologous sequence, the homology level is calculated as follows. In the
preceding scenario there are 20
non-matched, non-aligned amino acids in the sequence encoded by the extended
cDNA, 5'EST, or consensus
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contigated 5' EST. This represents 20% of the length of the amino acid
sequence encoded by the extended
cDNA, 5'EST, or consensus contigated 5' EST. If the remaining amino acids are
1005 identical between the
two sequences, the homology level would be 100%-20%=80% homology. No
adjustments are made if the
non-matched or non-aligned sequences are internal or under any other
conditions.
[0268] In addition to the above described methods, other protocols are
available to obtain extended
cDNAs using 5' ESTs or consensus contigated 5'ESTs as outlined in the
following paragraphs.
[0269] Extended cDNAs may be prepared by obtaining mRNA from the tissue, cell,
or organism of
interest using mRNA preparation procedures utilizing polyA selection
procedures or other techniques lrnown
to those skilled in the art. A first primer capable of hybridizing to the
polyA tail of the mRNA is hybridized
to the mRNA and a reverse transcription reaction is performed to generate a
first cDNA strand.
[0270] The first cDNA strand is hybridized to a second primer containing at
least 10 consecutive
nucleotides of the sequences of S>:?Q m NOs 24-13309 and 26596-52153.
Preferably, the primer comprises
at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive nucleotides from
the sequences of SEQ ~ NOs 24-
13309 and 26596-52153. In some embodiments, the primer comprises more than 30
nucleotides from the
1 S sequences of S>?Q ID NOs 24-13309 and 26596-52153. If it is desired to
obtain extended cDNAs containing
the full protein coding sequence, including the authentic translation
initiation site, the second primer used
contains sequences located upstream of the translation initiation site. The
second primer is extended to
generate a second cDNA strand complementary to the first cDNA strand.
Alternatively, RT-PCR may be
performed as described above using primers from both ends of the cDNA to be
obtained.
[0271] Extended cDNAs containing 5' fragments of the mRNA may be prepared by
hybridizing an
mRNA comprising the sequences of SEQ )D NOs. 24-13309 and 26596-52153 with a
primer comprising a
complementary to a fragment of an EST-related nucleic acid hybridizing the
primer to the mRNAs, and
reverse transcribing the hybridized primer to make a first cDNA strand from
the mRNAs. Preferably, the
primer comprises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive
nucleotides of the sequences
complementary to SEQ >D NOs. 24-13309 and 26596-52153.
[0272] Thereafter, a second cDNA strand complementary to the first cDNA strand
is synthesized.
The second cDNA strand may be made by hybridizing a primer complementary to
sequences in the first
cDNA strand to the first cDNA strand and extending the primer to generate the
second cDNA strand.
[0273] The double stranded extended cDNAs made using the methods described
above are isolated
and cloned. The extended cDNAs may be cloned into vectors such as plasmids or
viral vectors capable of
replicating in an appropriate host cell. For example, the host cell may be a
bacterial, mammalian, avian, or
insect cell.
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[0274] Techniques for isolating mRNA, reverse transcribing a primer hybridized
to mRNA to
generate a first cDNA strand, extending a primer to make a second cDNA strand
complementary to the first
cDNA strand, isolating the double stranded cDNA and cloning the double
stranded cDNA are well known to
those skilled in the art and are described in Current Protocols in Molecular
Biology, John Wiley & Sons, Inc.
1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor
Laboratory Press, 1989, the entire disclosures of which are incorporated
herein by reference.
[0275] Alternatively, other procedures may be used for obtaining full-length
cDNAs or extended
cDNAs. In one approach, full-length or extended cDNAs are prepared from mRNA
and cloned into double
stranded phagemids as follows. The cDNA library in the double stranded
phagemids is then rendered single
stranded by treatment with an endonuclease, such as the Gene II product of the
phage F1 and an exonuclease
(Chang et al., Gene 127:95-8, 1993). A biotinylated oligonucleotide comprising
the sequence of a fragment
of an EST-related nucleic acid is hybridized to the single stranded phagemids.
Preferably, the fragment
comprises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive
nucleotides of the sequences of SEQ ID
NOs. 24-13309 and 26596-52153.
(0276] Hybrids between the biotinylated oligonucleotide and phagemids are
isolated by incubating
the hybrids with streptavidin coated paramagnetic beads and retrieving the
beads with a magnet (Fry et al.,
Biotechniques, 13: 124-131, 1992). Thereafter, the resulting phagemids are
released from the beads and
converted into double stranded DNA using a primer specific for the 5' ES'T or
consensus contigated 5'EST
sequence used to design the biotinylated oligonucleotide. Alternatively,
protocols such as the Gene Trapper
kit (Gibco BRL;) may be used. The resulting double stranded DNA is transformed
into bacteria. Extended
cDNAs or full length cDNAs containing the 5' EST or consensus contigated 5'EST
sequence are identified
by colony PCR or colony hybridization.
EXAMPLE 19
Extended cDNAs and Full Length cDNAs
[0277] The procedure described in Example 17 was used to obtain extended cDNAs
or full-length
cDNAs derived from 5' ESTs in a variety of tissues. The following list
provides a few examples of thus
obtained cDNAs.
[0278] Using this procedure, the full length cDNA of SEQ ID NO.1 (internal
identification number
58-34-2-E7-FL2) was obtained. This cDNA encodes the signal peptide
MWWFQQGLSFLPSALVIWTSA
(SEQ )D N0.2) having a von Heijne score of 5.5.
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[0279] Using this approach, the full-length cDNA of SEQ lD N0.3 (internal
identification number
48-19-3-G1-FL,1) was obtained. 'this cDNA encodes the signal peptide
MKKVLLLITAIL,AVAVG (SEQ
1D NO. 4) having a von Heijne score of 8.2.
[0280] The full-length cDNA of SEQ )D NO.S (internal identification number 58-
35-2-F10-FL2)
was also obtained using this procedure. This cDNA encodes a signal peptide
LWLLFFLVTAIHA (SEQ )D
N0.6) having a von Heijne score of 10.7.
[0281] Furthermore, the polypeptides encoded by the extended or full-length
cDNAs may be
screened for the presence of known structural or functional motifs or for the
presence of signatures, small
amino acid sequences which are well conserved amongst the members of a protein
family. The results
obtained for the polypeptides encoded by a few full-length cDNAs derived from
5'ESTs that were screened
for the presence of known protein signatures and motifs using the Proscan
software from the GCG package
and the Prosite 15.0 database are provided below.
[0282] The protein of SEQ >D NO. 8 encoded by the full-length cDNA SEQ ID NO.
7 (internal
designation 78-8-3-E6-CLO_1C) and expressed in adult prostate belong to the
phosphatidylethanolamine
binding protein from which it exhibits the characteristic PROSITE signature.
Proteins from this
widespread family, from nematodes to fly, yeast, rodent and primate species,
bind hydrophobic ligands
such as phospholipids and nucleotides. They are mostly expressed in brain and
in testis and are thought
to play a role in cell growth and/or maturation, in regulation of the sperm
maturation, motility and in
membrane remodeling. They may act either through signal transduction or
through oxidoreduction
reactions (for a review see Schoentgen and Jolles, FEBS Letters, 369:22-26
(1995)). Taken together,
these data suggest that the protein of SEQ >D NO. 8 may play a role in cell
growth, maturation and in
membrane remodeling and/or may be related to male fertility. Thus, these
proteins may be useful in
diagnosing and/or treating cancer, neurodegenerative diseases, and/or
disorders related to male fertility
and sterility.
[0283] The protein of SEQ >Z7 NOs. 10 encoded by the extended cDNA SEQ >D NO.
9 (internal
designation 59-9-2-E6-FLO_1C) belong to the stomatin or band 7 family. The
human stomatin is an
integral membrane phosphoprotein thought to regulate the canon conductance by
interacting with other
proteins of the functional complex of the membrane skeleton (Gallagher and
Forget, J. Biol. Chem.,
270:26358-26363 (1995)). The protein of SEQ ID NO. 10 exhibits the PROSITE
signature typical for the
band 7 family signature. Taken together, these data suggest that the protein
of SEQ >D NO. 10 plays a
role in the regulation of ion transport, hence in the control of cellular
volume. This protein may find
applications in diagnosing and/or treating stomatocytosis and/or
cryohydrocytosis.
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[0285] The protein of SEQ >D NO. 12 encoded by the extended cDNA SEQ >D NO. 11
(internal
designation 19-10-1-C2-CL1 3) shows homology with the bovine subunit B14.SB of
the NADH-
ubiquinone oxidureductase complex (Arizmendi et al, FEBS Lett., 313 : 80-84
(1992) and Swissprot
accession number Q02827). This complex is the first of four complexes located
in the inner
mitochondria) membrane which make up the mitochondria) electron transport
chain. Complex I is
involved in the dehydrogenation of NADH and the transportation of electrons to
coenzyme Q. It is
composed of 7 subunits encoded by the mitochondria) genome and 34 subunits
encoded by the nuclear
genome. It is also thought to play a role in the regulation of apoptosis and
necrosis.
Mitochondriocytopathies due to complex I deficiency are frequently encountered
and affect tissues with a
high energy demand such as brain (mental retardation, convulsions, movement
disorders), heart
(cardiomyopathy, conduction disorders), kidney (Fanconi syndrome), skeletal
muscle (exercise
intolerance, muscle weakness, hypotonia) andlor eye (opthmaloplegia, ptosis,
cataract and retinopathy).
For a review on complex I see Smeitink et al., Hurn. Mol. Gent., 7 : 1573-1579
(1998). Taken together,
these data suggest that the protein of SEQ ID NO. 12 may be part of the
mitochondria) energy-generating
1 S system, probably as a subunit of the NADH-ubiquinone oxidoreductase
complex. Thus, this protein or
part therein, may find applications in diagnosing and/or treating several
disorders including, but not
limited to, brain disorders (mental retardation, convulsions, movement
disorders), heart disorders
(cardiomyopathy, conduction disorders), kidney disorders (Fanconi syndrome),
skeletal muscle disorders
(exercise intolerance, muscle weakness, hypotonia) andlor eye disorders
opthmalmoplegia, ptosis,
cataract and retinopathy).
[0286] The protein of SEQ >D N0.14 encoded by the extended cDNA SEQ 117 NO. 13
(internal
designation 77-13-1-C11-FL2 2C) exhibits an extensive homology with a murine
protein named MP1 for
MEK binding partner 1 (Genbank accession number AF082526). MP 1 was shown to
enhance enzymatic
activation of mitogen-activated protein (MAP) kinase cascade. The MAP kinase
pathway is one of the
important enzymatic cascade that is conserved among all eukaryotes from yeast
to human. This kind of
pathway is involved in vital functions such as the regulation of growth,
differentiation and apoptosis.
MPl probably acts by facilitating the interaction of the two sequentially
acting kinases MEK1 and ERK1
(Schaffer et al., Science, 281:1668-1671 (1998)). Taken together, these data
suggest that the protein of
SEQ ID NO. 14 may be involved in regulating protein-protein interaction in the
signal transduction
pathways. Thus, this protein may be useful in diagnosing and/or treating
several types of disorders
including, but not limited to, cancer, neurodegenerative diseases,
cardiovascular disorders, hypertension,
renal injury and repair and septic shock.
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[0287] Bacterial clones containing plasmids containing the full-length cDNAs
described above are
presently stored in the inventor's laboratories under the internal
identification numbers provided above. The
inserts may be recovered from the deposited materials by growing an aliquot of
the appropriate bacterial
clone in the appropriate medium. The plasmid DNA can then be isolated using
plasmid isolation procedures
familiar to those skilled in the art such as alkaline lysis minipreps or large
scale alkaline lysis plasmid
isolation procedures. If desired the plasmid DNA may be further enriched by
centrifugation on a cesium
chloride gradient, size exclusion chromatography, or anion exchange
chromatography. The plasmid DNA
obtained using these procedures may then be manipulated using standard cloning
techniques familiar to those
skilled in the art. Alternatively, a PCR can be done with primers designed at
both ends of the EST insertion.
The PCR product which corresponds to the 5'EST can then be manipulated using
standard cloning techniques
familiar to those skilled in the art.
[0288] Using any of the above described methods in section N, a plurality of
extended cDNAs
containing full-length protein coding sequences or portions of the protein
coding sequences may be provided
as cDNA libraries for subsequent evaluation of the encoded proteins or use in
diagnostic assays as described
below.
V. Expression of Proteins Encoded by EST-related nucleic acids
[0289] EST-related nucleic acids, fragments of EST-related nucleic acids,
positional segments of
EST-related nucleic acids, and fragments of positional segments of EST-related
nucleic acids may be used to
express the polypeptides which they encode. In particular, they may be used to
express EST-related
polypeptides, fi-agments of EST-related polypeptides, positional segments of
EST-related polypeptides, or
fragments of positional segments of EST-related polypeptides. In some
embodiments, the EST-related
nucleic acids, positional segments of EST-related nucleic acids, and fragments
of positional segments of
EST-related nucleic acids may be used to express the full polypeptide (i.e.
the signal peptide and the mature
polypeptide) of a secreted protein, the mature protein (i.e. the polypeptide
generated after cleavage of the
signal peptide). or the signal peptide of a secreted protein. If desired,
nucleic acids encoding the signal
peptide may be used to facilitate secretion of the expressed protein. It will
be appreciated that a plurality of
EST-related nucleic acids, fragments of EST-related nucleic acids, positional
segments of EST-related
nucleic acids, or fragments of positional segments of EST-related nucleic
acids may be simultaneously cloned
into expression vectors to create an expression library for analysis of the
encoded proteins as described
below.
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EXAMPLE 20
E~ression of the Proteins Encoded by the Genes Correspondin tg o the 5'ESTs or
Consensus Conti
c~ ~cT~
[0290] To express their encoded proteins the EST-related nucleic acids,
fragments of EST-related
nucleic acids, positional segments of EST-related nucleic acids, or fragments
of positional segments of EST-
related nucleic acids are cloned into a suitable expression vector. In some
instances, nucleic acids encoding
EST-related polypeptides, fragments of EST-related polypeptides, positional
segments of EST-related
polypeptides or fragments of positional segments of EST-related polypeptides
may be cloned into a suitable
expression vector.
[0291 ] In some embodiments, the nucleic acids inserted into the expression
vector may comprise
the coding sequence of a sequence selected from the group consisting of 24-
13309. In other embodiments,
the nucleic acids inserted into the expression vector may comprise may
comprise the full coding sequence
(i.e. the nucleotides encoding the signal peptide and the mature polypeptide)
of one of SEQ )D NOs. 4597-
6443. In some embodiments, the nucleic acid inserted into the expression
vector may comprise the
nucleotides of one of the sequences of SEQ )D NOs. 4597-6443 which encode the
mature polypeptide (i.e.
the nucleotides encoding the polypeptide generated after cleavage of the
signal peptide). In fiu-ther
embodiments, the nucleic acids inserted into the expression vector may
comprise the nucleotides of SEQ >D
NOs. 24-1027 and 4597-6443 which encode the signal peptide to facilitate
secretion of the expressed protein.
The nucleic acids inserted into the expression vectors may also contain
sequences upstream of the sequences
encoding the signal peptide, such as sequences which regulate expression
levels or sequences which confer
tissue specific expression.
[0292] The nucleic acid inserted into the expression vector may encode a
polypeptide comprising
the one of the sequences of SEQ ID NOs. 13310-26595. In some embodiments, the
nucleic acid inserted into
the expression vector may encode the full polypeptide sequence (i.e. the
signal peptide and the mature
polypeptide) included in one of SEQ )D NOs. 17883-19729. In other embodiments,
the nucleic acid inserted
into the expression vector may encode the mature polypeptide (i.e. the
polypeptide generated after cleavage
of the signal peptide) included in one of the sequences of SEQ ID NOs. 17883-
19729. In further
embodiments, the nucleic acids inserted into the expression vector may encode
the signal peptide included in
one of the sequences of 13310-14313 and 17883-19729.
[0293] The nucleic acid encoding the protein or polypeptide to be expressed is
operably linked to a
promoter in an expression vector using conventional cloning technology. The
expression vector may be any
of the mammalian, yeast, insect or bacterial expression systems known in the
art. Commercially available
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vectors and expression systems are available from a variety of suppliers
including Genetics Institute
(Cambridge, MA), Stratagene (La Jolla, C',alifornia), Promega (Madison,
Wisconsin), and Invitrogen (San
Diego, California). If desired, to enhance expression and facilitate proper
protein folding, the codon context
and codon pairing of.the sequence may be optimized for the particular
expression organism in which the
expression vector is introduced, as explained by Hatfield, et al., U.S. Patent
No. 5,082,767, incorporated
herein by this reference.
[0294] The following is provided as one exemplary method to express the
proteins encoded by the
nucleic acids described above. In some instances the nucleic acid encoding the
protein or polypeptide to be
expressed includes a methionine initiation codon and a polyA signal. If the
nucleic acid encoding the
polypeptide to be expressed lacks a methionine to serve as the initiation
site, an initiating methionine can be
introduced next to the first codon of the nucleic acid using conventional
techniques. Similarly, if the nucleic
acid encoding the protein or polypeptide to be expressed lacks a polyA signal,
this sequence can be added to
the construct by, for example, splicing out the polyA signal from pSGS
(Stratagene) using BgII and SaII
restriction endonuclease enzymes and incorporating it into the mammalian
expression vector pXTI
(Stratagene). pXTl contains the LTRs and a portion of the gag gene from
Moloney Murine Leukemia Virus.
The position of the LTRs in the construct allows efficient stable
transfection. The vector includes the Herpes
Simplex thymidine kinase promoter and the selectable neomycin gene. The
nucleic acid encoding the
polypeptide to be expressed is obtained by PCR from the bacterial vector using
oligonucleotide primers
complementary to the nucleic acid encoding the protein or polypeptide to be
expressed and containing
restriction endonuclease sequences for Pst I incorporated into the 5'primer
and BgIII at the 5' end of 3'
primer, taking care to ensure that the nucleic acid encoding the protein or
polypeptide to be expressed is
correctly positioned with respect to the poly A signal. The purified fragment
obtained from the resulting
PCR reacrion is digested with PstI, blunt ended with an exonuclease, digested
with Bgl II, purified and ligated
to pXTl, now containing a poly A signal and digested with BgIII.
[0295] The ligated product is transfected into mouse NIH 3T3 cells using
Lipofectin (Life
Technologies, Inc., Grand Island, New York) under conditions outlined in the
product specification. Positive
transfectants are selected after growing the transfected cells in 600 ~,g/ml
6418 (Sigma, St. Louis, Missouri).
[0296] Alternatively, the nucleic acid encoding the protein or polypeptide to
be expressed may be
cloned into pED6dpc2 as described above. The resulting pED6dpc2 constructs may
be transfected into a
suitable host cell, such as COS 1 cells. Methotrexate resistant cells are
selected and expanded. The
expressed protein or polypeptide may be isolated, purified, or enriched as
described above.
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[0297] To confirm expression of the desired protein or polypeptide, the
proteins or polypeptides
produced by cells containing a vector with a nucleic acid insert encoding the
protein or polypeptide are
compared to those lacking such an insert. The expressed proteins are detected
using techniques familiar to
those skilled in the art such as Coomassie blue or silver staining or using
antibodies against the protein or
polypeptide encoded by the nucleic acid insert. Antibodies capable of
specifically recognizing the protein of
interest may be generated using synthetic 15-mer peptides having a sequence
encoded by the appropriate
nucleic acid. The synthetic peptides are injected into mice to generate
antibody to the polypeptide encoded
by the nucleic acid.
[0298] If the proteins or polypeptides encoded by the nucleic acid inserts are
secreted, medium
prepared from the host cells or organisms containing an expression vector
which contains a nucleic acid insert
encoding the desired protein or polypeptide is compared to mdieum prepared
from the control cells or
organism. The presence of a band in medium from the cells containing the
nucleic acid insert which is absent
from preparations from the control cells indicates that the protein or
polypeptide encoded by the nucleic acid
insert is being expressed and secreted. Generally, the band corresponding to
the protein encoded by the
nucleic acid insert will have a mobility near that expected based on the
number of amino acids in the open
reading frame of the nucleic acid insert. However, the band may have a
mobility different than that expected
as a result of modifications such as glycosylation, ubiquitination, or
enzymatic cleavage.
[0299] Alternatively, if the protein expressed from the above expression
vectors does not contain
sequences directing its secretion, the proteins expressed from host cells
containing an expression vector with
an insert encoding a secreted protein or portion thereof can be compared to
the proteins expressed in control
host cells containing the expression vector without an insert. The presence of
a band in samples from cells
containing the expression vector with an insert which is absent in samples
from cells containing the
expression vector without an insert indicates that the desired protein or
portion thereof is being expressed.
Generally, the band will have the mobility expected for the secreted protein
or portion thereof. However, the
band may have a mobility different than that expected as a result of
modifications such as glycosylation,
ubiquitination, or enzymatic cleavage.
[0300] The expressed protein or polypeptide may be purified, isolated or
enriched using a variety of
methods. In some methods, the protein or polypeptide may be secreted into the
culture medium via a native
signal peptide or a heterologous signal peptide operably linked thereto. In
some methods, the protein or
polypeptide may be linked to a heterologous polypeptide which facilitates its
isolation, purification, or
enrichment such as a nickel binding polypeptide. The protein or polypeptide
may also be obtained by gel
electrophoresis, ion exchange chromatography, size chromatography, hplc, salt
precipitation,
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immunoprecipitation, a combination of any of the preceding methods, or any of
the isolation, purification, or
enrichment techniques familiar to those skilled in the art.
[0301] The protein encoded by the nucleic acid insert may also be purified
using standard
immunochromatography techniques using immunoaffinity chromatography with
antibodies directed against
the encoded protein or polypeptide as described in more detail below. If
antibody production is not possible,
the nucleic acid insert encoding the desired protein or polypeptide may be
incorporated into expression
vectors designed for use in purification schemes employing chimeric
polypeptides. In such strategies, the
coding sequence of the nucleic acid insert is ligated in frame with the gene
encoding the other half of the
chimera. The other half of the chimera may be ~3-globin or a nickel binding
polypeptide. A chromatography
matrix having antibody to (3-globin or nickel attached thereto is then used to
purify the chimeric protein.
Protease cleavage sites may be engineered between the (3-globin gene or the
nickel binding polypeptide and
the extended cDNA or portion thereof. Thus, the two polypeptides of the
chimera may be separated from one
another by protease digestion.
[0302] One useful expression vector for generating /3-globin chimerics is pSGS
(Stratagene), which
encodes rabbit /3-globin. Intron II of the rabbit ~i-globin gene facilitates
splicing of the expressed transcript,
and the polyadenylation signal incorporated into the construct increases the
level of expression. These
techniques as described are well known to those skilled in the art of
molecular biology. Standard methods are
published in methods texts such as Davis et al., (Basic Methods in Molecular
Biology, L.G. Davis, M.D.
Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986) and many of the
methods are available from
Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally
be produced from the
construct using in vitro translation systems such as the In vitro ExpressTM
Translation Kit (Stratagene).
[0303] Following expression and purification of the proteins or polypeptides
encoded by the nucleic
acid inserts, the purified proteins may be tested for the ability to bind to
the surface of various cell types as
described in Example 21 below. It will be appreciated that a plurality of
proteins expressed from these
nucleic acid inserts may be included in a panel of proteins to be
simultaneously evaluated for the activities
specifically described below, as well as other biological roles for which
assays for determining activity are
available.
EXAMPLE 21
Anal~is of Secreted Proteins or Polypeptides to Determine Whether they Bind to
the Cell Surface
[0304) The EST-related nucleic acids, fragments of EST-related nucleic acids,
positional segments
of EST-related nucleic acids, fragments of positional segments of EST-related
nucleic acids, nucleic acids
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encoding the EST-related polypeptides, nucleic acids encoding fragments of the
EST-related polypeptides,
nucleic acids encoding positional segments of EST-related polypeptides, or
nucleic acids encoding fragments
of positional segments of EST-related polypeptides are cloned into expression
vectors such as those described
in Example 20. The encoded proteins or polypeptides are purified, isolated, or
enriched as described above.
S Following purification, isolation, or enrichment, the proteins or
polypeptides are labeled using techniques
known to those skilled in the art. The labeled proteins or polypeptides are
incubated with cells or cell lines
derived from a variety of organs or tissues to allow the proteins to bind to
any receptor present on the cell
surface. Following the incubation, the cells are washed to remove non-
specifically bound proteins or
polypeptides. 'The specifically bound labeled proteins or polypeptides are
detected by autoradiography.
Alternatively, unlabeled proteins or polypeptides may be incubated with the
cells and detected with
antibodies having a detectable label, such as a fluorescent molecule, attached
thereto.
[0305] Specificity of cell surface binding may be analyzed by conducting a
competition analysis in
which various amounts of unlabeled protein or polypeptide are incubated along
with the labeled protein or
polypeptide. The amount of labeled protein or polypeptide bound to the cell
surface decreases as the amount
of competitive unlabeled protein or polypeptide increases. As a control,
various amounts of an unlabeled
protein or polypeptide unrelated to the labeled protein or polypeptide is
included in some binding reactions.
The amount of labeled protein or polypeptide bound to the cell surface does
not decrease in binding reactions
containing increasing amounts of unrelated unlabeled protein, indicating that
the protein or polypeptide
encoded by the nucleic acid binds specifically to the cell surface.
[0306] As discussed above, human proteins have been shown to have a number of
important
physiological effects and, consequently, represent a valuable therapeutic
resource. The human proteins or
polypeptides made as described above may be evaluated to determine their
physiological activities as
described below.
EXAMPLE 22
Assayin the Expressed Proteins or Polypeptides for Cytokine, Cell
Proliferation or Cell Differentiation
Activi
[0307] As discussed above, some human proteins act as cytokines or may affect
cellular
proliferation or differentiation. Many protein factors discovered to date,
including all known cytokines, have
exhibited activity in one or more factor dependent cell proliferation assays,
and hence the assays serve as a
convenient confirmation of cytokine activity. The activity of a protein or
polypeptide of the present invention
is evidenced by any one of a number of routine factor dependent cell
proliferation assays for cell lines
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including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+
(preB M+), 2E8, RBS,
DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7c and CMK. The proteins or polypeptides
prepared as described
above may be evaluated for their ability to regulate T cell or thymocyte
proliferation in assays such as those
described above or in the following references, which are incorporated herein
by reference: Current
Protocols in Immunology, Ed. by J.E. Coligan et al., Greene Publishing
Associates and Wiley-Interscience;
Takai et al. J Immunol. 137:3494-3500, 1986., Bertagnolli et al. J. Immunol.
145:1706-1712, 1990.,
Bertagnolli et al., Cellular Immunology 133:327-341, 1991. Bertagnolli, et al.
J. Lnmunol. 149:3778-3783,
1992; Bowman et al., J. Immunol. 152:1756-1761, 1994.
[0308] In addition, numerous assays for cytokine production and/or the
proliferation of spleen cells,
lymph node cells and thymocytes are known. These include the techniques
disclosed in Current Protocols
in Immunology. J.E. Coligan et al. Eds., 1:3.12.1-3.12.14, John Wiley and
Sons, Toronto. 1994; and
Schreiber, R.D. fn Current Protocols in Immunology., supra 1 : 6.8.1-6.8.8.
[0309] The proteins or polypeptides prepared as described above may also be
assayed for the ability
to regulate the proliferation and differentiation of hematopoietic or
lymphopoietic cells. Many assays for
such activity are familiar to those skilled in the art, including the assays
in the following references, which are
incorporated herein by reference: Bottomly et al., In Current Protocols in
Immunology., supra. 1 : 6.3.1-
6.3.12,; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al.,
Nature 36:690-692, 1988;
Greenberger et al., Proc. Natl. Acad. Sci. US.A. 80:2931-2938, 1983; Nordan,
R., In Current Protocols in
Immunology., supra. 1 : 6.6.1-6.6.5; Smith et al., Proc. Natl. Acad. Sci.
LLS.A. 83:1857-1861, 1986; Bennett
et al in Current Protocols in Immunology supra 1 : 6.15.1; Ciarletta et al In
Current Protocols in
Immunology. supra 1 : 6.13.1.
[0310] The proteins or polypeptides prepared as described above may also be
assayed for their
ability to regulate T-cell responses to antigens. Many assays for such
activity are familiar to those skilled in
the art, including the assays described in the following references, which are
incorporated herein by
reference: Chapter 3 (In vitro Assays for Mouse Lymphocyte Function), Chapter
6 (Cytokines and Their
Cellular Receptors) and Chapter 7, (Immunologic Studies in Humans) in Current
Protocols in Immunology
supra; Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980;
Weinberger et al., Eur. J. Immun.
11:405-411, 1981; Takai et al., J Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512,
1988.
(0311] Those proteins or polypeptides which exhibit cytokine, cell
proliferation, or cell
differentiation activity may then be formulated as pharmaceuticals and used to
treat clinical conditions in
which induction of cell proliferation or differentiation is beneficial.
Alternatively, as described in more detail
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below, nucleic acids encoding these proteins or polypeptides or nucleic acids
regulating the expression of
these proteins or polypeptides may be introduced into appropriate host cells
to increase or decrease the
expression of the proteins or polypeptides as desired.
EXAMPLE 23
Assay tg he Expressed Proteins or Polypeptides for Activity as Immune System
Regulators
[0312] The proteins or polypeptides prepared as described above may also be
evaluated for their
effects as immune regulators. For example, the proteins or polypeptides may be
evaluated for their activity to
influence thymocyte or splenocyte cytotoxicity. Numerous assays for such
activity are familiar to those
skilled in the art including the assays described in the following references,
which are incorporated herein by
reference: Chapter 3 (In vitro Assays for Mouse Lymphocyte Function 3.1-3.19)
and Chapter 7
(Immunologic studies in Humans) in Current Protocols in Immunology , J.E.
Coligan et al. Eds, Greene
Publishing Associates and Wiley-Interscience; Hemnann et al., Proc. Natl.
Acad. Sci. USA 78:2488-2492,
1981; Hemnann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 135:1564-1572, 1985;
Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Bowman et al.,
J. Virology 61:1992-1998; Bertagnolli et al. Cell. Immunol. 133:327-341, 1991;
Brown et al., J. Immunol.
153:3079-3092, 1994.
[0313] The proteins or polypeptides prepared as described above may also be
evaluated for their
effects on T-cell dependent immunoglobulin responses and isotype switching.
Numerous assays for such
activity are familiar to those skilled in the art, including the assays
disclosed in the following references,
which are incorporated herein by reference: Maliszewski, J. Immunol. 144:3028-
3033, 1990; Mond et al. in
Current Protocols in Immunology, 1 : 3.8.1-3.8.16, supra.
[0314] The proteins or polypeptides prepared as described above may also be
evaluated for their
effect on immure effector cells, including their effect on Thl cells and
cytotoxic lymphocytes. Numerous
assays for such activity are familiar to those skilled in the art, including
the assays disclosed in the following
references, which are incorporated herein by reference: Chapter 3 (In vitro
Assays for Mouse Lymphocyte
Function 3.1-3.19) and Chapter 7 (Immunologic Studies in Humans) in Current
Protocols in Immunology,
supra; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.; J Immunol.
140:508-512, 1988;
Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
[0315] The proteins or polypeptides prepared as described above may also be
evaluated for their
effect on dendritic cell mediated activation of naive T-cells. Numerous assays
for such activity are familiar to
those skilled in the art, including the assays disclosed in the following
references, which are incorporated
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herein by reference: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et
al., J. Exp. Med. 173:549-559,
1991; Macatonia et al., J. Immunol. 154:5071-5079, 1995; Porgador et al J.
Exp. Med 182:255-260, 1995;
Nair et al., J. Virol. 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al J. Exp.
Med 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation
94:797-807, 1994; and Inaba et
al., J. Exp. Med 172:631-640, 1990.
[0316] The proteins or polypeptides prepared as described above may also be
evaluated for their
influence on the lifetime of lymphocytes. Numerous assays for such activity
are familiar to those skilled in
the art, including the assays disclosed in the following references, which are
incorporated herein by reference:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia
7:659-670, 1993; Gorczyca et
al., Cancer Res. 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk, J. Immunol. 145:4037-
4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., Int. J.
Oncol. 1:639-648, 1992.
[0317] The proteins or polypeptides prepared as described above may also be
evaluated for their
influence on early steps of T-cell commitment and development. Numerous assays
for such activity are
familiar to those skilled in the art, including without limitation the assays
disclosed in the following
references, which are incorporated herein by references: Antica et al., Blood
84:111-117, 1994; Fine et al.,
Cell. Immunol. 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki
et al., Proc. Nat. Acad Sci.
USA 88:7548-7551, 1991.
[0318] Those proteins or polypeptides which exhibit activity as immune system
regulators activity
may then be formulated as pharmaceuticals and used to treat clinical
conditions in which regulation of
immune activity is beneficial. For example, the protein or polypeptide may be
useful in the treatment of
various immune deficiencies and disorders (including severe combined
immunodeficiency), e.g., in
regulating (up or down) growth and proliferation of T and/or B lymphocytes, as
well as effecting the cytolytic
activity of NK cells and other cell populations. These immune deficiencies may
be genetic or be caused by
viral (e.g., HIV) as well as bacterial or fungal infections, or may result
from autoimmune disorders. More
specifically, infectious diseases caused by viral, bacterial, fungal or other
infection may be treatable using the
protein or polypeptide including infections by HIV, hepatitis viruses, herpes
viruses, mycobacteria,
Leishmania spp., plamodium. and various fungal infections such as candidiasis.
Of course, in this regard, a
protein or polypeptide may also be useful where a boost to the immune system
generally may be desirable,
i.e., in the treatment of cancer.
[0319] Alternatively, the proteins or polypeptides prepared as described above
may be used in
treatment of autoimmune disorders including, for example, connective tissue
disease, multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary
inflammation, Guillain-Bane
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syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,
myasthenia gravis, graft-versus-host
disease and autoimmune inflammatory eye disease. Such a protein or polypeptide
may also to be useful in
the treatment of allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other
respiratory problems. _ Other conditions, in which immune suppression is
desired (including, for example,
organ transplantation), may also be treatable using the protein or
polypeptide.
[0320] Using the proteins or polypeptides of the invention it may also be
possible to regulate
immune responses either up or down. Down regulation may involve inhibiting or
blocking an immune
response akeady in progress or may involve preventing the induction of an
immune response. The functions
of activated T-cells may be inhibited by suppressing T cell responses or by
inducing specific tolerance in T
cells, or both. Immunosuppression of T cell responses is generally an active
non-antigen-specific process
which requires continuous exposure of the T cells to the suppressive agent.
Tolerance, which involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is
generally antigen-specific and persists after the end of exposure to the
tolerizing agent. Operationally,
tolerance can be demonstrated by the lack of a T cell response upon reexposure
to specific antigen in the
absence of the tolerizing agent.
[0321] Down regulating or preventing one or more antigen functions (including
without limitation
B lymphocyte antigen functions, such as, for example, B7 costimulation), e.g.,
preventing high level
lymphokine synthesis by activated T cells, will be useful in situations of
tissue, skin and organ transplantation
and in graft-versus-host disease (GVHD). For example, blockage of T cell
function should result in reduced
tissue destruction in tissue transplantation. Typically, in tissue
transplants, rejection of the transplant is
initiated through its recognition as foreign by T cells, followed by an immune
reaction that destroys the
transplant. The administration of a molecule which inhibits or blocks
interaction of a B7 lymphocyte antigen
with its natural ligand(s) on immune cells (such as a soluble, monomeric form
of a peptide having B7-2
activity alone or in conjunction with a monomeric form of a peptide having an
activity of another B
lymphocyte antigen (e.g., B7-l, B7-3) or blocking antibody), prior to
transplantation, can lead to the binding
of the molecule to the natural ligand(s) on the immune cells without
transmitting the corresponding
costimulatory signal. Blocking B lymphocyte antigen function in this matter
prevents cytokine synthesis by
immune cells, such as T cells, and thus acts as an immunosuppressant.
Moreover, the lack of costimulation
may also be sufficient to anergize the T cells, thereby inducing tolerance in
a subject. Induction of long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated administration of
these blocking reagents. To achieve sufficient immunosuppression or tolerance
in a subject, it may also be
necessary to black the function of a combination of B lymphocyte antigens.
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[0322] The efficacy of particular blocking reagents in preventing organ
transplant rejection or
GVHD can be assessed using animal models that are predictive of efficacy in
humans. Examples of
appropriate systems which can be used include allogeneic cardiac grafts in
rats and xenogeneic pancreatic
islet cell grafts in mice, both of which have been used to examine the
immunosuppressive effects of
CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science
257:789-792 (1992) and Turka et
al., Proc. Natl. .Acad. Sci USA, 89:11102-11105 (1992). In addition, marine
models of GVHD (see Paul ed.,
Fundamental Irnmunology, Raven Press, New York, 1989, pp. 846-847) can be used
to determine the effect
of blocking B lymphocyte antigen function in vivo on the development of that
disease.
[0323] Blocking antigen function may also be therapeutically useful for
treating autoimmune
diseases. Many autoimmune disorders are the result of inappropriate activation
of T cells that are reactive
against self tissue and which promote the production of cytokines and
autoantibodies involved in the
pathology of the diseases. Preventing the activation of autoreactive T cells
may reduce or eliminate disease
symptoms. Administration of reagents which block costimulation of T cells by
disrupting receptor/ligand
interactions of B lymphocyte antigens can be used to inhibit T cell activation
and prevent production of
autoantibodies or T cell-derived cytokines which potentially involved in the
disease process. Additionally,
blocking reagents may induce antigen-specific tolerance of autoreactive T
cells which could lead to long-term
relief from the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune disorders
can be determined using a number of well-characterized animal models of human
autoimmune diseases.
Examples include marine experimental autoimmune encephalitis, systemic lupus
erythmatosis in MRL/pr/pr
mice or NZB hybrid mice, marine autoimmuno collagen arthritis, diabetes
mellitus in OD mice and BB rats,
and marine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New
York, 1989, pp. 840-856).
[0324) Upregulation of an antigen function (preferably a B lymphocyte antigen
function), as a
means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses
may involve either enhancing an existing immune response or eliciting an
initial immune response as shown
by the following examples. For instance, enhancing an immune response through
stimulating B lymphocyte
antigen function may be useful in cases of viral infection. In addition,
systemic viral diseases such as
influenza, the common cold, and encephalitis might be alleviated by the
administration of stimulatory form of
B lymphocyte antigens systemically.
[0325] Alternatively, antiviral immune responses may be enhanced in an
infected patient by
removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-pulsed APCs either
expressing the proteins or polypeptides described above or together with a
stimulatory form of the protein or
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polypeptide and reintroducing the in vitro primed T cells into the patient.
The infected cells would now be
capable of delivering a costimulatory signal to T cells in vivo, thereby
activating the T cells.
[0326] In another application, upregulation or enhancement of antigen function
(preferably B
lymphocyte antigen function) may be useful in the induction of tumor immunity.
Tumor cells (e.g., sarcoma,
melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with one
of the above-described
nucleic acids encoding a protein or polypeptide can be administered to a
subject to overcome tumor-specific
tolerance in the subject. If desired, the tumor cell can be transfected to
express a combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an expression vector
directing the expression of a peptide having B7-2-like activity alone, or in
conjunction with a peptide having
B7-I-like activity and/or B7-3-like activity. The transfected tumor cells are
returned to the patient to result in
expression of the peptides on the surface of the transfected cell.
Alternatively, gene therapy techniques can
be used to target a tumor cell for transfection in vivo.
[0327] The presence of the protein or polypeptide encoded by the nucleic acids
described above
having the activity of a B lymphocyte antigens) on the surface of the tumor
cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the transfected tumor
cells. In addition, tumor cells which lack or which fail to reexpress
sufficient amounts of MHC class I or
MHC class II molecules can be transfected with nucleic acids encoding all or a
portion of (e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain and az
microglobulin or an MHC class II
L chain and an MHC class II /3 chain to thereby express MHC class I or MHC
class II proteins on the cell
surface, respectively. Expression of the appropriate MHC class I or class II
molecules in conjunction with a
peptide having the activity of a B lymphocyte antigen (e.g., B7-l, B7-2, B7-3)
induces a T cell mediated
immune response against the transfected tumor cell. Optionally, a nucleic acid
encoding an antisense
construct which blocks expression of an MHC class II associated protein, such
as the invariant chain, can also
be cotransfected with a DNA encoding a protein or polypeptide having the
activity of a B lymphocyte antigen
to promote presentation of tumor associated antigens and induce tumor specific
immunity. Thus, the
induction of a T cell mediated immune response in a human subject may be
sufficient to overcome tumor-
specific tolerance in the subject. Alternatively, as described in more detail
below, nucleic acids encoding
these immune system regulator proteins or polypeptides or nucleic acids
regulating the expression of such
proteins or polyPeptides may be introduced into appropriate host cells to
increase or decrease the expression
of the proteins as desired.
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EXAMPLE 24
Assay~ the E~c~ressed Proteins or Polypeptides for Hematopoiesis Regulating
Activity
[0328] The proteins or polypeptides encoded by the nucleic acids described
above may also be
evaluated for their hematopoiesis regulating activity. For example, the effect
of the proteins or polypeptides
on embryonic stem cell differentiation may be evaluated. Numerous assays for
such activity are familiar to
those skilled in the art, including the assays disclosed in the following
references, which are incorporated
herein by reference: Johansson et al. Cell. Biol. 15:141-151, 1995; Keller et
al., Mol. Cell. Biol. 13:473-486,
1993; McClanahan et al., Blood 81:2903-2915, 1993.
[0329] The proteins or polypeptides encoded by the nucleic acids described
above may also be
evaluated for their influence on the lifetime of stem cells and stem cell
differentiation. Numerous assays for
such activity are familiar to those skilled in the art, including the assays
disclosed in the following references,
which are incorporated herein by reference: Freshney, M.G. Methylcellulose
Colony Forming Assays, in
Culture of HematoQoietic Cells. R.I. Freshney, et al. Eds. pp. 265-268, Wiley-
Liss, Inc., New York, NY.
1994; Hirayama et al., Proc. Natl. .9cad. Sci. USA 89:5907-5911, 1992;
McNiece, LK. and Briddell, R.A.
Primitive Hematopoietic Colony Forming Cells with High Proliferative
Potential, in Culture of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss,
Inc., New York, NY. 1994; Neben
et al., Experimental Hematology 22:353-359, 1994; Ploemacher, R.E. Cobblestone
Area Forming Cell Assay,
In Culture of Hematopoietic Cells. R.I. Freshney, et al. Eds. pp. I-21, Wiley-
Liss, Inc., New York, NY. 1994;
Spooncer, E., Dexter, M. and Allen, T. Long Term Bone Marrow Cultures in the
Presence of Stromal Cells,
in Culture of Hematopoietic Cells. R.I. Freshney, et al. Eds. pp. 163-179,
Wiley-Liss, Inc., New York, NY.
1994; and Sutherland, H.J. Long Term Culture Initiating Cell Assay, in Culture
of Hematopoietic Cells. R.I.
Freshney, et al. Eds. pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
[0330] Those proteins or polypeptides which exhibit hematopoiesis regulatory
activity may then be
formulated as pharmaceuticals and used to treat clinical conditions in which
regulation of hematopoiesis is
beneficial. For example, a protein or polypeptide of the present invention may
be useful in regulation of
hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell
deficiencies. Even marginal
biological activity in support of colony forming cells or of factor-dependent
cell lines indicates involvement
in regulating hematopoiesis, e.g. in supporting the growth and proliferation
of erythroid progenitor cells alone
or in combination with other cytokines, thereby indicating utility, for
example, in treating various anemias or
for use in conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors
and/or erythroid cells; in supporting the growth and proliferation of myeloid
cells such as granulocytes and
monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in
conjunction with
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chemotherapy to prevent or treat consequent myelo-suppression; in supporting
the growth and proliferation of
megakaryocytes and consequently of platelets thereby allowing prevention or
treatment of various platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary to platelet
transfusions; and/or in_ supporting the growth and proliferation of
hematopoietic stem cells which are capable
of maturing to any and all of the above-mentioned hematopoietic cells and
therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with transplantion,
including, without limitation,
aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in
repopulating the stem cell
compartment post imadiation/chemotherapy, either in-vivo or ex-vivo (i.e., in
conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation (homologous
or heterologous)) as normal
cells or genetically manipulated for gene therapy. Alternatively, as described
in more detail below, nucleic
acids encoding these proteins or polypeptides or nucleic acids regulating the
expression of these proteins or
polypeptides may be introduced into appropriate host cells to increase or
decrease the expression of the
proteins as desired.
EXAMPLE 25
Assayin tg~ he E.x~ressed Proteins or Polype~tides for Regulation of Tissue
Growth
[0331] The proteins or polypeptides encoded by the nucleic acids described
above may also be
evaluated for their effect on tissue growth. Numerous assays for such activity
are familiar to those skilled in
the art, including the assays disclosed in International Patent Publication
No. W095/16035, International
Patent Publication No. W095/05846 and International Patent Publication No.
W091/07491, which are
incorporated herein by reference.
[0332] Assays for wound healing activity include, without limitation, those
described in: Winter,
Epidermal Wound Healing, pps. 71-112 (Maibach, H1 and Rovee, DT, eds.), Year
Book Medical Publishers,
Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-
84 (1978) which are
incorporated herein by reference.
[0333] Those proteins or polypeptides which are involved in the regulation of
tissue growth may
then be formulated as pharmaceuticals and used to treat clinical conditions in
which regulation of tissue
growth is beneficial. For example, a protein or polypeptide may have utility
in compositions used for bone,
cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as
well as for wound healing and
tissue repair and replacement, and in the treatment of burns, incisions and
ulcers.
[0334] A protein or polypeptide encoded by the nucleic acids described above
which induces
cartilage and/or bone growth in circumstances where bone is not normally
formed, has application in the
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healing of bone fractures and cartilage damage or defects in humans and other
animals. Such a preparation
employing a protein or polypeptide of the invention may have prophylactic use
in closed as well as open
fracture reduction and also in the improved fixation of artificial joints. De
novo bone synthesis induced by an
osteogenic agent contributes to the repair of congenital, trauma induced, or
oncologic resection induced
craniofacial defects, and also is useful in cosmetic plastic surgery.
[0335] A protein or polypeptide of this invention may also be used in the
treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment to attract bone-forming
cells, stimulate growth of bone-forming cells or induce differentiation of
progenitors of bone-forming cells.
A protein of the invention may also be useful in the treatment of osteoporosis
or osteoarthritis, such as
through stimulation of bone and/or cartilage repair or by blocking
inflammation or processes of tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory processes.
[0336] Another category of tissue regeneration activity that may be
attributable to the proteins or
polypeptides encoded by the nucleic acids described above is tendon/ligament
formation. A protein or
polypeptide encoded by the nucleic acids described above, which induces
tendon/ligament-like tissue or other
tissue formation in circumstances where such tissue is not normally formed,
has application in the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and other animals.
Such a preparation employing a tendon/ligament-like tissue inducing protein
may have prophylactic use in
preventing damage to tendon or ligament tissue, as well as use in the improved
fixation of tendon or ligament
to bone or other tissues, and in repairing defects to tendon or ligament
tissue. De novo tendon/ligament-like
tissue formation induced by a protein or polypeptide of the present invention
contributes to the repair of
tendon or ligaments defects of congenital, traumatic or other origin and is
also useful in cosmetic plastic
surgery for attachment or repair of tendons or ligaments. The proteins or
polypeptides of the present
invention may provide an environment to attract tendon- or ligament-forming
cells, stimulate growth of
tendon- or ligament-forming cells, induce differentiation of progenitors of
tendon- or ligament-forming cells,
or induce growth of tendon/ligament cells or progenitors ex vivo for return in
vivo to effect tissue repair. The
proteins or polypeptides of the invention may also be useful in the treatment
of tendinitis, carpal tunnel
syndrome and other tendon or ligament defects. The therapeutic compositions
may also include an
appropriate matrix and/or sequestering agent as a carrier as is well known in
the art.
[0337] The proteins or polypeptides of the present invention may also be
useful for proliferation of
neural cells and for regeneration of nerve and brain tissue, i.e., for the
treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and traumatic
disorders, which involve
degeneration, death or trauma to neural cells or nerve tissue. More
specifically, a protein or polypeptide may
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be used in the treatment of diseases of the peripheral nervous system, such as
peripheral nerve injuries,
peripheral neuropathy and localized neuropathies, and central nervous system
diseases, such as Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager syndrome. Further
conditions which maybe treated in accordance with the present invention
include mechanical and traumatic
disorders, such as spinal cord disorders, head trauma and cerebrovascular
diseases such as stroke. Peripheral
neuropathies resulting from chemotherapy or other medical therapies may also
be treatable using a protein or
polypeptide of the invention.
[0338] Proteins or polypeptides of the invention may also be useful to promote
better or faster
closure of non-healing wounds, including without limitation pressure ulcers,
ulcers associated with vascular
insufficiency, surgical and traumatic wounds, and the like.
[0339] It is expected that a protein or polypeptide of the present invention
may also exhibit activity
for generation or regeneration of other tissues, such as organs (including,
for example, pancreas, liver,
intestine, kidney, skin, endothelium) muscle (smooth, skeletal or cardiac) and
vascular (including vascular
endothelium) tissue, or for promoting the growth of cells comprising such
tissues. Part of the desired effects
may be by inhibition or modulation of fibrotic scarring to allow normal tissue
to generate. A protein or
polypeptide of the invention may also exhibit angiogenic activity.
[0340] A protein or polypeptide of the present invention may also be useful
for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues, and conditions
resulting from systemic cytokine damage.
[0341] A protein or polypeptide of the present invention may also be useful
for promoting or
inhibiting differentiation of tissues described above from precursor tissues
or cells; or for inhibiting the
growth of tissues described above.
[0342] Alternatively, as described in more detail below, nucleic acids
encoding tissue growth
regulating activity proteins or polypeptides or nucleic acids regulating the
expression of such proteins or
polypeptides may be introduced into appropriate host cells to increase or
decrease the expression of the
proteins as desired.
EXAMPLE 2fi
Assayin tg he E~ressed Proteins or Polypeptides for Regulation of Reproductive
Hormones
[0343] The proteins or polypeptides of the present invention may also be
evaluated for their ability
to regulate reproductive hormones, such as follicle stimulating hormone.
Numerous assays for such activity
are familiar to those skilled in the art, including the assays disclosed in
the following references, which are
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incorporated herein by reference: Vale et al., Endocrinol. 91:562-572, 1972;
Ling et al., Nature 321:779-782,
1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986. Chapter 6.12 in Current Protocols in
Immunology, J.E. Coligan et
al. Eds. Greene Publishing Associates and Wiley-Intersciece ; Taub et al. J.
Clin. Invest. 95:1370-1376,
1995; Lind et al. APMIS 103:140-146, 1995; Muller et al. Eur. J. Immunol.
25:1744-1748; Gruber et al. J.
Immunol. 152:5860-5867, 1994; Johnston et al., Jlmmunol. 153:1762-1768, 1994.
[0344] Those proteins or polypeptides which exhibit activity as reproductive
hormones or regulators
of cell movement may then be formulated as pharmaceuticals and used to treat
clinical conditions in which
regulation of reproductive hormones are beneficial. For example, a protein or
polypeptide may exhibit
activin- or inhibin-related activities. Inhibins are characterized by their
ability to inhibit the release of follicle
stimulating hormone (FSH), while activins are characterized by their ability
to stimulate the release of FSH.
Thus, a protein or polypeptide of the present invention, alone or in
heterodimers with a member of the inhibin
_ family, may be useful as a contraceptive based on the ability of inhibins to
decrease fertility in female
mammals and decrease spermatogenesis in male mammals. Administration of
sufficient amounts of other
inhibins can induce infertility in these mammals. Alternatively, the protein
or polypeptide of the invention, as
a homodimer or as a heterodimer with other protein subunits of the irihibin-B
group, may be useful as a
fertility inducing therapeutic, based upon the ability of activin molecules in
stimulating FSH release from
cells of the anterior pituitary. See, for example, United States Patent
4,798,885, the disclosure of which is
incorporated herein by reference. A protein or polypeptide of the invention
may also be useful for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the lifetime
reproductive performance of domestic animals such as cows, sheep and pigs.
[0345] Alternatively, as described in more detail below, nucleic acids
encoding reproductive
hormone regulating activity proteins or polypeptides or nucleic acids
regulating the expression of such
proteins or polypeptides may be introduced into appropriate host cells to
increase or decrease the expression
of the proteins or polypeptides as desired.
EXAMPLE 27
Assaying_the Expressed Proteins or Polypeptides For Chemotactic/Chemokinetic
Activity
[0346] The proteins or polypeptides of the present invention may also be
evaluated for
chemotactic/chemokinetic activity. For example, a protein or polypeptide of
the present invention may have
chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian
cells, including, for example,
monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or endothelial cells.
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Chemotactic and chemokinetic proteins or polypeptides can be used to mobilize
or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
or polypeptides provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in treatment of localized
infections. For example, attraction of lymphocytes, monocytes or neutrophils
to tumors or sites of infection
may result in improved immune responses against the tumor or infecting agent.
[0347] A protein or polypeptide has chemotactic activity for a particular cell
population if it can
stimulate, directly or indirectly, the directed orientation or movement of
such cell population. Preferably, the
protein or polypeptide has the ability to directly stimulate directed movement
of cells. Whether a particular
protein or polypeptide has chemotactic activity for a population of cells can
be readily determined by
employing such protein or polypeptide in any known assay for cell chemotaxis.
[0348] The activity of a protein or polypeptide of the invention may, among
other means, be
measured by the following methods:
[0349] Assays for chemotactic activity (which will identify proteins or
polypeptides that induce or
prevent chemotaxis) consist of assays that measure the ability of a protein or
polypeptide to induce the
migration of cells across a membrane as well as the ability of a protein or
polypeptide to induce the adhesion
of one cell population to another cell population. Suitable assays for
movement and adhesion include,
without limitation, those described in: Current Protocols in Immunology, Ed by
J.E. Coligan, A.M.
Kruisbeek, D.I-l. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-
Interscience, Chapter 6.12: 6.12.1-6.12.28; Taub et al. J Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS
103:140-146, 1995; Mueller et al., Eur. J. Immunol. 25:1744-1748; Gruber et
al. J. Immunol. 152:5860-5867,
1994; Johnston et al. J. Immunol., 153:1762-1768, 1994.
EXAMPLE 28
Assaying the Expressed Proteins or Polypeptides for Regulation of Blood
Clotting
[0350] The proteins or polypeptides of the present invention may also be
evaluated for their effects
on blood clotting. Numerous assays for such activity are familiar to those
skilled in the art, including the
assays disclosed in the following references, which are incorporated herein by
reference: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;
Humphrey et al.,
Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
[0351] Those proteins or polypeptides which are involved in the regulation of
blood clotting may
then be formulated as pharmaceuticals and used to treat clinical conditions in
which regulation of blood
clotting is beneficial. For example, a protein or polypeptide of the invention
may also exhibit hemostatic or
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thrombolytic activity. As a result, such a protein or polypeptide is expected
to be useful in treatment of
various coagulations disorders (including hereditary disorders, such as
hemophiliac) or to enhance
coagulation and other hemostatic events in treating wounds resulting from
trauma, surgery or other causes. A
protein or polypeptide of the invention may also be useful for dissolving or
inhibiting formation of
thromboses and for treatment and prevention of conditions resulting therefrom
(such as infarction of cardiac
and central nervous system vessels (e.g., stroke)). Alternatively, as
described in more detail below, nucleic
acids encoding blood clotting activity proteins or polypeptides or nucleic
acids regulating the expression of
such proteins or polypeptides may be introduced into appropriate host cells to
increase or decrease the
expression of the proteins or polypeptides as desired.
EXAMPLE 29
Ass~ing the Expressed Proteins or Polypeptides for Involvement in
Receptor/Ligand Interactions
[0352] The proteins or polypeptides of the present invention may also be
evaluated for their
involvement in receptor/ligand interactions. Numerous assays for such
involvement are familiar to those
skilled in the art:, including the assays disclosed in the following
references, which are incorporated herein by
reference: Chapter 7. 7.28.1-7.28.22 in Current Protocols in Immunology, J.E.
Coligan et al. Eds. Greene
Publishing Associates and Wiley-Interscience; Takai et al., Proc. Natl. Acad.
Sci. USA 84:6864-6868, 1987;
Bierer et al., .I Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160, 1989;
Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell
80:661-670, 1995; Gyuris et al.,
Cel175:791-803, 1993.
[0353] For example, the proteins or polypeptides of the present invention may
also demonstrate
activity as receptors, receptor ligands or inhibitors or agonists of
receptor/ligand interactions. Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their ligands, receptor kinases
and their ligands, receptor phosphatases and their ligands, receptors involved
in cell-cell interactions and their
ligands (including without limitation, cellular adhesion molecules (such as
selectins, integrins and their
ligands) and receptor/ligand pairs involved in antigen presentation, antigen
recognition and development of
cellular and humoral immune responses). Receptors and ligands are also useful
for screening of potential
peptide or small molecule inhibitors of the relevant receptor/ligand
interaction. A protein or polypeptide of
the present invention (including, without limitation, fragments of receptors
and ligands) may be useful as
inhibitors of receptor/ligand interactions. Alternatively, as described in
more detail below, nucleic acids
encoding proteins or polypeptides involved in receptor/ligand interactions or
nucleic acids regulating the
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expression of such proteins or polypeptides may be introduced into appropriate
host cells to increase or
decrease the expression of the proteins or polypeptides as desired.
EXAMPLE 30
Assay_in_g the Proteins or Polypeptides for Anti-Inflammatory Activity
[0354] The proteins or polypephdes of the present invention may also be
evaluated for anti-
inflammatory activity. The anti-inflammatory activity may be achieved by
providing a stimulus to cells
involved in the inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by stimulating or
suppressing production of other
factors which more directly inhibit or promote an inflammatory response.
Proteins or polypeptides
exhibiting such activities can be used to treat inflammatory conditions
including chronic or acute
conditions, including without limitation inflammation associated with
infection (such as septic shock,
sepsis or systemic inflammatory response syndrome), ischemia-reperfusioninury,
endotoxin lethality,
arthritis, complement-mediated hyperacute rejection, nephritis, cytokine- or
chemokine-induced lung
injury, inflammatory bowel disease, Crohn's disease or resulting from over
production of cytokines such
as TNF or IL-1. Proteins or polypeptides of the invention may also be useful
to treat anaphylaxis and
hypersensitivity to an antigenic substance or material. Alternatively, as
described in more detail below,
nucleic acids encoding anti-inflammatory activity proteins or polypeptides or
nucleic acids regulating the
expression of such proteins or polypeptides may be introduced into appropriate
host cells to increase or
decrease the expression of the proteins or polypeptides as desired.
EXAMPLE 31
Assaying the Expressed Proteins or Polypeptides for Tumor Inhibition Activity
[0355) The proteins or polypeptides of the present invention may also be
evaluated for tumor
inhibition activity. In addition to the activities described above for
immunological treatment or prevention of
tumors, a protein or polypeptide of the invention may exhibit other anti-tumor
activities. A protein or
polypeptide may inhibit tumor growth directly or indirectly (such as, for
example, via ADCC). A protein or
polypeptide may exhibit its tumor inhibitory activity by acting on tumor
tissue or tumor precursor tissue, by
inhibiting formation of tissues necessary to support tumor growth (such as,
for example, by inhibiting
angiogenesis), by causing production of other factors, agents or cell types
which inhibit tumor growth, or by
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suppressing, eliminating or inhibiting factors, agents or cell types which
promote tumor growth. .
Alternatively, as described in more detail below, nucleic acids encoding
proteins or polypeptides with tumor
inhibition activity or nucleic acids regulating the expression of such
proteins or polypeptides may be
introduced into appropriate host cells to increase or decrease the expression
of the proteins or polypeptides as
desired.
[0356] A protein or polypeptide of the invention may also exhibit one or more
of the following
additional activities or effects: inhibiting the growth, infection or function
of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other parasites;
effecting (suppressing or enhancing)
bodily characteristics, including, without limitarion, height, weight, hair
color, eye color, skin, fat to lean ratio
or other tissue pigmentation, or organ or body part size or shape (such as,
for example, breast augmentation
or diminution, change in bone form or shape); effecting biorhythms or
circadian cycles or rhythms; effecting
the fertility of male or female subjects; effecting the metabolism,
catabolism, anabolism, processing,
utilization, storage or elimination of dietary fat, lipid, protein,
carbohydrate, vitamins, minerals, cofactors or
other nutritional factors or component(s); effecting behavioral
characteristics, including, without limitation,
appetite, libido, stress, cognition (including cognitive disorders),
depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain reducing
effects; promoting differentiation
and growth of embryonic stem cells in lineages other than hematopoietic
lineages; hormonal or endocrine
activity; in the case of enzymes, correcting deficiencies of the enzyme and
treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for example,
psoriasis); immunoglobulin-like
activity (such as, for example, the ability to bind antigens or complement);
and the ability to act as an antigen
in a vaccine composition to raise an immune response against such protein or
another material or entity which
is cross-reactive with such protein. Alternatively, as described in more
detail below, nucleic acids encoding
proteins or polypeptides involved in any of the above mentioned activities or
nucleic acids regulating the
expression of such proteins may be introduced into appropriate host cells to
increase or decrease the
expression of the proteins or polypeptides as desired.
EXAMPLE 32
Identification of Proteins or Polypeptides which Interact with Proteins or
Polype~tides of the Present
Invention
[0357] Proteins or polypeptides which interact with the proteins or
polypeptides of the present
invention, such as receptor proteins, may be identified using two hybrid
systems such as the Matchmaker
Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the
manual accompanying the kit
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which is incorporated herein by reference, nucleic acids encoding the proteins
or polypeptides of the present
invention, are inserted into an expression vector such that they are in frame
with DNA encoding the DNA
binding domain of the yeast transcriptional activator GAL4. cDNAs in a cDNA
library which encode
proteins or polypeptides which might interact with the proteins or
polypeptides of the present invention are
inserted into a second expression vector such that they are in frame with DNA
encoding the activation
domain of GAI_4. The two expression plasmids are transformed into yeast and
the yeast are plated on
selection medium which selects for expression of selectable markers on each of
the expression vectors as well
as GAL4 dependent expression of the HIS3 gene. Transformants capable of
growing on medium lacking
histidine are screened for GAL4 dependent lacZ expression. Those cells which
are positive in both the
histidine selection and the lacZ assay contain plasmids encoding proteins or
polypeptides which interact with
the proteins or polypeptides of the present invention.
[0358] Alternatively, the system described in Lustig et al., Methods in
Enrymology 283: 83-99
(1997), the disclosure of which is incorporated herein by reference, may be
used for identifying molecules
which interact with the proteins or polypeptides of the present invention. In
such systems, in vitro
1 S transcription reactions are performed on a pool of vectors containing
nucleic acid inserts which encode the
proteins or polypeptides of the present invention. The nucleic acid inserts
are cloned downstream of a
promoter which drives in vitro transcription. The resulting pools of mRNAs are
introduced into Xenopus
laevis oocytes. 'The oocytes are then assayed for a desired activity.
[0359) Alternatively, the pooled in vitro transcription products produced as
described above may be
translated in vitro. The pooled in vitro translation products can be assayed
for a desired activity or for
interaction with a known protein or polypeptide.
[0360] Proteins, polypeptides or other molecules interacting with proteins or
polypeptides of the
present invention can be found by a variety of additional techniques. In one
method, affinity columns
containing the protein or polypeptide of the present invention can be
constructed. In some versions, of
this method the affinity column contains chimeric proteins in which the
protein or polypeptide of the
present invention is fused to glutathione S-transferase. A mixture of cellular
proteins or pool of expressed
proteins as described above and is applied to the affinity column. Molecules
interacting with the protein
or polypeptide attached to the column can then be isolated and analyzed on 2-D
electrophoresis gel as
described in Ramunsen et al. Electrophoresis, 18, 588-598 (1997), the
disclosure of which is
incorporated herein by reference. Alternatively, the molecules retained on the
affinity column can be
purified by electrophoresis based methods and sequenced. The same method can
be used to isolate
antibodies, to screen phage display products, or to screen phage display human
antibodies.
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[0361] Molecules interacting with the proteins or polypeptides of the present
invention can also
be screened by using an Optical Biosensor as described in Edwards &
Leatherbarrow, Analytical
Biochemistry, 246, 1-6 (1997), the disclosure of which is incorporated herein
by reference. The main
advantage of the method is that it allows the determination of the association
rate between the protein or
polypeptide and other interacting molecules. Thus, it is possible to
specifically select interacting
molecules with a high or low association rate. Typically a target molecule is
linked to the sensor surface
(through a carboxymethl dextran matrix) and a sample of test molecules is
placed in contact with the
target molecules. The binding of a test molecule to the target molecule causes
a change in the refractive
index and/ or thickness. This change is detected by the Biosensor provided it
occurs in the evanescent
field (which extend a few hundred nanometers from the sensor surface). In
these screening assays, the
target molecule can be one of the proteins or polypeptides of the present
invention and the test sample can
be a collection of proteins, polypeptides or other molecules extracted from
tissues or cells, a pool of
expressed proteins, combinatorial peptide andl or chemical libraries, or phage
displayed peptides. The
tissues or cells from which the test molecules are extracted can originate
from any species.
[0362] In other methods, a target protein or polypeptide is immobilized and
the test population is
a collection of unique proteins or polypeptides of the present invention.
[0363] To study the interaction of the proteins or polypeptides of the present
invention with
drugs, the microdialysis coupled to HPLC method described by Wang et al.,
Chromatographia, 44, 205-
208(1997) or the affinity capillary electrophoresis method described by Busch
et al., J. Chromatogr.
777:311-328 ( 1997), the disclosures of which are incorporated herein by
reference can be used.
[0364] The system described in U.S. Patent No. 5,654,150, the disclosure of
which is incorporated
herein by reference, may also be used to identify molecules which interact
with the proteins or polypeptides
of the present invention. In this system, pools of nucleic acids encoding the
proteins or polypeptides of the
present invention are transcribed and translated in vitro and the reaction
products are assayed for interaction
with a lrnown polypeptide or antibody.
[0365] It will be appreciated by those skilled in the art that the proteins or
polypeptides of the
present invention may be assayed for numerous activities in addition to those
specifically enumerated above.
For. example, the expressed proteins or polypeptides may be evaluated for
applications involving control and
regulation of inflammation, tumor proliferation or metastasis, infection, or
other clinical conditions. In
addition, the proteins or polypeptides may be useful as nutritional agents or
cosmetic agents.
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Epitopes and Antibody Fusions
[0366] A preferred embodiment of the present invention is directed to epitope-
bearing
polypeptides and epitope-bearing polypeptide fragments. These epitopes may be
"antigenic epitopes" or
both an "antigenic epitope" and an "immunogenic epitope". An "immunogenic
epitope" is defined as a
part of a protein that elicits an antibody response in vivo when the
polypeptide is the immunogen. On the
other hand, a region of polypeptide to which an antibody binds is defined as
an "antigenic determinant" or
"antigenic epitope." The number of immunogenic epitopes of a protein generally
is less than the number
of antigenic epitopes. See, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci.
USA 81:39984002. It is
particularly noted that although a particular epitope may not be immunogenic,
it is nonetheless useful
since antibodies can be made in vitro to any epitope.
[0367] An epitope can comprise as few as 3 amino acids in a spatial
conformation which is
unique to the epitope. Generally an epitope consists of at least 6 such amino
acids, and more often at least
8-10 such amino acids. In preferred embodiment, antigenic epitopes comprise a
number of amino acids
that is any integer between 3 and 50. Fragments which function as epitopes may
be produced by any
conventional means. See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985),
further described in U.S. Patent No. 4,631,211. Methods for determining the
amino acids which make up
an epitope include x-ray crystallography, 2-dimensional nuclear magnetic
resonance, and epitope
mapping, e.g., the Pepscan method described by H. Mario Geysen et al. (1984);
Proc. Natl. Acad. Sci.
U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No.
WO 84/03506.
Another example is the algorithm of Jameson and Wolf, Comp. Appl. Biosci.
4:181-186 (1988) (said
references incorporated by reference in their entireties). The Jameson-Wolf
antigenic analysis, for
example, may be performed using the computer program PROTEAN, using default
parameters (Version
3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison,
WI.
[0368] Antigenic epitopes are useful, for example, to raise antibodies,
including monoclonal
antibodies, that specifically bind the epitope. (See, for instance, Wilson et
al., Cell 37:767-778 (1984);
Sutcliffe, J. G. et al., Science 219:660-666 (1983).)
[0369] Similarly, immunogenic epitopes can be used to induce antibodies
according to methods
well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et
al., supra; Chow, M. et al.,
Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen.
Virol. 66:2347-2354 (1985).) A
preferred immunogenic epitope includes the secreted protein. The immunogenic
epitopes may be
presented together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or
mouse) or, if it is long enough (at least about 25 amino acids), without a
carrier. However, immunogenic
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epitopes comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies
capable of binding to, at the very least, linear epitopes in a denatured
polypeptide (e.g., in Western
blotting.)
[0370] Epitope-bearing polypeptides of the present invention are used to
induce antibodies
according to methods well known in the art including, but not limited to, in
vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe, et al., supra;
Wilson, et al., supra, and
Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If in vivo immunization is
used, animals may be
immunized with free peptide; however, anti-peptide antibody titer may be
boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH)
or tetanus toxoid. For
instance, peptides containing cysteine residues may be coupled to a carrier
using a linker such as
-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may
be coupled to carriers
using a more general linking agent such as glutaraldehyde. Animals such as
rabbits, rats and mice are
immunized with either free or carrier-coupled peptides, for instance, by
intraperitoneal and/or intradermal
injection of emulsions containing about 100 figs of peptide or carrier protein
and Freund's adjuvant.
Several booster injections may be needed, for instance, at intervals of about
two weeks, to provide a
useful titer of anti-peptide antibody which can be detected, for example, by
ELISA assay using free
peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in
serum from an immunized
animal may be increased by selection of anti-peptide antibodies, for instance,
by adsorption to the peptide
on a solid support and elution of the selected antibodies according to methods
well known in the art.
[0371] As one of skill in the art will appreciate, and discussed above, the
polypeptides of the
present invention comprising an immunogenic or antigenic epitope can be fused
to heterologous
polypeptide sequences. For example, the polypeptides of the present invention
may be fused with the
constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof
(CH1, CH2, CH3, any
combination thereof including both entire domains and portions thereof)
resulting in chimeric
polypeptides. 'These fusion proteins facilitate purification, and show an
increased half life in vivo. This
has been shown, e.g., for chimeric proteins consisting of the first two
domains of the human
CD4-polypeptide and various domains of the constant regions of the heavy or
light chains of mammalian
immunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988) Nature
331:84-86. Fusion proteins
that have a disulfide-linked dimeric structure due to the IgG portion can also
be more efficient in binding
and neutralizing other molecules than monomeric polypeptides or fragments
thereof alone. See, e.g.,
Fountoulakis et al. (1995) J. Biochem. 270:3958-3964. Nucleic acids encoding
the above epitopes can
also be recombined with a gene of interest as an epitope tag to aid in
detection and purification of the
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expressed polypeptide.
[0372] Additional fusion proteins of the invention may be generated through
the techniques of
gene-shuffling, motif shuffling, exon-shuffling, or codon-shuffling
(collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to modulate the activities of
polypeptides of the present
invention thereby effectively generating agonists and antagonists of the
polypeptides. See, for example,
U.S. Patent Nos.: 5,605,793; 5,811,238; 5,834,252; 5,837,458; and Fatten,
P.A., et al., Curr. Opinion
Biotechnol. 8:724-733 (1997); Harayama, S., Trends Biotechnol. 16(2):76-82
(1998); Hansson, L.O., et
al. J. Mol. Biol. 287:265-276 (1999); and Lorenzo, M.M. and Blasco, R.,
Biotechniques 24(2):308-313
(1998) (each of these documents are hereby incorporated by reference). In one
embodiment, one or more
components, motifs, sections, parts, domains, fragments, etc., of coding
polynucleotides of the invention,
or the polypeptides encoded thereby may be recombined with one or more
components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous molecules.
Antibodies
[0373] The present invention further relates to antibodies and T-cell antigen
receptors (TCR)
which specifically bind the polypeptides of the present invention. The
antibodies of the present invention
include IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and
IgA2), IgD, IgE, or IgM,
and IgY. As used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including
single-chain whole antibodies, and antigen-binding fragments thereof. In a
preferred embodiment the
antibodies are human antigen binding antibody fragments of the present
invention include, but are not
limited to, Fab, Fab' F(ab)2 and F(ab')2, Fd, single-chain Fvs (scFv), single-
chain antibodies, disulfide-
linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The
antibodies may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human, murine, rabbit, goat,
guinea pig, camel, horse, or chicken.
[0374) Antigen-binding antibody fragments, including single-chain antibodies,
may comprise the
variable regions) alone or in combination with the entire or partial of the
following: hinge region, CH1,
CH2, and CH3 domains. Also included in the invention are any combinations of
variable regions) and
hinge region, C'H1, CH2, and CH3 domains. The present invention further
includes chimeric, humanized,
and human monoclonal and polyclonal antibodies which specifically bind the
polypeptides of the present
invention. The present invention further includes antibodies which are anti-
idiotypic to the antibodies of
the present invention.
[0375] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of
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greater multispecificity. Multispecific antibodies may be specific for
different epitopes of a polypeptide
of the present invention or may be specific for both a polypeptide of the
present invention as well as for
heterologous compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. {1991) J.
Immunol. 147:60-69; US
Patents Nos.: 5,573,920; 4,474,893; 5,601,819; 4,714,681; 4,925,648; Kostelny,
S.A. et al. (1992) J.
Immunol. 148:1547-1553.
[0376] In some embodiments, the antibodies may be capable of specifically
binding to a protein or
polypeptide encoded by EST-related nucleic acids, fragments of EST-related
nucleic acids, positional
segments of ES'T-related nucleic acids or fragments of positional segments of
EST-related nucleic acids. In
some embodiments, the antibody may be capable of binding an antigenic
determinant or an epitope in a
protein or polypeptide encoded by EST-related nucleic acids, fragments of EST-
related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids.
[0377] In other embodiments, the antibodies may be capable of specifically
binding to an EST-
related polypeptide, fragment of an EST-related polypeptide, positional
segment of an EST-related
polypeptide or fragment of a positional segment of an EST-related polypeptide.
In some embodiments, the
antibody may be capable of binding an antigenic determinant or an epitope in
an EST-related polypeptide,
fragment of an EST-related polypeptide, positional segment of an EST-related
polypeptide or fragment of a
positional segment of an EST-related polypeptide.
(0378] Antibodies of the present invention may be described or specified in
terms of the
epitope(s) or portions) of a polypeptide of the present invention which are
recognized or specifically
bound by the antibody. In the case of secreted proteins, the antibodies may
specifically bind a full-length
protein encoded by a nucleic acid of the present invention, a mature protein
(i.e. the protein generated by
cleavage of the signal peptide) encoded by a nucleic acid of the present
invention, or a signal peptide encoded
by a nucleic acid of the present invention. Moreover, the epitope(s) or
polypeptide portions) may be
specified as described herein, e.g., by N-terminal and C-terminal positions,
by size in contiguous amino
acid residues, or listed in the Tables and sequence listing. Antibodies which
specifically bind any epitope
or polypeptide of the present invention may also be excluded. Therefore, the
present invention includes
antibodies that specifically bind polypeptides of the present invention, and
allows for the exclusion of the
same.
[0379] Antibodies of the present invention may also be described or specified
in terms of their
cross-reactivity. Antibodies that do not bind any other analog, ortholog, or
homolog of the polypeptides
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of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less
than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less
than 65%, less than 60%, less
than 55%, and less than 50% identity (as calculated using methods known in the
art and described herein)
to a polypeptide of the present invention are also included in the present
invention. Further included in
the present invention are antibodies which only bind polypeptides encoded by
polynucleotides which
hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as
described herein). Antibodies of the present invention may also be described
or specified in terms of their
binding affinity. Preferred binding affinities include those with a
dissociation constant or Kd less than
5X10-6M, 10-6M, 5X10~'M, 10-'M, 5X10-8M, 10-~M, 5X10-9M, 10-~M, 5X10-
'°M, 10-'°M, 5X10"M,
10-"M, 5X10-''M, 10-'zM, 5X10-'3M, 10-'3M, 5X10-'4M, 10-'~M, 5X10-"M, and 10-
'SM.
[0380] Antibodies of the present invention have uses that include, but are not
limited to, methods
known in the art to purify, detect, and target the polypeptides of the present
invention including both in
vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the present
invention in biological samples. See, e.g., Harlow et al., ANTIBODIES: A
LABORATORY MANUAL,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference
in the entirety).
[0381] The antibodies of the present invention may be used either alone or in
combination with
other compositions. The antibodies may further be recombinantly fused to a
heterologous polypeptide at
the N- or C-tezminus or chemically conjugated (including covalent and non-
covalent conjugations) to
polypeptides or other compositions. For example, antibodies of the present
invention may be
recombinantly fused or conjugated to molecules useful as labels in detection
assays and effector
molecules such as heterologous polypeptides, drugs, ar toxins. See, e.g., WO
92/08495; WO 91/14438;
WO 89/12624; US Patent 5,314,995; and EP 0 396 387.
[0382] The antibodies of the present invention may be prepared by any suitable
method known
in the art. For example, a polypeptide of the present invention or an
antigenic fragment thereof can be
administered to an animal in order to induce the production of sera containing
polyclonal antibodies. The
term "monoclonal antibody" is not limited to antibodies produced through
hybridoma technology. The
term "antibody" refers to a polypeptide or group of polypeptides which are
comprised of at least one
binding domain, where a binding domain is formed from the folding of variable
domains of an antibody
molecule to form three-dimensional binding spaces with an internal surface
shape and charge distribution
complementary to the features of an antigenic determinant of an antigen.,
which allows an immunological
reaction with the antigen. The term "monoclonal antibody" refers to an
antibody that is derived from a
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single clone, including eukaryotic, prokaryotic, or phage clone, and not the
method by which it is
produced. Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art
including the use of hybridoma, recombinant, and phage display technology.
[0383) Hybridoma techniques include those known in the art (See, e.g., Harlow
et al.,
ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988);
Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-<~ELL HYBR)DOMAS 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference in their
entireties). Fab and F(ab')2
fragments may be produced, for example, from hybridoma-produced antibodies by
proteolytic cleavage,
using enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2 fragments).
[0384] Alternatively, antibodies of the present invention can be produced
through the application
of recombinant DNA technology or through synthetic chemistry using methods
known in the art. For
example, the antibodies of the present invention can be prepared using various
phage display methods
known in the art. In phage display methods, functional antibody domains are
displayed on the surface of
a phage particle which carries polynucleotide sequences encoding them. Phage
with a desired binding
property are selected from a repertoire or combinatorial antibody library
(e.g. human or murine) by
selecting directly with antigen, typically antigen bound or captured to a
solid surface or bead. Phage used
in these methods are typically filamentous phage including fd and M13 with
Fab, Fv or disulfide
stabilized Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein.
Examples of phage display methods that can be used to make the antibodies of
the present invention
include those disclosed in Brinkman U. et al. (1995) J. Immunol. Methods
182:41-50; Ames, R.S. et al.
(1995) J. Immunol. Methods 184:177-186; Kettleborough, C.A. et al. (1994) Eur.
J. Immunol. 24:952-
958; Persic, L. et al. (1997) Gene 187 9-18; Burton, D.R. et al. (1994)
Advances in Immunology 57:191-
280; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236;
WO 95/15982; WO 95/20401; and US Patents 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908,
5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727
and 5,733,743 (said
references incorporated by reference in their entireties).
[0385) As described in the above references, after phage selection, the
antibody coding regions
from the phage can be isolated and used to generate whole antibodies,
including human antibodies, or any
other desired antigen binding fragment, and expressed in any desired host
including mammalian cells,
insect cells, plant cells, yeast, and bacteria. For example, techniques to
recombinantly produce Fab, Fab'
F(ab)2 and F(ab')2 fragments can also be employed using methods known in the
art such as those
disclosed in WO 92/22324; Mullinax, R.L. et al. (1992) BioTechniques 12(6):864-
869; and Sawai, H. et
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al. (1995) AJRI 34:26-34; and Better, M. et al. (1988) Science 240:1041-1043
(said references
incorporated by reference in their entireties).
[0386] Examples of techniques which can be used to produce single-chain Fvs
and antibodies
include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et al.
(1991) Methods in
Enzymology 203:46-88; Shu, L. et al. (1993) PNAS 90:7995-7999; and Skerra, A.
et al. (1988) Science
240:1038-1040. For some uses, including in vivo use of antibodies in humans
and in vitro detection
assays, it may be preferable to use chimeric, humanized, or human antibodies.
Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al.,
BioTechniques 4:214 (1986); Gillies, S.D. et al. (1989) J. Immunol. Methods
125:191-202; and US Patent
5,807,715. Antibodies can be humanized using a variety of techniques including
CDR-grafting (EP 0 239
400; WO 91/09967; US Patent 5,530,101; and 5,585,089), veneering or
resurfacing (EP 0 592 106; EP 0
519 596; Padlan E.A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka
G.M. et al. (1994)
Protein Engineering 7(6):805-814; Roguska M.A. et al. (1994) PNAS 91:969-973),
and chain shuffling
(US Patent 5,565,332). Human antibodies can be made by a variety of methods
known in the art
including phage display methods described above. See also, US Patents
4,444,887, 4,716,111, 5,545,806,
and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO
96/33735; and WO
91/10741 (said references incorporated by reference in their entireties).
[0387] Further included in the present invention are antibodies recombinantly
fused or
chemically conjugated (including both covalently and non-covalently
conjugations) to a polypeptide of
the present invention. The antibodies may be specific for antigens other than
polypeptides of the present
invention. For example, antibodies may be used to target the polypeptides of
the present invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the polypeptides of the present
invention to antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the
polypeptides of the present invention may also be used in in vitro
immunoassays and purification methods
using methods known in the art. See e.g., Harbor et al., supra, and WO
93/21232; EP 0 439 095;
Naramura, M. et al. (1994) Immunol. Lett. 39:91-99; US Patent 5,474,981;
Gillies, S.O. et al. (1992)
PNAS 89:1428-1432; Fell, H.P. et al. (1991) J. Immunol. 146:2446-2452 (said
references incorporated by
reference in their entireties).
[0388] The present invention further includes compositions comprising the
polypeptides of the
present invention fused or conjugated to antibody domains other than the
variable regions. For example,
the polypeptides of the present invention may be fused or conjugated to an
antibody Fc region, or portion
thereof. The antibody portion fused to a polypeptide of the present invention
may comprise the hinge
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region, CH 1 domain, CH2 domain, and CH3 domain or any combination of whole
domains or portions
thereof. The polypeptides of the present invention may be fused or conjugated
to the above antibody
portions to increase the in vivo half life of the polypeptides or for use in
immunoassays using methods
known in the art. The polypephdes may also be fused or conjugated to the above
antibody portions to
form multimers. For example, Fc portions fused to the polypeptides of the
present invention can form
dimers through disulfide bonding between the Fc portions. Higher multimeric
forms can be made by
fusing the polypeptides to portions of IgA and IgM. Methods for fusing or
conjugating the polypeptides
of the present invention to antibody portions are known in the art. See e.g.,
US Patents Nos. 5,336,603;
5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 0 307 434, EP 0 367
166; WO 96/04388, WO
91/06570; Ashkenazi, A. et al. (1991) PNAS 88:10535-10539; Zheng, X.X. et al.
(1995) J. Immunol.
154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (said references
incorporated by
reference in their entireties).
[0389] The invention fiu-ther relates to antibodies which act as agonists or
antagonists of the
polypeptides of the present invention. For example, the present invention
includes antibodies which
disrupt the receptor/ligand interactions with the polypeptides of the
invention either partially or fully.
Included are both receptor-specific antibodies and ligand-specific antibodies.
Included are receptor-
specific antibodies which do not prevent ligand binding but prevent receptor
activation. Receptor
activation (i.e., signaling) may be determined by techniques described herein
or otherwise known in the
art. Also include are receptor-specific antibodies which both prevent ligand
binding and receptor
activation. Likewise, included are neutralizing antibodies which bind the
ligand and prevent binding of
the ligand to the receptor, as well as antibodies which bind the ligand,
thereby preventing receptor
activation, but do not prevent the ligand from binding the receptor. Further
included are antibodies which
activate the receptor. These antibodies may act as agonists for either all or
less than all of the biological
activities affected by ligand-mediated receptor activation. The antibodies may
be specified as agonists or
antagonists for biological activities comprising specific activities disclosed
herein. The above antibody
agonists can be made using methods known in the art. See e.g., WO 96/40281; US
Patent 5,811,097;
Deng, B. et al. (1998) Blood 92(6):1981-1988; Chen, Z. et al. (1998) Cancer
Res. 58(16):3668-3678;
Harrop, J.A. et al. (1998) J. Immunol. 161(4):1786-1794; Zhu, Z. et al. (1998)
Cancer Res. 58(15):3209-
3214; Yoon, D.Y. et al. (1998) J. Immunol. 160(7):3170-3179; Prat, M. et al.
(1998) J. Cell. Sci.
111(Pt2):237-247; Pitard, V. et al. (1997) J. Immunol. Methods 205(2):177-190;
Liautard, J. et al. (1997)
Cytokinde 9(4):233-241; Carlson, N.G. et al. (1997) J. Biol. Chem.
272(17):11295-11301; Taryman, R.E.
et al. (1995) Neuron 14(4):755-762; Muller, Y.A. et a1. (1998) Structure
6(9):1153-1167; Bartunek, P. et
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al. (1996) Cytokine 8(1):14-20 (said references incorporated by reference in
their entireties).
[0383] As discussed above, antibodies of the polypeptides of the invention
can, in turn, be
utilized to generate anti-idiotypic antibodies that "mimic" polypeptides of
the invention using techniques
well known to those.skilled in the art. See, e.g. Greenspan and Bona, FASEB J.
7(5):437-444 (1989);
Nissinoff, J. Immunol. 147(8):2429-2438 (1991). For example, antibodies which
bind to and
competitively inhibit polypeptide multimerization or binding of a polypeptide
of the invention to ligand
can be used to generate anti-idiotypes that "mimic" the polypeptide
multimerization or binding domain
and, as a consequence, bind to and neutralize polypeptide or its ligand. Such
neutralization anti-idiotypic
antibodies can be used to bind a polypeptide of the invention or to bind its
ligands/receptors, and thereby
block its biological activity,
EXAMPLE 33
Production of an Antibody to a Human Polypeptide or Protein
[0384] The above described EST-related nucleic acids, fragments of EST-related
nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids or nucleic; acids encoding EST-related polypeptides, fragments of EST-
related polypeptides, positional
segments of EST-related polypeptides or fragments of positional segments of
EST-related polypeptides are
operably linked to promoters and introduced into cells as described above.
[0385] In the case of secreted proteins, nucleic acids encoding the full
protein (i.e. the mature
protein and the signal peptide), nucleic acids encoding the mature protein
(i.e. the protein generated by
cleavage of the signal peptide), or nucleic acids encoding the signal peptide
are operably linked to promoters
and introduced into cells as described above.
[0386] The encoded proteins or polypeptides are then substantially purified or
isolated as described
above. The concentration of protein in the final preparation is adjusted, for
example, by concentration on an
Amicon filter device, to the level of a few ~g/ml. Monoclonal or polyclonal
antibody to the protein or
polypeptide can then be prepared as follows:
1. Monoclonal Antibody Production by H~bridoma Fusion
[0387] Monoclonal antibody to epitopes of any of the proteins or polypeptides
identified and
isolated as described can be prepared from murine hybridomas according to the
classical method of Kohler,
and Milstein, .Nature 256:495 (1975) or derivative methods thereof. Briefly, a
mouse is repetitively
inoculated with a few micrograms of the selected protein or peptides derived
therefrom over a period of a few
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weeks. The mouse is then sacrificed, and the antibody producing cells of the
spleen isolated. The spleen
cells are fused by means of polyethylene glycol with mouse myeloma cells, and
the excess unfused cells
destroyed by growth of the system on selective media comprising aminopterin
(HAT media). The
successfully fused cells are diluted and aliquots of the dilution placed in
wells of a microtiter plate where
growth of the culture is continued. Antibody-producing clones are identified
by detection of antibody in the
supernatant fluid of the wells by immunoassay procedures, such as Elisa, as
originally described by Engvall,
Meth. Enzymol. 70:419 (1980), the disclosure of which is incorporated herein
by reference and derivative
methods thereof. Selected positive clones can be expanded and their monoclonal
antibody product harvested
for use. Detailed procedures for monoclonal antibody production are described
in Davis, L. et al. in Basic
Methods in Molecular Biology Elsevier, New York. Section 21-2, the disclosure
of which is incorporated
herein by reference.
2. Polyclonal Antibody Production by Immunization
[0388] Polyclonal antiserum containing antibodies to heterogenous epitopes of
a single protein or
polypeptide can be prepared by immunizing suitable animals with the expressed
protein or peptides derived
therefrom, which can be unmodified or modified to enhance immunogenicity.
Effective polyclonal antibody
production is affected by many factors related both to the antigen and the
host species. For example, small
molecules tend to be less immunogenic than others and may require the use of
carriers and adjuvant. Also,
host animals' response vary depending on site of inoculations and doses, with
both inadequate or excessive
doses of antigen resulting in low titer antisera. Small doses (ng level) of
antigen administered at multiple
intradermal sites appears to be most reliable. An effective immunization
protocol for rabbits can be found in
Vaitukaitis. et al., J. Clin. Endocrinol. Metab. 33:988-991 (1971), the
disclosure of which is incorporated
herein by reference.
[0389] Booster injections can be given at regular intervals, and antiserum
harvested when antibody
titer thereof, as determined semi-quantitatively, for example, by double
immunodiffusion in agar against
known concentrations of the antigen, begins to fall. See, for example,
Ouchterlony, et al., Chap. 19 in:
Handbook of ~;xperimental Immunology D. Wier (ed) Blackwell (1973) , the
disclosure of which is
incorporated herein by reference. Plateau concentration of antibody is usually
in the range of 0.1 to 0.2
mg/ml of serum (about 12 p,M). Affinity of the antisera for the antigen is
determined by preparing
competitive binding curves, as described, for example, by Fisher, D., Chap. 42
in: Manual of Clinical
Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol.,
Washington, D.C. (1980) , the
disclosure of which is incorporated herein by reference.
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[0390] Antibody preparations prepared according to either of the above
protocols are useful in a
variety of contexts. In particular, the antibodies may be used in
immunoaffinity chromatography techniques
such as those described below to facilitate large scale isolation,
purification, or enrichment of the proteins or
polypeptides encoded. by EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of positional segments of EST-related nucleic acids or for the
isolation, purification or enrichment
of EST-related polypeptides, fragments of EST-related polypeptides, positional
segments of EST-related
polypeptides or fragments of positional segments of EST-related polypeptides.
[0391] In the case of secreted proteins, the antibodies may be used for the
isolation, purification, or
enrichment of the full protein (i.e. the mature protein and the signal
peptide), the mature protein (i.e. the
protein generated by cleavage of the signal peptide), or the signal peptide
are operably linked to promoters
and introduced into cells as described above.
[0392] Additionally, the antibodies may be used in immunoaffinity
chromatography techniques
such as those described below to isolate, purify, or enrich polypeptides which
have been linked to the proteins
or polypeptides encoded by EST-related nucleic acids, positional segments of
EST-related nucleic acids or
fragments of positional segments of EST-related nucleic acids or to isolate,
purify, or enrich EST-related
polypeptides, fragments of EST-related polypeptides, positional segments of
EST-related polypeptides or
fragments of positional segments of EST-related polypeptides.
[0393] The antibodies may also be used to determine the cellular localization
of polypeptides
encoded by the proteins or polypeptides encoded by EST-related nucleic acids,
positional segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids or the cellular
localization of EST-related polypeptides, fragments of EST-related
polypeptides, positional segments of
EST-related polypeptides or fragments of positional segments of EST-related
polypeptides.
[0394] In addition, the antibodies may also be used to determine the cellular
localization of
polypeptides which have been linked to the proteins or polypeptides encoded by
EST-related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids or polypeptides which have been linked EST-related polypeptides,
fragments of EST-related
polypeptides, positional segments of EST-related polypeptides or fragments of
positional segments of EST-
related polypeptides .
[0395] The antibodies may also be used in quantitative immunoassays which
determine
concentrations of antigen-bearing substances in biological samples; they may
also used semi-quantitatively or
qualitatively to identify the presence of antigen in a biological sample or to
identify the type of tissue present
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in a biological sample. The antibodies may also be used in therapeutic
compositions for killing cells
expressing the protein or reducing the levels of the protein in the body.
VI. Use of 5'ESTs and Consensus Contigated 5' ESTs or Seguences Obtainable
Therefrom or
Portions Thereof as Reagents
[0396] The EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids may be used as
reagents in isolation
procedures, diagnostic assays, and forensic procedures. For example, sequences
from the EST-related
nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
1 0 related nucleic acids, may be detectably labeled and used as probes to
isolate other sequences capable of
hybridizing to them. In addition, the he EST-related nucleic acids, positional
segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
may be used to design PCR
primers to be used in isolation, diagnostic, or forensic procedures.
1. Use of EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional set menu of EST-related nucleic acids in isolation, diagnostic and
forensic procedures
EXAMPLE 34
Preparation of 1?CR Primers and Amplification of DNA
[0397] The EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids may be used to
prepare PCR primers for a
variety of applications, including isolation procedures for cloning nucleic
acids capable of hybridizing to such
sequences, diagnostic techniques and forensic techniques. In some embodiments,
the PCR primers at least
10, 15, 18, 20, 23, 25, 28, 30, 40, or 50 nucleotides in length. In some
embodiments, the PCR primers may
be more than 30 bases in length. It is preferred that the primer pairs have
approximately the same G/C ratio,
so that melting temperatures are approximately the same. A variety of PCR
techniques are familiar to those
skilled in the art. For a review of PCR technology, see Molecular Cloning to
Genetic Engineering White,
B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa 1997, the
disclosure of which is
incorporated herein by reference. In each of these PCR procedures, PCR primers
on either side of the nucleic
acid sequences to be amplified are added to a suitably prepared nucleic acid
sample along with dNTPs and a
thermostable polymerise such as Taq polymerise, Pfu polymerise, or Vent
polymerise. The nucleic acid in
the sample is denatured and the PCR primers are specifically hybridized to
complementary nucleic acid
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sequences in the sample. The hybridized primers are extended. Thereafter,
another cycle of denaturation,
hybridization, and extension is initiated. The cycles are repeated multiple
times to produce an amplified
fragment containing the nucleic acid sequence between the primer sites.
EXAMPLE 35
Use of the EST-related nucleic acids positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids as probes
[0398] Probes derived from EST-related nucleic acids, positional segments of
EST-related nucleic
acids or fragments of positional segments of EST-related nucleic acids may be
labeled with detectable labels
familiar to those skilled in the art, including radioisotopes and non-
radioactive labels, to provide a detectable
probe. The detectable probe may be single stranded or double stranded and may
be made using techniques
known in the art, including in vitro transcription, nick translation, or
kinase reactions. A nucleic acid sample
containing a sequence capable of hybridizing to the labeled probe is contacted
with the labeled probe. If the
nucleic acid in the sample is double stranded, it may be denatured prior to
contacting the probe. In some
applications, the nucleic acid sample may be immobilized on a surface such as
a nitrocellulose or nylon
membrane. T7ie nucleic acid sample may comprise nucleic acids obtained from a
variety of sources,
including genomic DNA, cDNA libraries, RNA, or tissue samples.
[0399] Procedures used to detect the presence of nucleic acids capable of
hybridizing to the
detectable probe include well known techniques such as Southern blotting,
Northern blotting, dot blotting,
colony hybridization, and plaque hybridization. In some applications, the
nucleic acid capable of hybridizing
to the labeled probe may be cloned into vectors such as expression vectors,
sequencing vectors, or in vitro
transcription vectors to facilitate the characterization and expression of the
hybridizing nucleic acids in the
sample. For example, such techniques may be used to isolate and clone
sequences in a genomic library or
eDNA library which are capable of hybridizing to the detectable probe as
described in Example 18 above.
[0400] PCR primers made as described in Example 34 above may be used in
forensic analyses,
such as the DNA fingerprinting techniques described in Examples 36-40 below.
Such analyses may utilize
detectable probes or primers based on the sequences of the EST-related nucleic
acids, positional segments of
EST-related nucleic acids or fragments of positional segments of EST-related
nucleic acids.
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EXAMPLE 36
Forensic Matching by DNA Sequencing
[0401] In one exemplary method, DNA samples are isolated from forensic
specimens of, for
example, hair, semen; blood or skin cells by conventional methods. A panel of
PCR primers based on a
number of the EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids is then utilized in
accordance with Example 34 to amplify
DNA of approximately 100-200 bases in length from the forensic specimen.
Corresponding sequences are
obtained from z test subject. Each of these identification DNAs is then
sequenced using standard techniques,
and a simple database comparison determines the differences, if any, between
the sequences from the subject
IO and those from the sample. Statistically significant differences between
the suspect's DNA sequences and
those from the sample conclusively prove a lack of identity. This lack of
identity can be proven, for example,
with only one sequence. Identity, on the other hand, should be demonstrated
with a large number of
sequences, all matching. Preferably, a minimum of 50 statistically identical
sequences of 100 bases in length
are used to prove identity between the suspect and the sample.
EXAMPLE 37
Positive Identification by DNA Sequencing
[0402] The technique outlined in the previous example may also be used on a
larger scale to
provide a unique fingerprint-type identification of any individual. In this
technique, primers are prepared
from a large number of EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of positional segments of EST-related nucleic acids. Preferably, 20
to 50 different primers are
used. These primers are used to obtain a corresponding number of PCR-generated
DNA segments from the
individual in question in accordance with Example 34. Each of these DNA
segments is sequenced, using the
methods set forth in Example 3C. The database of sequences generated through
this procedure uniquely
identifies the individual from whom the sequences were obtained. The same
panel of primers may then be
used at any later time to absolutely correlate tissue or other biological
specimen with that individual.
EXAMPLE 38
Southern Blot Forensic Identification
(0403] The procedure of Example 37 is repeated to obtain a panel of at least
10 amplified sequences
from an individual and a specimen. Preferably, the panel contains at least 50
amplified sequences. More
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preferably, the panel contains 100 amplified sequences. In some embodiments,
the panel contains 200
amplified sequences. This PCR-generated DNA is then digested with one or a
combination of, preferably,
four base specific restriction enzymes. Such enzymes are commercially
available and known to those of skill
in the art. After digestion, the resultant gene fragments are size separated
in multiple duplicate wells on an
agarose gel and transferred to nitrocellulose using Southern blotting
techniques well known to those with skill
in the art. For a review of Southern blotting see Davis et al. (Basic Methods
in Molecular Biology, 1986,
Elsevier Press. pp 62-65) , the disclosure of which is incorporated herein by
reference.
[0404] A panel of probes based on the sequences of the EST-related nucleic
acids, positional
segments of ES'T-related nucleic acids or fragments of positional segments of
EST-related nucleic acids are
IO radioactively or colorimetrically labeled using methods known in the art,
such as nick translation or end
labeling, and hybridized to the Southern blot using techniques known in the
art (Davis et al., supra).
Preferably, the probe is at least 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50,
75, 100, 150, 200, 300, 400 or 500
nucleotides in length. Preferably, the probes are at least 10, 12, 15, 18, 20,
25, 28, 30, 35, 40, 50, 75, 100,
150, 200, 300, 400 or 500 nucleotides in length. In some embodiments, the
probes are oligonucleotides
which are 40 nucleotides in length or less.
[0405] Preferably, at least 5 to 10 of these labeled probes are used, and more
preferably at least
about 20 or 30 are used to provide a unique pattern. The resultant bands
appearing from the hybridization of
a large sample of EST-related nucleic acids, positional segments of EST-
related nucleic acids or fragments of
positional segments of EST-related nucleic acids will be a unique identifier.
Since the restriction enzyme
cleavage will be different for every individual, the band pattern on the
Southern blot will also be unique.
Increasing the number of probes will provide a statistically higher level of
confidence in the identification
since there will be an increased number of sets of bands used for
identification.
EXAMPLE 39
Dot Blot Identification Procedure
[0406] Another technique for identifying individuals using the EST-related
nucleic acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids
disclosed herein utilizes a dot blot hybridization technique.
[0407] Genomic DNA is isolated from nuclei of subject to be identified. Probes
are prepared that
correspond to at least 10, preferably 50 sequences from the EST-related
nucleic acids, positional segments of
EST-related nucleic acids or fragments of positional segments of EST-related
nucleic acids. The probes are
used to hybridize to the genomic DNA through conditions known to those in the
art. The oligonucleotides
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are end labeled with P3'' using polynucleotide kinase (Pharmacia). Dot Blots
are created by spotting the
genomic DNA onto nitrocellulose or the like using a vacuum dot blot manifold
(BioRad, Richmond
California). The nitrocellulose filter containing the genomic sequences is
baked or LTV linked to the filter,
prehybridized and hybridized with labeled probe using techniques known in the
art (Davis et al., supra). The
3zP labeled DNA fragments are sequentially hybridized with successively
stringent conditions to detect
minimal differences between the 30 by sequence and the DNA.
Tetramethylammonium chloride is useful for
identifying clones containing small numbers of nucleotide mismatches (Wood et
al., Proc. Natl. Acad. Sci.
USA 82(6):1585-1588 (1985)) which is hereby incorporated by reference. A
unique pattern of dots
distinguishes one individual from another individual.
[0408] EST-related nucleic acids, positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids can be used as probes in the
following alternative
fingerprinting technique. In some embodiments, the probes are oligonucleotides
which are 40 nucleotides in
length or less.
[0409] Preferably, a plurality of probes having sequences from different EST-
related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids are used in the alternative fingerprinting technique. Example 40 below
provides a representative
alternative fingerprinting procedure in which the probes are derived from EST-
related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids.
EXAMPLE 40
Alternative "Fin~er~rint" Identification Technique
(0410] Oligonucleotides are prepared from a large number, e.g. 50, 100, or
200, EST-related nucleic
acids, positional segments of EST-related nucleic acids or fragments of
positional segments of EST-related
nucleic acids using commercially available oligonucleotide services such as
Genset, Paris, France.
Preferably, the oligonucleotides are at least 10, 15, 18, 20, 23, 25 28, or 30
nucleotides in length. However,
in some embodiments, the oligonucleotides may be more than 30 nucleotides in
length.
[0411] Cell samples from the test subject are processed for DNA using
techniques well known to
those with skill in the art. The nucleic acid is digested with restriction
enzymes such as EcoRI and XbaI.
Following digestion, samples are applied to wells for electrophoresis. The
procedure, as known in the art,
may be modified to accommodate polyacrylamide electrophoresis, however in this
example, samples
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containing S ug of DNA are loaded into wells and separated on 0.8% agarose
gels. The gels are transferred
onto nitrocellulose using standard Southern blotting techniques.
[0412] 10 ng of each of the oligonucleotides are pooled and end-labeled with
P3''. The
nitrocellulose is prehybridized with blocking solution and hybridized with the
labeled probes. Following
hybridization and washing, the nitrocellulose filter is exposed to X-Omat AR X-
ray film. The resulting
hybridization pattern will be unique for each individual.
[0413] It is additionally contemplated within this example that the number of
probe sequences used
can be varied for additional accuracy or clarity.
[0414] In addition to their applications in forensics and identification, EST-
related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids may be mapped to their chromosomal locations. Example 41 below describes
radiation hybrid (RH)
mapping of human chromosomal regions using EST-related nucleic acids,
positional segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids. Example 42 below
describes a representative procedure for mapping EST-related nucleic acids,
positional segments of EST-
1 S related nucleic acids or fragments of positional segments of EST-related
nucleic acids to their locations on
human chromosomes. Example 43 below describes mapping of EST-related nucleic
acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids on
metaphase chromosomes by Fluorescence In Situ Hybridization (FISH).
2. Use of EST-related nucleic acids, positional seQrnents of EST-related
nucleic acids or fragments of
positional se~rnents of EST-related nucleic acids in Chromosome Mapping
EXAMPLE 41
Radiation hybrid mapping of EST-related nucleic acids, positional segments of
EST-related nucleic acids
or fragments ofpositional segments of EST-related nucleic acids to the human
genome
[0415] Radiation hybrid (RH) mapping is a somatic cell genetic approach that
can be used for high
resolution mapping of the human genome. In this approach, cell lines
containing one or more human
chromosomes are lethally irradiated, breaking each chromosome into fragments
whose size depends on the
radiation dose. These fragments are rescued by fusion with cultured rodent
cells, yielding subclones
containing different portions of the human genome. This technique is described
by Benham et al. (Genomics
4:509-517, 1989) and Cox et al., (Science 250:245-250, 1990), the entire
contents of which are hereby
incorporated by reference. The random and independent nature of the subclones
permits efficient mapping of
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any human genome marker. Human DNA isolated from a panel of 80-100 cell lines
provides a mapping
reagent for ordering EST-related nucleic acids, positional segments of EST-
related nucleic acids or fragments
of positional segments of EST-related nucleic acids. In this approach, the
frequency of breakage between
markers is used to measure distance, allowing construction of fine resolution
maps as has been done using
conventional ESTs (Schuler et al., Science 274:540-546, 1996, hereby
incorporated by reference).
[0416] RH mapping has been used to generate a high-resolution whole genome
radiation hybrid
map of human chromosome 17q22-q25.3 across the genes for growth hormone (GH)
and thymidine kinase
(TK) (Foster et al., Genomics 33:185-192, 1996), the region surrounding the
Gorlin syndrome gene
(Obermayr et al., Eur. J. Hum. Genet. 4:242-245, 1996), 60 loci covering the
entire short arm of chromosome
12 (Raeymaekers et al., Genomics 29:170-178, 1995), the region of human
chromosome 22 containing the
neurofibromatosis type 2 locus (Frazer et al., Genomics 14:574-584, 1992) and
13 loci on the long arm of
chromosome 5 I Warnngton et al., Genomics 11:701-708, 1991).
EXAMPLE 42
1 S Mapping of EST-related nucleic acids positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids to Human Chromosomes using
PCR technigues
[0417] EST-related nucleic acids, positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids may be assigned to human
chromosomes using PCR based
methodologies. In such approaches, oligonucleotide primer pairs are designed
from EST-related nucleic
acids, positional segments of EST-related nucleic acids or fragments of
positional segments of EST-related
nucleic acids to minimize the chance of amplifying through an intron.
Preferably, the oligonucleotide
primers are 18-23 by in length and are designed for PCR amplification. The
creation of PCR primers from
lrnown sequences is well known to those with skill in the art. For a review of
PCR technology see Erlich. in
PCR Technology; Principles and Applications for DNA Amplification. 1992. W.H.
Freeman and Co., New
York, the disclosure of which is incorporated herein by reference.
[0418] The primers are used in polymerase chain reactions (PCR) to amplify
templates from total
human genomic DNA. PCR conditions are as follows: 60 ng of genomic DNA is used
as a template for PCR
with 80 ng of each oligonucleotide primer, 0.6 unit of Taq polymerase, and 1
~Cu of a 32P-labeled
deoxycytidine triphosphate. The PCR is performed in a microplate thermocycler
(Techne) under the
following conditions: 30 cycles of 94°C, 1.4 min; 55°C, 2 min;
and 72°C, 2 min; with a final extension at
72°C for 10 min. The amplified products are analyzed on a 6%
polyacrylamide sequencing gel and
visualized by autoradiography. If the length of the resulting PCR product is
identical to the distance between
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the ends of the primer sequences in the 5'EST from which the primers are
derived, then the PCR reaction is
repeated with DNA templates from two panels of human-rodent somatic cell
hybrids, BIOS PCRable DNA
(BIOS Corporation) and NIGMS Human-Rodent Somatic Cell Hybrid Mapping Panel
Number 1 (NIGMS,
Camden, N~.
[0419] PCR is used to screen a series of somatic cell hybrid cell lines
containing defined sets of
human chromosomes for the presence of a given 5'EST. DNA is isolated from the
somatic hybrids and used
as starting templates for PCR-reactions using the primer pairs from the EST-
related nucleic acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids.
Only those somatic cell hybrids with chromosomes containing the human gene
corresponding to the EST-
related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional segments of
EST-related nucleic acids will yield an amplified fragment. The 5'ESTs are
assigned to a chromosome by
analysis of the segregation pattern of PCR products from the somatic hybrid
DNA templates. The single
human chromosome present in all cell hybrids that give rise to an amplified
fragment is the chromosome
containing that EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
1 S positional segments of EST-related nucleic acids. For a review of
techniques and analysis of results from
somatic cell gene mapping experiments. (See Ledbetter et al., Genomics 6:475-
481 (1990). , the disclosure
of which is incorporated herein by reference.)
[0420] Alternatively, the EST-related nucleic acids, positional segments of
EST-related nucleic
acids or fragments of positional segments of EST-related nucleic acids may be
mapped to individual
chromosomes using FISH as described in Example 43 below.
EXAMPLE 43
Mappin of EST-related nucleic acids positional segments of EST-related nucleic
acids or fra~nents of
positional segments of EST-related nucleic acids to Chromosomes Using
Fluorescence In Situ
Hybridization
[0421] Fluorescence in situ hybridization allows the EST-related nucleic
acids, positional segments
of EST-related nucleic acids or fragments of positional segments of EST-
related nucleic acids to be mapped
to a particular location on a given chromosome. The chromosomes to be used for
fluorescence in situ
hybridization techniques may be obtained from a variety of sources including
cell cultures, tissues, or whole
blood.
[0422] In a preferred embodiment, chromosomal localization of EST-related
nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
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acids are obtained by FISH as described by Cherif et al. (Proc. Natl. Acad.
Sci. U.S.A., 87:6639-6643, 1990) ,
the disclosure of which is incorporated herein by reference. Metaphase
chromosomes are prepared from
phytohemagglutinin (PHA)-stimulated blood cell donors. PHA-stimulated
lymphocytes from healthy males
are cultured for 72 h in RPMI-1640 medium. For synchronization, methotrexate
(10 ~.M) is added for 17 h,
followed by addition of 5-bromodeoxyuridine (5-BrdU, 0.1 mM) for 6 h. Colcemid
(1 ~g/ml) is added for
the last 15 min before harvesting the cells. Cells are collected, washed in
RPMI, incubated with a hypotonic
solution of KCl (75 mM) at 37°C for 15 min and fixed in three changes
of methanol:acetic acid (3:1). The
cell suspension is dropped onto a glass slide and air dried. The EST-related
nucleic acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids is
labeled with biotin-16 dUTP by nick translation according to the
manufacturer's instructions (Bethesda
Research Laboratories, Bethesda, MD), purified using a Sephadex G-50 column
(Pharmacia, Upsala,
Sweden) and precipitated. Just prior to hybridization, the DNA pellet is
dissolved in hybridization buffer
(50% formamide, 2 X SSC, 10% dextran sulfate, 1 mg/ml sonicated salmon sperm
DNA, pH 7) and the
probe is denatured at 70°C for 5-10 min.
[0423 Slides kept at -20°C are treated for 1 h at 37°C with
RNase A (100 ~,g/ml), rinsed three times
in 2 X SSC and dehydrated in an ethanol series. Chromosome preparations are
denatured in 70% formamide,
2 X SSC for 2 min at 70°C, then dehydrated at 4°C. The slides
are treated with proteinase K (10 ~g/100 ml
in 20 mM Tris-HCI, 2 mM CaCl2) at 37°C for 8 min and dehydrated. The
hybridization mixture containing
the probe is placed on the slide, covered with a coverslip, sealed with rubber
cement and incubated overnight
in a humid chamber at 37°C. Alter hybridization and post-hybridization
washes, the biotinylated probe is
detected by avidin-FTTC and amplified with additional layers of biotinylated
goat anti-avidin and avidin-
FTTC. For chromosomal localization, fluorescent R-bands are obtained as
previously described (Cherif et al.,
supra.). The slides are observed under a LEICA fluorescence microscope
(DMRXA). Chromosomes are
counterstained with propidium iodide and the fluorescent signal of the probe
appears as two symmetrical
yellow-green spots on both chromatids of the fluorescent R-band chromosome
(red). Thus, a particular EST-
related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional segments of
EST-related nucleic acids may be localized to a particular cytogenetic R-band
on a given chromosome.
[0424] Once the EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids have been
assigned to particular chromosomes
using the techniques described in Examples 41-43 above, they may be utilized
to construct a high resolution
map of the chromosomes on which they are located or to identify the
chromosomes in a sample.
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EXAMPLE 44
Use of EST-related nucleic acids positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids to Construct or Expand
Chromosome Maps
[0425] Chromosome mapping involves assigning a given unique sequence to a
particular
chromosome as described above. Once the unique sequence has been mapped to a
given chromosome, it is
ordered relative to other unique sequences located on the same chromosome. One
approach to chromosome
mapping utilizes a series of yeast artificial chromosomes (YACs) bearing
several thousand long inserts
derived from the chromosomes of the organism from which the EST-related
nucleic acids, positional
segments of ES'T-related nucleic acids or fragments of positional segments of
EST-related nucleic acids are
obtained. This approach is described in Ramaiah Nagaraja et al., Genome
Research 7:210-222, March 1997,
the disclosure of which is incorporated herein by reference. Briefly, in this
approach each chromosome is
broken into overlapping pieces which are inserted into the YAC vector. The YAC
inserts are screened using
PCR or other methods to determine whether they include the EST-related nucleic
acids, positional segments
of EST-related nucleic acids or fragments of positional segments of EST-
related nucleic acids whose position
is to be determined. Once an insert has been found which includes the S'EST,
the insert can be analyzed by
PCR or other methods to determine whether the insert also contains other
sequences lrnown to be on the
chromosome or in the region from which the EST-related nucleic acids,
positional segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
was derived. This process can
be repeated for each insert in the YAC library to determine the location of
each of the EST-related nucleic
acids, positional segments of EST-related nucleic acids or fragments of
positional segments of EST-related
nucleic acids relative to one another and to other known chromosomal markers.
In this way, a high resolution
map of the distribution of numerous unique markers along each of the
organism's chromosomes may be
obtained.
[0426] As described in Example 45 below EST-related nucleic acids, positional
segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids may also be used to
identify genes associated with a particular phenotype, such as hereditary
disease or drug response.
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3 Use of EST-related nucleic acids positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids Gene Identification
EXAMPLE 45
Identification of genes associated with hereditar,r diseases or drug response
[0427] This example illustrates an approach useful for the association of EST-
related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids with particular phenotypic characteristics. In this example, a
particular EST-related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids is used as a test probe to associate that EST-related nucleic acids,
positional segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
with a particular phenotypic
characteristic.
[0428] EST-related nucleic acids, positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids are mapped to a particular
location on a human
chromosome using techniques such as those described in Examples 41 and 42 or
other techniques known in
1 S the art. A search of Mendelian Inheritance in Man (V. McKusick, Mendelian
Inheritance in Man (available
on line through Johns Hopkins University Welch Medical Library) reveals the
region of the human
chromosome which contains the EST-related nucleic acids, positional segments
of EST-related nucleic acids
or fragments of positional segments of EST-related nucleic acids to be a very
gene rich region containing
several known genes and several diseases or phenotypes for which genes have
not been identified. The gene
corresponding to this EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of pasitional segments of EST-related nucleic acids thus becomes an
immediate candidate for each
of these genetic diseases.
[0429] Cells from patients with these diseases or phenotypes are isolated and
expanded in culture.
PCR primers from the EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of positional segments of EST-related nucleic acids are used to
screen genomic DNA, mRNA or
cDNA obtained from the patients. EST-related nucleic acids, positional
segments of EST-related nucleic
acids or fragments of positional segments of EST-related nucleic acids that
are not amplified in the patients
can be positively associated with a particular disease by further analysis.
Alternatively, the PCR analysis
may yield fralnnents of different lengths when the samples are derived from an
individual having the
phenotype assaciated with the disease than when the sample is derived from a
healthy individual, indicating
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that the gene containing the EST-related nucleic acids, positional segments of
EST-related nucleic acids or
fragments of positional segments of EST-related nucleic acids may be
responsible for the genetic disease.
VII Use of EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids to Construct
Vectors and Uses
Thereof
[0430] The present EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of positional segments of EST-related nucleic acids may also be used
to construct secretion vectors
capable of directing the secretion of the proteins encoded by genes therein.
Such secretion vectors may
facilitate the purification or enrichment of the proteins encoded by genes
inserted therein by reducing the
number of background proteins from which the desired protein must be purified
or enriched. Exemplary
secretion vectors are described in Example 46 below.
1. Construction of Vectors and Uses Thereof
EXAMPLE 46
Construction of Secretion Vectors
[0431] The secretion vectors of the present invention include a promoter
capable of directing gene
expression in the host cell, tissue, or organism of interest. Such promoters
include the Rous Sarcoma Virus
promoter, the SV40 promoter, the human cytomegalovirus promoter, and other
promoters familiar to those
skilled in the art.
[0432] A signal sequence from one of the EST-related nucleic acids, positional
segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids is operably linked to
the promoter such that the mRNA transcribed from the promoter will direct the
translation of the signal
peptide. Preferably, the signal sequence is from one of the nucleic acids of
SEQ )D NOs.24-13309. The host
cell, tissue, or organism may be any cell, tissue, or organism which
recognizes the signal peptide encoded by
the signal sequence in the EST-related nucleic acids, positional segments of
EST-related nucleic acids or
fragments of positional segments of EST-related nucleic acids. Suitable hosts
include mammalian cells,
tissues or organisms, avian cells, tissues, or organisms, insect cells,
tissues or organisms, or yeast.
(0433] In addition, the secretion vector contains cloning sites for inserting
genes encoding the
proteins which are to be secreted. The cloning sites facilitate the cloning of
the insert gene in frame with the
signal sequence such that a fusion protein in which the signal peptide is
fused to the protein encoded by the
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inserted gene is expressed from the mRNA transcribed from the promoter. The
signal peptide directs the
extracellular secretion of the fusion protein.
[0434] The secretion vector may be DNA or RNA and may integrate into the
chromosome of the
host, be stably maintained as an extrachromosomal replicon in the host, be an
artificial chromosome, or be
transiently present in the host. Preferably, the secretion vector is
maintained in multiple copies in each host
cell. As used herein, multiple copies means at least 2, 5, 10, 20, 25, SO or
more than 50 copies per cell. In
some embodiments, the multiple copies are maintained extrachromosomally. In
other embodiments, the
multiple copies result from amplification of a chromosomal sequence.
[0435] Many nucleic acid backbones suitable for use as secretion vectors are
known to those skilled
in the art, including retroviral vectors, SV40 vectors, Bovine Papilloma Virus
vectors, yeast integrating
plasmids, yeast episomal plasmids, yeast artificial chromosomes, human
artificial chromosomes, P element
vectors, baculo~rirus vectors, or bacterial plasmids capable of being
transiently introduced into the host.
[0436] The secretion vector may also contain a polyA signal such that the
polyA signal is located
downstream of the gene inserted into the secretion vector.
[0437] After the gene encoding the protein for which secretion is desired is
inserted into the
secretion vector, the secretion vector is introduced into the host cell,
tissue, or organism using calcium
phosphate precipitation, DEAF-Dextran, electroporation, liposome-mediated
transfection, viral particles or as
naked DNA. The protein encoded by the inserted gene is then purified or
enriched from the supernatant
using conventional techniques such as ammonium sulfate precipitation,
immunoprecipitation,
immunoaffinitychromatography, size exclusion chromatography, ion exchange
chromatography, and HPLC.
Alternatively, the secreted protein may be in a sufficiently enriched or pure
state in the supernatant or growth
media of the host to permit it to be used for its intended purpose without
further enrichment.
[0438] The signal sequences may also be inserted into vectors designed for
gene therapy. In such
vectors, the signal sequence is operably linked to a promoter such that mRNA
transcribed from the promoter
encodes the signal peptide. A cloning site is located downstream of the signal
sequence such that a gene
encoding a protein whose secretion is desired may readily be inserted into the
vector and fused to the signal
sequence. The vector is introduced into an appropriate host cell. The protein
expressed from the promoter is
secreted extracellularly, thereby producing a therapeutic effect.
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EXAMPLE 47
Fusion Vectors
[0439] The EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positionai segments of EST-related nucleic acids may be used to
construct fusion vectors for the
expression of chimeric polypeptides. The chimeric polypeptides comprise a
first polypeptide portion and a
second polypeptide portion. In the fusion vectors of the present invention,
nucleic acids encoding the first
polypeptide portion and the second polypeptide portion are joined in frame
with one another so as to generate
a nucleic acid encoding the chimeric polypeptide. The nucleic acid encoding
the chimeric polypeptide is
operably linked to a promoter which directs the expression of an mRNA encoding
the chimeric polypeptide.
The promoter may be in any of the expression vectors described herein
including those described in
Examples 20 and 46.
[0440] Preferably, the fusion vector is maintained in multiple copies in each
host cell. In some
embodiments, the multiple copies are maintained extrachromosomally. In other
embodiments, the multiple
copies result from amplification of a chromosomal sequence.
(0441) The first polypeptide portion may comprise any of the polypeptides
encoded by the EST-
related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional segments of
EST-related nucleic acids. In some embodiments, the first polypeptide portion
may be one of the EST-
related polypeptides, fragments of EST-related polypeptides, positional
segments of EST-related
polypeptides, or fragments of positional segments of EST-related polypeptides.
[0442] The second polypeptide portion may comprise any polypeptide of
interest. In some
embodiments, the second polypeptide portion may comprise a polypeptide having
a detectable enzymatic
activity such as green fluorescent protein or (3 galactosidase. Chimeric
polypeptides in which the second
polypeptide portion comprises a detectable polypeptide may be used to
determine the intracellular
localization of the first polypeptide portion. In such procedures, the fusion
vector encoding the chimeric
polypeptide is introduced into a host cell under conditions which facilitate
the expression of the chimeric
polypeptide. Where appropriate, the cells are treated with a detection reagent
which is visible under the
microscope following a catalytic reaction with the detectable polypeptide and
the cellular location of the
detection reagent is determined. For example, if the polypeptide having a
detectable enzymatic activity is (i
galactosidase, the cells may be treated with Xgal. Alternatively, where the
detectable polypeptide is directly
detectable without the addition of a detection reagent, the intracellular
location of the chimeric polypeptide is
determined by performing microscopy under conditions in which the dectable
polypeptide is visible. For
example, if the detectable polypeptide is green fluorescent protein or a
modified version thereof, microscopy
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is performed by exposing the host cells to light having an appropriate
wavelength to cause the green
fluorescent protein or modified version thereof to fluoresce.
[0443] Alternatively, the second polypeptide portion may comprise a
polypeptide whose isolation,
purification, or enrichment is desired. In such embodiments, the isolation,
purification, or enrichment of the
second polypeptide portion may be achieved by performing the immunoaffmity
chromatography procedures
described below using an immunoaffinity column having an antibody directed
against the first polypeptide
portion coupled thereto.
[0444] The proteins encoded by the EST-related nucleic acids, positional
segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
or the EST-related
polypeptides, fragments of EST-related polypeptides, positional segments of
EST-related polypeptides, or
fragments of positional segments of EST-related polypeptides may also be used
to generate antibodies as
explained in Examples 20 and 33 in order to identify the tissue type or cell
species from which a sample is
derived as described in Example 48.
EXAMPLE 48
Identification of Tissue Types or Cell Species by Means of
Labeled Tissue Specific Antibodies
[0445] Identification of specific tissues is accomplished by the visualization
of tissue specific
antigens by means of antibody preparations according to Examples 20 and 33
which are conjugated, directly
or indirectly to a detectable marker. Selected labeled antibody species bind
to their specific antigen binding
partner in tissue sections, cell suspensions, or in extracts of soluble
proteins from a tissue sample to provide a
pattern for qualitative or semi-qualitative interpretation.
[0446] Antisera for these procedures must have a potency exceeding that of the
native preparation,
and for that reason, antibodies are concentrated to a mg/ml level by isolation
of the gamma globulin fraction,
for example, by ion-exchange chromatography or by ammonium sulfate
fractionation. Also, to provide the
most specific antisera, unwanted antibodies, for example to common proteins,
must be removed from the
gamma globulin fraction, for example by means of insoluble immunoabsorbents,
before the antibodies are
labeled with the marker. Either monoclonal or heterologous antisera is
suitable for either procedure.
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1. Immunohistochemical Techniques
[0447] Purified, high-titer antibodies, prepared as described above, are
conjugated to a detectable
marker, as described, for example, by Fudenberg, H., Chap. 26 in: Basic' 503
Clinical Immunology, 3'~ Ed.
Lange, Los Altos, California (1980) or Rose, et al., Chap. 12 in: Methods in
ImmunodiagNOsis, 2d Ed. John
Wiley and Sons, New York ( 1980), the disclosures of which are incorporated
herein by reference.
(0448] A fluorescent marker, either fluorescein or rhodamine, is preferred,
but antibodies can also
be labeled with an enzyme that supports a color producing reaction with a
substrate, such as horseradish
peroxidase. Markers can be added to tissue-bound antibody in a second step, as
described below.
Alternatively, the specific antitissue antibodies can be labeled with ferritin
or other electron dense particles,
and localization of the ferritin coupled antigen-antibody complexes achieved
by means of an electron
microscope. In yet another approach, the antibodies are radiolabeled, with,
for example ''''I, and detected by
overlaying the antibody treated preparation with photographic emulsion.
[0449] Preparations to carry out the procedures can comprise monoclonal or
polyclonal antibodies
to a single protein or peptide identified as specific to a tissue type, for
example, brain tissue, or antibody
preparations to several antigenically distinct tissue specific antigens can be
used in panels, independently or
in mixtures, as required.
[0450] Tissue sections and cell suspensions are prepared for
immunohistochemical examination
according to common histological techniques. Multiple cryostat sections (about
4 Vim, unfixed) of the
unlrnown tissue and known control, are mounted and each slide covered with
different dilutions of the
antibody preparation. Sections of known and unknown tissues should also be
treated with preparations to
provide a positive control, a negative control, for example, pre-immune sera,
and a control for non-specific
staining, for example, buffer.
[0451 ] Treated sections are incubated in a humid chamber for 30 min at room
temperature, rinsed,
then washed in buffer for 30-45 min. Excess fluid is blotted away, and the
marker developed.
[0452] If the tissue specific antibody was not labeled in the first
incubation, it can be labeled at this
time in a second antibody-antibody reaction, for example, by adding
fluorescein- or enzyme-conjugated
antibody against the immunoglobulin class of the antiserum-producing species,
for example, fluorescein
labeled antibody to mouse IgG. Such labeled sera are commercially available.
[0453] The antigen found in the tissues by the above procedure can be
quantified by measuring the
intensity of color or fluorescence on the tissue section, and calibrating that
signal using appropriate standards.
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2. Identification of Tissue Specific Soluble Proteins
[0454] The visualization of tissue specific proteins and identification of
unknown tissues from that
procedure is carried out using the labeled antibody reagents and detection
strategy as described for
immunohistochemistry; however the sample is prepared according to an
electrophoretic technique to
distribute the proteins extracted from the tissue in an orderly array on the
basis of molecular weight for
detection.
[0455] A tissue sample is homogenized using a Virtis apparatus; cell
suspensions are disrupted by
Dounce homogenization or osmotic lysis, using detergents in either case as
required to disrupt cell
membranes, as is the practice in the art. Insoluble cell components such as
nuclei, microsomes, and
membrane fragments are removed by ultracentrifugation, and the soluble protein-
containing fraction
concentrated if necessary and reserved for analysis.
[0456] A sample of the soluble protein solution is resolved into individual
protein species by
conventional SDS polyacrylamide electrophoresis as described, for example, by
Davis,L. et al., Section 19-2
in: Basic Methods in Molecular Biology (P. Leder, ed), Elsevier, New York
(1986), the disclosure of which is
incorporated herein by reference, using a range of amounts of polyacrylamide
in a set of gels to resolve the
entire molecular weight range of proteins to be detected in the sample. A size
marker is run in parallel for
purposes of estimating molecular weights of the constituent proteins. Sample
size for analysis is a convenient
volume of from 5 to 55 ~,1, and containing from about I to 100 ~g protein. An
aliquot of each of the resolved
proteins is transferred by blotting to a nitrocellulose filter paper, a
process that maintains the pattern of
resolution. Multiple copies are prepared. The procedure, known as Western Blot
Analysis, is well described
in Davis, L. et al., .supra Section 19-3. One set of nitrocellulose blots is
stained with Coomassie Blue dye to
visualize the entire set of proteins for comparison with the antibody bound
proteins. The remaining
nitrocellulose filters are then incubated with a solution of one or more
specific antisera to tissue specific
proteins prepared as described in Examples 20 and 33. In this procedure, as in
procedure A above,
appropriate positive and negative sample and reagent controls are run.
[0457] In either procedure described above a detectable label can be attached
to the primary tissue
antigen-primary antibody complex according to various strategies and
permutations thereof. In a
straightforward approach, the primary specific antibody can be labeled;
alternatively, the unlabeled complex
can be bound by a labeled secondary anti-IgG antibody. In other approaches,
either the primary or secondary
antibody is conjugated to a biotin molecule, which can, in a subsequent step,
bind an avidin conjugated
marker. According to yet another strategy, enzyme labeled or radioactive
protein A, which has the property
of binding to any IgG, is bound in a final step to either the primary or
secondary antibody.
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EXAMPLE 49
Immunohistochemical Localization of Polypeptides
[0458] The antibodies prepared as described in Examples 20 and 33 above may be
utilized to
determine the cellular location of a polypeptide. The polypeptide may be any
of the polypeptides encoded by
EST-related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional
segments of EST-related nucleic acids or the polypeptide may be one of the EST-
related polypeptides,
fragments of EST-related polypeptides, positional segments of EST-related
polypeptides, or fragments of
positional segments of EST-related polypeptides. In some embodiments, the
polypeptide may be a chimeric
polypeptide such as those encoded by the fusion vectors of Example 47.
[0459] Cells expressing the polypeptide to be localized are applied to a
microscope slide and fixed
using any of the procedures typically employed in immunohistochemical
localization techniques, including
the methods described in Current Protocols in Molecular Biology, John Wiley
and Sons, Inc. 1997.
Following a washing step, the cells are contacted with the antibody. In some
embodiments, the antibody is
conjugated to a detectable marker as described above to facilitate detection.
Alternatively, in some
embodiments, after the cells have been contacted with an antibody to the
polypeptide to be localized, a
secondary antibody which has been conjugated to a detectable marker is placed
in contact with the antibody
against the polypeptide to be localized.
[0460] Thereafter, microscopy is performed under conditions suitable for
visualizing the cellular
location of the polypeptide.
[0461] The visualization of tissue specific antigen binding at levels above
those seen in control
tissues to one or more tissue specific antibodies, directed against the
polypeptides encoded by EST-related
nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
related nucleic acids or antibodies against the EST-related polypeptides,
fragments of EST-related
polypeptides, positional segments of EST-related polypeptides, or fragments of
positional segments of EST-
related polypeptides, can identify tissues of unknown origin, for example,
forensic samples, or differentiated
tumor tissue that has metastasized to foreign bodily sites.
[0462] The antibodies of Example 20 and 33 may also be used in the
immunoaffinity
chromatography techniques described below to isolate, purify or enrich the
polypeptides encoded by the
EST-related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional
segments of ES'T-related nucleic acids or to isolate, purify or enrich EST-
related polypeptides, fragments of
EST-related polypeptides, positional segments of EST-related polypeptides, or
fragments of positional
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segments of EST-related polypeptides. The immunoaffinity chromatography
techniques described below
may also be used to isolate, purify or enrich polypeptides which have been
linked to the polypeptides
encoded by the EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids or to isolate, purify or
enrich polypeptides which have been
linked to EST-related polypeptides, fragments of EST-related polypeptides,
positional segments of EST-
related polypeptides, or fragments of positional segments of EST-related
polypeptides.
EXAMPLE 50
Immunoaffinity ChromatoQraphy
(0463] Antibodies prepared as described above are coupled to a support.
Preferably, the antibodies
are monoclonal antibodies, but polyclonal antibodies may also be used. The
support may be any of those
typically employed in immunoaffinity chromatography, including Sepharose CL-4B
(Pharmacia, Piscataway,
NJ), Sepharose CL-2B (Pharmacia, Piscataway, NJ), Affi-gel 10 (Biorad,
Richmond, CA), or glass beads.
[0464] The antibodies may be coupled to the support using any of the coupling
reagents typically
used in immunoaffinity chromatography, including cyanogen bromide. After
coupling the antibody to the
support, the support is contacted with a sample which contains a target
polypeptide whose isolation,
purification or enrichment is desired. The target polypeptide may be a
polypeptide encoded by the EST-
related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional segments of
EST-related nucleic acids or the target polypeptide may be one of the EST-
related polypeptides, fragments of
EST-related polypeptides, positional segments of EST-related polypeptides, or
fragments of positional
segments of EST-related polypeptides. The target polypeptides may also be
polypeptides which have been
linked to the polypeptides encoded by the EST-related nucleic acids,
positional segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
or the target polypeptides may
be polypeptides which have been linked to EST-related polypeptides, fragments
of EST-related polypeptides,
positional segments of EST-related polypeptides, or fragments of positional
segments of EST-related
polypeptides using the fusion vectors described above.
[0465] Preferably, the sample is placed in contact with the support for a
sufficient amount of time
and under appropriate conditions to allow at least 50% of the target
polypeptide to specifically bind to the
antibody coupled to the support.
[0466] Thereafter, the support is washed with an appropriate wash solution to
remove polypeptides
which have non-specifically adhered to the support. The wash solution may be
any of those typically
employed in irrununoaffinity chromatography, including PBS, Tris-lithium
chloride buffer (O.1M lysine base
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and O.SM lithium chloride, pH 8.0), Tris-hydrochloride buffer (O.OSM Tris-
hydrochloride, pH 8.0), or
Tris/Triton/NaCI buffer (50mM Tris.cl, pH 8.0 or 9.0, 0.1 '% Triton X-100, and
O.SMNaCI).
[0467] After washing, the specifically bound target polypeptide is eluted from
the support using the
high pH or low pH elution solutions typically employed in immunoaffinity
chromatography. In particular,
the elution solutions may contain an eluant such as triethanolamine,
diethylamine, calcium chloride, sodium
thiocyanate, potassium bromide, acetic acid, or glycine. In some embodiments,
the elution solution may also
contain a detergent such as Triton X-I00 or octyl-[3-D-glucoside.
[0468] The EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids may also be used
to clone sequences located
upstream of the 5'ESTs which are capable of regulating gene expression,
including promoter sequences,
enhancer sequences, and other upstream sequences which influence transcription
or translation levels. Once
identified and cloned, these upstream regulatory sequences may be used in
expression vectors designed to
direct the expression of an inserted gene in a desired spatial, temporal,
developmental, or quantitative fashion.
Example 51 describes a method for cloning sequences upstream of the EST-
related nucleic acids, positional
segments of ES'T-related nucleic acids or fragments of positional segments of
EST-related nucleic acids.
2. Identification of upstream sequences with promotin or re ,ulatory
activities
EXAMPLE Sl.
Use of EST-related nucleic acids positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids to Clone Upstream Sequences
from Genomic DNA
[0469] Sequences derived from EST-related nucleic acids, positional segments
of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
may be used to isolate the
promoters of the corresponding genes using chromosome walking techniques. In
one chromosome walking
technique, which utilizes the GenomeWalker' - kit available from Clontech,
five complete genomic DNA
samples are each digested with a different restriction enzyme which has a 6
base recognition site and leaves a
blunt end. Following digestion, oligonucleotide adapters are ligated to each
end of the resulting genomic
DNA fragments.
[0470] For each of the five genomic DNA libraries, a first PCR reaction is
performed according to
the manufacturer's instructions (which are incorporated herein by reference)
using an outer adapter primer
provided in the kit and an outer gene specific primer. The gene specific
primer should be selected to be
specific for 5' I?ST of interest and should have a melting temperature,
length, and location in the EST-related
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nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
related nucleic acids which is consistent with its use in PCR reactions. Each
first PCR reaction contains Sng
of genomic DNA, 5 pl of lOX Tth reaction buffer, 0.2 mM of each dNTP, 0.2 pM
each of outer adapter
primer and outer gene. specific primer, 1.1 mM of Mg(OAc)z, and 1 pl of the
Tth polymerase SOX mix in a
total volume of 50 pl. The reaction cycle for the first PCR reaction is as
follows: 1 min at 94°C / 2 sec at
94°C, 3 min at 72°C (7 cycles) / 2 sec at 94°C, 3 min at
67°C (32 cycles) / S min at 67°C.
[0471] The product of the first PCR reaction is diluted and used as a template
for a second PCR
reaction according to the manufacturer's instructions using a pair of nested
primers which are located
internally on the amplicon resulting from the first PCR reaction. For example,
5 pl of the reaction
product of the first PCR reaction mixture may be diluted 180 times. Reactions
are made in a 50 pl volume
having a composition identical to that of the first PCR reaction except the
nested primers are used. The
first nested primer is specific for the adapter, and is provided with the
GenomeWalker -! kit. The second
nested primer is specific for the particular EST-related nucleic acids,
positional segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
for which the promoter is to
be cloned and should have a melting temperature, length, and location in the
EST-related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids which is consistent with its use in PCR reactions. The reaction
parameters of the second PCR
reaction are as follows: 1 min at 94°C / 2 sec at 94°C, 3 min at
72°C (6 cycles) / 2 sec at 94°C, 3 min at
67°C (25 cycles) / 5 min at - 67°C. The product of the second
PCR reaction is purified, cloned, and
sequenced using standard techniques.
[0472] Alternatively, two or more human genomic DNA libraries can be
constructed by using two
or more restriction enzymes. The digested genomic DNA is cloned into vectors
which can be converted into
single stranded, circular, or linear DNA. A biotinylated oligonucleotide
comprising at least 15 nucleotides
from the EST-related nucleic acids, positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids sequence is hybridized to the
single stranded DNA.
Hybrids between the biotinylated oligonucleotide and the single stranded DNA
containing the EST-related
nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
related nucleic acids are isolated as described above. Thereafter, the single
stranded DNA containing the
EST-related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional
segments of EST-related nucleic acids is released from the beads and converted
into double stranded DNA
using a primer specific for the EST-related nucleic acids, positional segments
of EST-related nucleic acids or
fragments of positional segments of EST-related nucleic acids or a primer
corresponding to a sequence
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included in the cloning vector. The resulting double stranded DNA is
transformed into bacteria. cDNAs
containing the I?ST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids are identified by colony PCR
or colony hybridization.
[04737]Once_the upstream genomic sequences have been cloned and sequenced as
described above,
prospective promoters and transcription start sites within the upstream
sequences may be identified by
comparing the sequences upstream of the EST-related nucleic acids, positional
segments of EST-related
nucleic acids or fragments of positional segments of EST-related nucleic acids
with databases containing
known transcription start sites, transcription factor binding sites, or
promoter sequences.
[0474) In addition, promoters in the upstream sequences may be identified
using promoter reporter
vectors as described in Example 52.
EXAMPLE 52
Identification of Promoters in Cloned Upstream Sequences
[0475] The genomic sequences upstream of the EST-related nucleic acids,
positional segments of
EST-related nucleic acids or fragments of positional segments of EST-related
nucleic acids are cloned into a
suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer,
p(3ga1-Basic, p(3ga1-Enhancer,
or pEGFP-1 Promoter Reporter vectors available from Clontech. Briefly, each of
these promoter reporter
vectors include multiple cloning sites positioned upstream of a reporter gene
encoding a readily assayable
protein such as secreted alkaline phosphatase, (3 galactosidase, or green
fluorescent protein. The sequences
upstream of the EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids are inserted into the cloning
sites upstream of the reporter
gene in both orientations and introduced into an appropriate host cell. The
level of reporter protein is assayed
and compared to the level obtained from a vector which lacks an insert in the
cloning site. The presence of an
elevated expression level in the vector containing the insert with respect to
the control vector indicates the
presence of a promoter in the insert. If necessary, the upstream sequences can
be cloned into vectors which
contain an enhancer for augmenting transcription levels from weak promoter
sequences. A significant level
of expression above that observed with the vector lacking an insert indicates
that a promoter sequence is
present in the inserted upstream sequence.
[0476] Appropriate host cells for the promoter reporter vectors may be chosen
based on the results
of the above described determination of expression patterns of the EST-related
nucleic acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids. For
example, if the expression pattern analysis indicates that the mRNA
corresponding to a particular EST-related
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nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
related nucleic acids is expressed in fibroblasts, the promoter reporter
vector may be introduced into a human
fibroblast cell line.
[0477] Promoter sequences within the upstream genomic DNA may be further
defined by
constructing nested deletions in the upstream DNA using conventional
techniques such as Exonuclease III
digestion. The resulting deletion fragments can be inserted into the promoter
reporter vector to determine
whether the deletion has reduced or obliterated promoter activity. In this
way, the boundaries of the
promoters may be defined. If desired, potential individual regulatory sites
within the promoter may be
identified using site directed mutagenesis or linker scanning to obliterate
potential transcription factor binding
sites within the promoter individually or in combination. T'he effects of
these mutations on transcription
levels may be determined by inserting the mutations into the cloning sites in
the promoter reporter vectors.
EXAMPLE 53
Cloning and Identification of Promoters
[0479] Using the method described in Example 51 above with 5' ESTs, sequences
upstream of
several genes were obtained. Using the primer pairs GGG AAG ATG GAG ATA GTA
TTG CCT G (SEQ
ID NO.15) and CTG CCA TGT ACA TGA TAG AGA GAT TC (SEQ )D N0.16), the promoter
having the
internal designation P13H2 (SEQ >D N0.17) was obtained.
[0480] Using the primer pairs GTA CCA GGGG ACT GTG ACC ATT GC (SEQ ID N0.18)
and
CTG TGA CCA TTG CTC CCA AGA GAG (SEQ ID N0.19), the promoter having the
internal designation
P15B4 (SEQ ID N0.20) was obtained.
[0481] Using the primer pairs CTG GGA TGG AAG GCA CGG TA (SEQ ID N0.21) and
GAG
ACC ACA CAG CTA GAC AA (SEQ 117 N0.22), the promoter having the internal
designation P29B6
(SEQ ID N0.23) was obtained.
[0482] Figure 4 provides a schematic description of the promoters isolated and
the way they are
assembled with the corresponding 5' tags. The upstream sequences were screened
for the presence of motifs
resembling transcription factor binding sites or known transcription start
sites using the computer program
MatInspector release 2.0, August 1996.
[0483] Figure 5 describes the transcription factor binding sites present in
each of these promoters.
The columns labeled matrice provides the name of the Matlnspector matrix used.
The column labeled
position provides the 5' position of the promoter site. Numeration of the
sequence starts from the
transcription site as determined by matching the genomic sequence with the 5'
EST sequence. The column
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labeled "orientation" indicates the DNA strand on which the site is found,
with the + strand being the coding
strand as determined by matching the genomic sequence with the sequence of the
5' EST. The column
labeled "score" provides the MatInspector score found for this site. The
column labeled "length" provides
the length of the site in nucleotides. The column labeled "sequence" provides
the sequence of the site found.
[0484] Bacterial clones containing plasmids containing the promoter sequences
described above
described above are presently stored in the inventor's laboratories under the
internal identification numbers
provided above. The inserts may be recovered from the deposited materials by
growing an aliquot of the
appropriate bacterial clone in the appropriate medium. The plasmid DNA can
then be isolated using plasmid
isolation procedures familiar to those skilled in the art such as alkaline
lysis minipreps or large scale alkaline
lysis plasmid isolation procedures. if desired the plasmid DNA may be further
enriched by centrifugation on
a cesium chloride gradient, size exclusion chromatography, or anion exchange
chromatography. The
plasmid DNA obtained using these procedures may then be manipulated using
standard cloning techniques
familiar to those skilled in the art. Alternatively, a PCR can be done with
primers designed at both ends of
the inserted EST-related nucleic acids, positional segments of EST-related
nucleic acids or fragments of
positional segments of EST-related nucleic acids. The PCR product which
corresponds to the EST-related
nucleic acids, positional segments of EST-related nucleic acids or fragments
of positional segments of EST-
related nucleic acids can then be manipulated using standard cloning
techniques familiar to those skilled in
the art.
[0485) The promoters and other regulatory sequences located upstream of the
EST-related nucleic
acids, positional segments of EST-related nucleic acids or fragments of
positional segments of EST-related
nucleic acids may be used to design expression vectors capable of directing
the expression of an inserted gene
in a desired spatial, temporal, developmental, or quantitative manner. A
promoter capable of directing the
desired spatial, temporal, developmental, and quantitative patterns may be
selected using the results of the
expression analysis described above. For example, if a promoter which confers
a high level of expression in
muscle is desired, the promoter sequence upstream of EST-related nucleic
acids, positional segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids derived from an
mRNA which are expressed at a high level in muscle, as determined by the
methods above, may be used in
the expression vector.
[0486] Preferably, the desired promoter is placed near multiple restriction
sites to facilitate the
cloning of the desired insert downstream of the promoter, such that the
promoter is able to drive expression of
the inserted gene. The promoter may be inserted in conventional nucleic acid
backbones designed for
extrachromosomal replication, integration into the host chromosomes or
transient expression. Suitable
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backbones for the present expression vectors include retroviral backbones,
backbones from eukaryotic
episomes such as SV40 or Bovine Papilloma Virus, backbones from bacterial
episomes, or artificial
chromosomes.
[0487] Preferably, the expression vectors also include a polyA signal
downstream of the multiple
restriction sites for directing the polyadenylation of mRNA transcribed from
the gene inserted into the
expression vector.
[0488] Following the identification of promoter sequences using the procedures
of Examples 51-53,
proteins which interact with the promoter may be identified as described in
Example 54 below.
EXAMPLE 54
Identification of Proteins Which Interact with Promoter SecFuences Upstream
Re~ulatory Seguences, or
mRNA
[0489] Sequences within the promoter region which are likely to bind
transcription factors may be
identified by homology to known transcription factor binding sites or through
conventional mutagenesis or
deletion analyses of reporter plasmids containing the promoter sequence. For
example, deletions may be
made in a reporter plasmid containing the promoter sequence of interest
operably linked to an assayable
reporter gene. The reporter plasmids carrying various deletions within the
promoter region are transfected
into an appropriate host cell and the effects of the deletions on expression
levels is assessed. Transcription
factor binding sites within the regions in which deletions reduce expression
levels may be further localized
using site directed mutagenesis, linker scanning analysis, or other techniques
familiar to those skilled in the
art.
[0490] Nucleic acids encoding proteins which interact with sequences in the
promoter may be
identified using one-hybrid systems such as those described in the manual
accompanying the Matchmaker
One-Hybrid System kit available from Clontech (Catalog No. K1603-1), the
disclosure of which is
incorporated herein by reference. Briefly, the Matchmaker One-hybrid system is
used as follows. The target
sequence for which it is desired to identify binding proteins is cloned
upstream of a selectable reporter gene
and integrated into the yeast genome. Preferably, multiple copies of the
target sequences are inserted into the
reporter plasmid in tandem. A library comprised of fusions between cDNAs to be
evaluated for the ability to
bind to the promoter and the activation domain of a yeast transcription
factor, such as GAL4, is transformed
into the yeast strain containing the integrated reporter sequence. The yeast
are plated on selective media to
select cells expressing the selectable marker linked to the promoter sequence.
The colonies which grow on
the selective media contain genes encoding proteins which bind the target
sequence. The inserts in the genes
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encoding the fusion proteins are further characterized by sequencing. In
addition, the inserts may be inserted
into expression vectors or in vitro transcription vectors. Binding of the
polypeptides encoded by the inserts to
the promoter DNA may be confirmed by techniques familiar to those skilled in
the art, such as gel shift
analysis or DNAse protection analysis.
VIII Use of EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids in Gene Therapy
[0491] The present invention also comprises the use of EST-related nucleic
acids, positional
segments of EST-related nucleic acids or fragments of positional segments of
EST-related nucleic acids in
gene therapy strategies, including antisense and triple helix strategies as
described in Examples 55 and 56
below. In antisense approaches, nucleic acid sequences complementary to an
mRNA are hybridized to the
mRNA intracellularly, thereby blocking the expression of the protein encoded
by the mRNA. The antisense
sequences may prevent gene expression through a variety of mechanisms. For
example, the antisense
sequences may inhibit the ability of ribosomes to translate the mRNA.
Alternatively, the antisense sequences
may block transport of the mRNA from the nucleus to the cytoplasm, thereby
limiting the amount of mRNA
available for translation. Another mechanism through which antisense sequences
may inhibit gene
expression is by interfering with mRNA splicing. In yet another strategy, the
antisense nucleic acid may be
incorporated in a ribozyme capable of specifically cleaving the target mRNA.
EXAMPLE 55
Preparation and Use of Antisense Oli~onucleotides
[0492] The antisense nucleic acid molecules to be used in gene therapy may be
either DNA or RNA
sequences. They may comprise a sequence complementary to the sequence of the
EST-related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids. The antisense nucleic acids should have a length and melting
temperature sufficient to permit
formation of an intracellular duplex with sufficient stability to inhibit the
expression of the mRNA in the
duplex. Strategies for designing antisense nucleic acids suitable for use in
gene therapy are disclosed in
Green et al., Ann. Rev. Biochem. 55:569-597 (1986) and Izant and Weintraub,
Cell 36:1007-1015 (1984),
which are hereby incorporated by reference.
[0493] In some strategies, antisense molecules are obtained from a nucleotide
sequence encoding a
protein by reversing the orientation of the coding region with respect to a
promoter so as to transcribe the
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opposite strand from that which is normally transcribed in the cell. The
antisense molecules may be
transcribed using in vitro transcription systems such as those which employ T7
or SP6 polymerase to
generate the transcript. Another approach involves transcription of the
antisense nucleic acids in vivo by
operably linking DNA containing the antisense sequence to a promoter in an
expression vector.
[0494] Alternatively, oligonucleotides which are complementary to the strand
normally transcribed
in the cell may be synthesized in vitro. Thus, the antisense nucleic acids are
complementary to the
corresponding mRNA and are capable of hybridizing to the mRNA to create a
duplex. In some
embodiments, the antisense sequences may contain modified sugar phosphate
backbones to increase stability
and make them less sensitive to RNase activity. Examples of modifications
suitable for use in antisense
strategies are described by Rossi et al., Pharmacol. Ther. 50(2):245-254,
(1991) which is hereby incorporated
by reference.
[0495] Various types of antisense oligonucleotides complementary to the
sequence of the EST-
related nucleic acids, positional segnnents of EST-related nucleic acids or
fragments of positional segments of
EST-related nucleic acids may be used. In one preferred embodiment, stable and
semi-stable antisense
1 S oligonucleotides described in International Application No. PCT
W094/23026, hereby incorporated by
reference, are used. In these molecules, the 3' end or both the 3' and 5' ends
are engaged in intramolecular
hydrogen bonding between complementary base pairs. These molecules are better
able to withstand
exonuclease attacks and exhibit increased stability compared to conventional
antisense oligonucleotides.
[0496] In another preferred embodiment, the antisense oligodeoxynucleotides
against herpes
simplex virus types 1 and 2 described in International Application No. WO
95/04141, hereby incorporated by
reference, are used.
[0497] In yet another preferred embodiment, the covalently cross-linked
antisense oligonucleotides
described in International Application No. WO 96/31523 (hereby incorporated by
reference) are used. These
double- or single-stranded oligonucleotides comprise one or more,
respectively, inter- or intra-
oligonucleotide covalent cross-linkages, wherein the linkage consists of an
amide bond between a primary
amine group of one strand and a carboxyl group of the other strand or of the
same strand, respectively, the
primary amine group being directly substituted in the 2' position of the
strand nucleotide monosaccharide
ring, and the carboxyl group being carried by an aliphatic spacer group
substituted on a nucleotide or
nucleotide analog of the other strand or the same strand, respectively.
[0498] The antisense oligodeoxynucleotides and oligonucleotides disclosed in
International
Application No. WO 92/18522, incorporated by reference, may also be used.
These molecules are stable to
degradation and contain at least one transcription control recognition
sequence which binds to control
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proteins and are effective as decoys therefore. These molecules may contain
"hairpin" structures, "dumbbell"
structures, "modified dumbbell" structures, "cross-linked" decoy structures
and "loop" structures.
[0499] In another preferred embodiment, the cyclic double-stranded
oligonucleotides described in
European Patent Application No. 0 572 287 A2, hereby incorporated by reference
are used. These ligated
oligonucleotide "dumbbells" contain the binding site for a transcription
factor and inhibit expression of the
gene under control of the transcription factor by sequestering the factor.
[0500] Use of the closed antisense oligonucleotides disclosed in International
Application No. WO
92/19732, hereby incorporated by reference, is also contemplated. Because
these molecules have no free
ends, they are more resistant to degradation by exonucleases than are
conventional oligonucleotides. These
oligonucleotides may be multifunctional, interacting with several regions
which are not adjacent to the target
mRNA.
[0501] The appropriate level of antisense nucleic acids required to inhibit
gene expression may be
determined using in vitro expression analysis. The antisense molecule may be
introduced into the cells by
diffusion, injection, infection or transfection using procedures known in the
art. For example, the antisense
nucleic acids can be introduced into the body as a bare or naked
oligonucleotide, oligonucleotide
encapsulated in lipid, oligonucleotide sequence encapsidated by viral protein,
or as an oligonucleotide
operably linked to a promoter contained in an expression vector. The
expression vector may be any of a
variety of expression vectors known in the art, including retroviral or viral
vectors, vectors capable of
extrachromosomal replication, or integrating vectors. The vectors may be DNA
or RNA.
[0502] The antisense molecules are introduced onto cell samples at a number of
different
concentrations preferably between 1x10~'°M to 1x10''M. Once the minimum
concentration that can
adequately control gene expression is identified, the optimized dose is
translated into a dosage suitable for use
in vivo. For example, an inhibiting concentration in culture of 1x10''
translates into a dose of approximately
0.6 mg/kg bodyweight. Levels of oligonucleotide approaching 100 mg/kg
bodyweight or higher may be
possible after testing the toxicity of the oligonucleotide in laboratory
animals. It is additionally contemplated
that cells from the vertebrate are removed, treated with the antisense
oligonucleotide, and reintroduced into
the vertebrate.
[0503] It is further contemplated that the antisense oligonucleotide sequence
is incorporated into a
ribozyme sequence to enable the antisense to specifically bind and cleave its
target mRNA. For technical
applications of ribozyme and antisense oligonucleotides see Rossi et al.,
supra.
[0504] In a preferred application of this invention, the polypeptide encoded
by the gene is first
identified, so that the effectiveness of antisense inhibition on translation
can be monitored using techniques
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that include but are not limited to antibody-mediated tests such as RIAs and
ELISA, functional assays, or
radiolabeling.
[0505] T'he EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids may also be used
in gene therapy approaches
based on intracellular triple helix formation. Triple helix oligonucleotides
are used to inhibit transcription
from a genome. They are particularly useful for studying alterations in cell
activity as it is associated with a
particular gene. The EST-related nucleic acids, positional segments of EST-
related nucleic acids or
fragments of positional segments of EST-related nucleic acids of the present
invention or, more preferably, a
portion of those sequences, can be used to inhibit gene expression in
individuals having diseases associated
with expression of a particular gene. Similarly, the EST-related nucleic
acids, positional segments of EST-
related nucleic acids or fragments of positional segments of EST-related
nucleic acids can be used to study
the effect of inhibiting transcription of a particular gene within a cell.
Traditionally, homopurine sequences
were considered the most useful for triple helix strategies. However,
homopyrimidine sequences can also
inhibit gene expression. Such homopyrimidine oligonucleotides bind to the
major groove at
homopurine:homopyrimidine sequences. Thus, both types of sequences from the
EST-related nucleic acids,
positional segments of EST-related nucleic acids or fragments of positional
segments of EST-related nucleic
acids are contemplated within the scope of this invention.
EXAMPLE 56
Preparation and use of Triple Helix Probes
[0506] The sequences of the EST-related nucleic acids, positional segments of
EST-related nucleic
acids or fragments of positional segments of EST-related nucleic acids are
scanned to identify 10-mer to 20-
mer homopyrimidine or homopurine stretches which could be used in triple-helix
based strategies for
inhibiting gene expression. Following identification of candidate
homopyrimidine or homopurine stretches,
their efficiency in inhibiting gene expression is assessed by introducing
varying amounts of oligonucleotides
containing the candidate sequences into tissue culture cells which normally
express the target gene. The
oligonucleotides may be prepared on an oligonucleotide synthesizer or they may
be purchased commercially
from a company specializing in custom oligonucleotide synthesis, such as
GENSET, Paris, France.
[0507) The oligonucleotides may be introduced into the cells using a variety
of methods known to
those skilled in the art, including but not limited to calcium phosphate
precipitation, DEAE-Dextran,
electroporation, liposome-mediated transfection or native uptake.
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[0508] Treated cells are monitored for altered cell function or reduced gene
expression using
techniques such as Northern blotting, RNase protection assays, or PCR based
strategies to monitor the
transcription levels of the target gene in cells which have been treated with
the oligonucleotide. The cell
functions to be monitored are predicted based upon the homologies of the
target genes corresponding to the
EST-related nucleic acids, positional segments of EST-related nucleic acids or
fragments of positional
segments of EST-related nucleic acids from which the oligonucleotide were
derived with known gene
sequences that have been associated with a particular function. The cell
functions can also be predicted based
on the presence of abnormal physiologies within cells derived from individuals
with a particular inherited
disease, particularly when the EST-related nucleic acids, positional segments
of EST-related nucleic acids or
fragments of positional segments of EST-related nucleic acids are associated
with the disease using
techniques described herein.
[0509] The oligonucleotides which are effective in inhibiting gene expression
in tissue culture cells
may then be introduced in vivo using the techniques described above and in
Example 55 at a dosage
calculated based on the in vitro results, as described in Example 55.
[0510] In some embodiments, the natural (beta) anomers of the oligonucleotide
units can be
replaced with alpha anomers to render the oligonucleotide more resistant to
nucleases. Further, an
intercalating agent such as ethidium bromide, or the like, can be attached to
the 3' end of the alpha
oligonucleotide to stabilize the triple helix. For information on the
generation of oligonucleotides suitable for
triple helix formation see Griffin et al. (Science 245:967-971 (1989), which
is hereby incorporated by this
reference).
EXAMPLE 57
Use of EST-related nucleic acids positional segments of EST-related nucleic
acids or fragments of
positional segments of EST-related nucleic acids to express an Encoded Protein
in a Host Or anism
[0511] The EST-related nucleic acids, positional segments of EST-related
nucleic acids or
fragments of positional segments of EST-related nucleic acids may also be used
to express an encoded
protein or polypeptide in a host organism to produce a beneficial effect. In
addition, nucleic acids encoding
the EST-related polypeptides, positional segments of EST-related polypeptides
or fragments of positional
segments of EST-related polypeptides may be used to express the encoded
protein or polypeptide in a host
organism to produce a beneficial effect.
[0512] In such procedures, the encoded protein or polypeptide may be
transiently expressed in the
host organism or stably expressed in the host organism. 'The encoded protein
or polypeptide may have any of
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the activities described above. The encoded protein or polypeptide may be a
protein or polypeptide which the
host organism lacks or, alternatively, the encoded protein may augment the
existing levels of the protein in
the host organism.
[0513] In some embodiments in which the protein or polypeptide is secreted,
nucleic acids encoding
the full length protein (i.e. the signal peptide and the mature protein), or
nucleic acids encoding only the
mature protein ( i.e. the protein generated when the signal peptide is cleaved
off) is introduced into the host
organism.
[0514] The nucleic acids encoding the proteins or polypeptides may be
introduced into the host
organism using a variety of techniques known to those of skill in the art. For
example, the extended cDNA
may be injected into the host organism as naked DNA such that the encoded
protein is expressed in the host
organism, thereby producing a beneficial effect.
[0515] Alternatively, the nucleic acids encoding the protein or polypeptide
may be cloned into an
expression vector downstream of a promoter which is active in the host
organism. The expression vector
may be any of the expression vectors designed for use in gene therapy,
including viral or retroviral vectors.
The expression vector may be directly introduced into the host organism such
that the encoded protein is
expressed in the host organism to produce a beneficial effect. In another
approach, the expression vector may
be introduced into cells in vitro. C'.ells containing the expression vector
are thereafter selected and introduced
into the host organism, where they express the encoded protein or polypeptide
to produce a beneficial effect.
EXAMPLE 58
Use of Signal Peptides To Import Proteins Into Cells
[0516] The short core hydrophobic region (h) of signal peptides encoded by the
sequences of SEQ
ID NOs. 24-1027 and 4597-6443 may also be used as a earner to import a peptide
or a protein of interest, so-
called cargo, into tissue culture cells (Lin et al., J. Biol. Chem., 270:
14225-14258 (1995); Du et al., J.
Peptide Res., 51: 235-243 (1998); Rojas et al., Nature Biotech., 16: 370-375
(1998)).
[0517] When cell permeable peptides of limited size (approximately up to 25
amino acids) are to be
translocated across cell membrane, chemical synthesis may be used in order to
add the h region to either the
C-terminus or the N-terminus to the cargo peptide of interest. Alternatively,
when longer peptides or proteins
are to be imported into cells, nucleic acids can be genetically engineered,
using techniques familiar to those
skilled in the art, in order to link the extended cDNA sequence encoding the h
region to the 5' or the 3' end of
a DNA sequence coding for a cargo polypeptide. Such genetically engineered
nucleic acids are then
translated either in vitro or in vivo after transfection into appropriate
cells, using conventional techniques to
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produce the resulting cell permeable polypeptide. Suitable hosts cells are
then simply incubated with the cell
permeable polypeptide which is then translocated across the membrane.
[0518] This method may be applied to study diverse intracellular functions and
cellular processes.
For instance, it has been used to probe functionally relevant domains of
intracellular proteins and to examine
protein-protein interactions involved in signal transduction pathways (L,in et
al., .supra; Lin et al., J. Biol.
Chem., 271: 5305-5308 (1996); Rojas et al., J. Biol. Chem., 271: 27456-27461
(1996); Liu et al., Proc. Natl.
Acad. Sci. USA, 93: 11819-11824 (1996); Rojas et al., Biach. Biophys. Res.
Commun., 234: 675-680 (1997)).
[0519] Such techniques may be used in cellular therapy to import proteins
producing therapeutic
effects. For instance, cells isolated from a patient may be treated with
imported therapeutic proteins and then
re-introduced into the host organism.
[0520] Alternatively, the h region of signal peptides of the present invention
could be used in
combination with a nuclear localization signal to deliver nucleic acids into
cell nucleus. Such
oligonucleotides may be antisense oligonucleotides or oligonucleotides
designed to form triple helixes, as
described above, in order to inhibit processing and maturation of a target
cellular RNA.
EXAMPLE 59
Computer Embodiments
(0521] As used herein the term "cDNA codes of SEQ 1D NOs. 24-13309 and 26596-
52153"
encompasses the nucleotide sequences of SEQ m NOs. 24-13309 and 26596-52153,
fragments of SEQ ID
NOs. 24-13309 and 26596-52153, nucleotide sequences homologous to SEQ ID NOs.
24-13309 and
26596-52153 or homologous to fragments of SEQ ID NOs. 24-13309 and 26596-
52153, and sequences
complementary to all of the preceding sequences. The fragments include
fragments of SEQ ID NOs. 24-
13309 and 26596-52153 comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30,
35, 40, 50, 75, 100, 150, 200,
300, 400, 500, 1000 or 2000 consecutive nucleotides of SEQ ID NOs. 24-13309
and 26596-52153.
Preferably, the fragments are novel fragments. Preferably the fragments
include polynucleotides described in
Tables IVa and IVb, polynucleotides described in Tables IVa and IVb updated,
or fragments thereof
comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100,
150, 200, 300, 400, 500, 1000 or
2000 consecutive nucleotides of the polynucleotides described in Tables IVa
and IVb, or polynucleotides
described in Tables IVa and IVb updated. Homologous sequences and fragments of
SEQ B7 NOs. 24-13309
and 26596-52153 refer to a sequence having at least 99%, 98%, 97%, 96%, 95%,
90%, 85%, 80%, or 75%
homology to these sequences. Homology may be determined using any of the
computer programs and
parameters described in Example 17, including BLAST2N with the default
parameters or with any modified
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parameters. Homologous sequences also include RNA sequences in which uridines
replace the thymines in
the cDNA codes of SEQ ID NOs. 24-13309 and 26596-52153. The homologous
sequences may be
obtained using any of the procedures described herein or may result from the
correction of a sequencing error
as described above. Preferably the homologous sequences and fragments of SEQ
>D NOs. 24-13309 and
26596-52153 include polynucleotides described in Tables IVa and IVb,
polynucleotides described in Tables
IVa and IVb updated, or fragments comprising at least 8, 10, 12, 15, 18, 20,
25, 28, 30, 35, 40, 50, 75, 100,
150, 200, 300, 400, 500, 1000 or 2000 consecutive nucleorides of the
polynucleotides described in Tables
IVa and IVb, or polynucleotides described in Tables IVa and IVb updated. It
will be appreciated that the
cDNA codes of SEQ )D NOs. 24-13309 and 26596-52153 can be represented in the
traditional single
character format (See the inside back cover of Styer, Lubert. Biochemistry,
3rd edition. W. H Freeman & Co.,
New York.) or in any other format which records the identity of the
nucleotides in a sequence.
[0522] As used herein the term "polypeptide codes of SEQ ID NOS. 13310-26595"
encompasses
the polypeptide sequence of SEQ ID NOs. 13310-26595 which are encoded by the
cDNAs of SEQ 117 NOs.
24-13309, polypeptide sequences homologous to the polypeptides of SEQ )D NOS.
13310-26595, or
fragments of any of the preceding sequences. Homologous polypeptide sequences
refer to a polypeptide
sequence having at least 99%, 98%, 97°ro, 96%, 95%, 90%, 85%, 80%, 75%
homology to one of the
polypeptide sequences of SEQ ID NOS. 13310-26595. Homology may be determined
using any of the
computer programs and parameters described herein, including FASTA with the
default parameters or with
any modified parameters. The homologous sequences may be obtained using any of
the procedures
described herein or may result from the correction of a sequencing error as
described above. The polypeptide
fragments comprise at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75,
100, 150 or 200 consecutive amino
acids of the polypeptides of SEQ ID NOS. 13310-26595. Preferably, the
fragments are novel fragments.
Preferably, the fragments include polypeptides encoded by the polynucleotides
described in Tables IVa and
Nb, polynucleotides described in Tables IVa and Nb updated, or fragments
thereof comprising at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of the
polypeptides encoded by the
polynucleotides described in Tables IVa and IVb, or polynucleotides described
in Tables IVa and IVb
updated. It will be appreciated that the polypeptide codes of the SEQ ID NOS.
13310-26595 can be
represented in the traditional single character format or three letter format
(See the inside back cover of
Starrier, Lubert. Biochemistry, 3'd edition. W. H Freeman & Co., New York.) or
in any other format which
relates the identity of the polypeptides in a sequence.
[0523] It will be appreciated by those skilled in the art that the cDNA codes
of SEQ ID NOs. 24-
13309 and 26596-52153 and polypeptide codes of SEQ ID NOS. 13310-26595 can be
stored, recorded,
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and manipulated on any medium which can be read and accessed by a computer. As
used herein, the words
"recorded" and "stored" refer to a process for storing information on a
computer medium. A skilled artisan
can readily adopt any of the presently known methods for recording information
on a computer readable
medium to generate manufactures comprising one or more of the eDNA codes of
SEQ ID NOs. 24-13309
and 26596-52153, one or more of the polypeptide codes of SEQ ID NOS. 13310-
26595. Another aspect of
the present invention is a computer readable medium having recorded thereon at
least 2, 5, 10, 15, 20, 25, 30,
or 50 cDNA codes of SEQ ID NOs. 24-13309 and 26596-52153. Another aspect of
the present invention is
a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20,
25, 30, or 50 polypeptide codes
of SEQ ID NOS. 13310-26595.
[0524] Computer readable media include magnetically readable media, optically
readable media,
electronically readable media and magnetic/optical media. For example, the
computer readable media may
be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk
(DVD), Random Access
Memory (RAM), or Read Only Memory (ROM) as well as other types of other media
known to those skilled
in the art.
[0525] Embodiments of the present invention include systems, particularly
computer systems which
store and manipulate the sequence information described herein. One example of
a computer system 100 is
illustrated in block diagram form in Figure 6. As used herein, "a computer
system" refers to the hardware
components, software components, and data storage components used to analyze
the nucleotide sequences of
the cDNA codes of SEQ ID NOs. 24-13309 and 26596-52153, or the amino acid
sequences of the
polypeptide codes of SEQ ID NOS. 13310-26595. In one embodiment, the computer
system 100 is a Sun
Enterprise 1000 server (Sun Microsystems, Palo Alto, CA). The computer system
100 preferably includes a
processor for processing, accessing and manipulating the sequence data. The
processor 105 can be any well-
known type of central processing unit, such as the Pentium III from Intel
Corporation, or similar processor
from Sun, Motorola, Compaq or International Business Machines.
[0526] Preferably, the computer system 100 is a general purpose system that
comprises the
processor 105 and one or more internal data storage components 110 for storing
data, and one or more data
retrieving devices for retrieving the data stored on the data storage
components. A skilled artisan can readily
appreciate that any one of the currently available computer systems are
suitable.
[0527] In one particular embodiment, the computer system 100 includes a
processor 105 connected
to a bus which is connected to a main memory 115 (preferably implemented as
RAM) and one or more
internal data storage devices 110, such as a hard drive and/or other computer
readable media having data
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recorded thereon. In some embodiments, the computer system 100 further
includes one or more data
retrieving device 118 for reading the data stored on the internal data storage
devices 110.
[0528] The data retrieving device 118 may represent, for example, a floppy
disk drive, a compact
disk drive, a magnetic tape drive, etc. In some embodiments, the internal data
storage device 110 is a
removable computer readable medium such as a floppy disk, a compact disk, a
magnetic tape, etc. containing
control logic and/or data recorded thereon. The computer system 100 may
advantageously include or be
programmed by appropriate software for reading the control logic and/or the
data from the data storage
component once inserted in the data retrieving device.
[0529] The computer system 100 includes a display 120 which is used to display
output to a
computer user. It should also be noted that the computer system 100 can be
linked to other computer systems
125a-c in a network or wide area network to provide centralized access to the
computer system 100.
[0530] Software for accessing and processing the nucleotide sequences of the
eDNA codes of SEQ
ID NOs. 24-13309 and 26596-52153, or the amino acid sequences of the
polypeptide codes of SEQ ID
NOS. 13310-26595 (such as search tools, compare tools, and modeling tools
etc.) may reside in main
memory 115 during execution.
[0531] In some embodiments, the computer system 100 may further comprise a
sequence comparer
for comparing the above-described cDNA codes of SEQ ID NOs. 24-13309 and 26596-
52153 or
polypeptide codes of SEQ )D NOS. 13310-26595 stored on a computer readable
medium to reference
nucleotide or polypeptide sequences stored on a computer readable medium. A
"sequence comparer" refers
to one or more programs which are implemented on the computer system 100 to
compare a nucleotide or
polypeptide sequence with other nucleotide or polypeptide sequences and/or
compounds including but not
limited to peptides, peptidomimetics, and chemicals stored within the data
storage means. For example, the
sequence comparer may compare the nucleotide sequences of the cDNA codes of
SEQ >D NOs. 24-13309
and 26596-52153, or the amino acid sequences of the polypeptide codes of SEQ
1D NOS. 13310-26595
stored on a computer readable medium to reference sequences stored on a
computer readable medium to
identify homologies, motifs implicated in biological function, or structural
motifs. The various sequence
comparer programs identified elsewhere in this patent specification are
particularly contemplated for use in
this aspect of the invention.
[0532] Figure 7 is a flow diagram illustrating one embodiment of a process 200
for comparing a
new nucleotide or protein sequence with a database of sequences in order to
determine the homology levels
between the new sequence and the sequences in the database. The database of
sequences can be a private
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database stored within the computer system 100, or a public database such as
GENBANK, PIR or
SWISSPROT that is available through the Internet.
[0533] The process 200 begins at a start state 201 and then moves to a state
202 wherein the new
sequence to be compared is stored to a memory in a computer system 100. As
discussed above, the memory
could be any type of memory, including RAM or an internal storage device.
[0534] The process 200 then moves to a state 204 wherein a database of
sequences is opened for
analysis and comparison. The process 200 then moves to a state 206 wherein the
first sequence stored in the
database is read into a memory on the computer. A comparison is then performed
at a state 210 to determine
if the first sequence is the same as the second sequence. It is important to
note that this step is not limited to
performing an exact comparison between the new sequence and the first sequence
in the database. Well-
known methods are known to those of skill in the art for comparing two
nucleotide or protein sequences, even
if they are not identical. For example, gaps can be introduced into one
sequence in order to raise the
homology level between the two tested sequences. The parameters that control
whether gaps or other
features are introduced into a sequence during comparison are normally entered
by the user of the computer
system.
[0535] Once a comparison of the two sequences has been performed at the state
210, a
determination is made at a decision state 210 whether the two sequences are
the same. Of course, the term
"same" is not limited to sequences that are absolutely identical. Sequences
that are within the homology
parameters entered by the user will be marked as "same" in the process 200.
[0536] If a determination is made that the two sequences are the same, the
process 200 moves to a
state 214 wherein the name of the sequence from the database is displayed to
the user. This state notifies the
user that the sequence with the displayed name fulfills the homology
constraints that were entered. Once the
name of the stored sequence is displayed to the user, the process 200 moves to
a decision state 218 wherein a
determination is made whether more sequences exist in the database. If no more
sequences exist in the
database, then the process 200 terminates at an end state 220. However, if
more sequences do exist in the
database, then the process 200 moves to a state 224 wherein a pointer is moved
to the next sequence in the
database so that it can be compared to the new sequence. In this manner, the
new sequence is aligned and
compared with every sequence in the database.
[0537] It should be noted that if a determination had been made at the
decision state 212 that the
sequences were not homologous, then the process 200 would move immediately to
the decision state 218 in
order to determine if any other sequences were available in the database for
comparison.
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[0538] Accordingly, one aspect of the present invention is a computer system
comprising a
processor, a data storage device having stored thereon a nucleic acid code of
SEQ >D NOs. 24-13309 and
26596-52153 or a polypeptide code of SEQ ID NOS. 13310-26595, a data storage
device having
retrievably stored thereon reference nucleotide sequences or polypeptide
sequences to be compared to the
nucleic acid code of SEQ ID NOs. 24-13309 and 26596-52153 or polypeptide code
of SEQ ID NOS.
13310-26595 and a sequence comparer for conducting the comparison. The
sequence comparer may
indicate a homology level between the sequences compared or identify
structural motifs in the above
described nucleic acid code of SEQ ID NOs. 24-13309 and 26596-52153 and
polypeptide codes of SEQ
)D NOS. 13310-26595 or it may identify structural motifs in sequences which
are compared to these
cDNA codes and polypeptide codes. In some embodiments, the data storage device
may have stored
thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cDNA
codes of SEQ )D NOs. 24-
13309 and 26596-52153 or polypeptide codes of SEQ >D NOS. 13310-26595.
[0539] Another aspect of the present invention is a method for determining the
level of homology
between a nucleic acid code of SEQ ID NOs. 24-13309 and 26596-52153 and a
reference nucleotide
sequence, comprising the steps of reading the nucleic acid code and the
reference nucleotide sequence
through the use of a computer program which determines homology levels and
determining homology
between the nucleic acid code and the reference nucleotide sequence with the
computer program. The
computer program may be any of a number of computer programs for determining
homology levels,
including those specifically enumerated herein, including BLAST2N with the
default parameters or with any
modified parameters. The method may be implemented using the computer systems
described above. The
method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the
above described cDNA codes
of SEQ ID NOs. 24-13309 and 26596-52153 through use of the computer program
and determining
homology between the cDNA codes and reference nucleotide sequences .
(0540] Figure 8 is a flow diagram illustrating one embodiment of a process 250
in a computer
for determining whether two sequences are homologous. The process 250 begins
at a start state 252 and
then moves to a state 254 wherein a first sequence to be compared is stored to
a memory. The second
sequence to be compared is then stored to a memory at a state 256. The process
250 then moves to a state
260 wherein the first character in the first sequence is read and then to a
state 262 wherein the first
character of the second sequence is read. It should be understood that if the
sequence is a nucleotide
sequence, then the character would normally be either A, T, C, G or U. If the
sequence is a protein
sequence, then it should be in the single letter amino acid code so that the
first and sequence sequences
can be easily compared.
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[0541] A determination is then made at a decision state 264 whether the two
characters are the
same. If they are the same, then the process 250 moves to a state 268 wherein
the next characters in the
first and second sequences are read. A determination is then made whether the
next characters are the
same. If they are, then the process 250 continues this loop until two
characters are not the same. If a
determination is made that the next two characters are not the same, the
process 250 moves to a decision
state 274 to determine whether there are any more characters either sequence
to read.
[0542] If there aren't any more characters to read, then the process 250 moves
to a state 276
wherein the level of homology between the first and second sequences is
displayed to the user. The level
of homology is determined by calculating the profragment of characters between
the sequences that were
the same out of the total number of sequences in the first sequence. Thus, if
every character in a first 100
nucleotide sequence aligned with a every character in a second sequence, the
homology level would be
100%.
[0543] Alternatively, the computer program may be a computer program which
compares the
nucleotide sequences of the cDNA codes of the present invention, to reference
nucleotide sequences in order
to determine whether the nucleic acid code of SEQ ID NOs. 24-13309 and 26596-
52153 differs from a
reference nucleic acid sequence at one or more positions. Optionally such a
program records the length and
identity of inserted, deleted or substituted nucleotides with respect to the
sequence of either the reference
polynucleotide or the nucleic acid code of SEQ 1D NOs. 24-13309 and 26596-
52153. In one embodiment,
the computer program may be a program which determines whether the nucleotide
sequences of the cDNA
codes of SEQ B7 NOs. 24-13309 and 26596-52153 contain a biallelic marker or
single nucleotide
polymorphism (SNP) with respect to a reference nucleotide sequence. This
single nucleotide polymorphism
may comprise a single base substitution, insertion, or deletion, while this
biallelic marker may comprise
about one to ten consecutive bases substituted, inserted or deleted.
[0544) Another aspect of the present invention is a method for determining the
level of homology
between a polypeptide code of SEQ ID NOS. 13310-26595 and a reference
polypeptide sequence,
comprising the steps of reading the polypeptide code of SEQ 1D NOS. 13310-
26595 and the reference
polypeptide sequence through use of a computer program which determines
homology levels and
determining homology between the polypeptide code and the reference
polypeptide sequence using the
computer program.
[0545] Accordingly, another aspect of the present invention is a method for
determining whether a
nucleic acid code of SEQ 1D NOs. 24-13309 and 26596-52153 differs at one or
more nucleotides from a
reference nucleotide sequence comprising the steps of reading the nucleic acid
code and the reference
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nucleotide sequence through use of a computer program which identifies
differences between nucleic acid
sequences and identifying differences between the nucleic acid code and the
reference nucleotide sequence
with the computer program. In some embodiments, the computer program is a
program which identifies
single nucleotide polymorphisms. The method may be implemented by the computer
systems described
above and the method illustrated in Figure 8. The method may also be performed
by reading at least 2, 5, 10,
15, 20, 25, 30, or 50 of the cDNA codes of SEQ ID NOs. 24-13309 and 26596-
52153 and the reference
nucleotide sequences through the use of the computer program and identifying
differences between the
cDNA codes and the reference nucleotide sequences with the computer program.
[0546] In other embodiments the computer based system may further comprise an
identifier for
identifying features within the nucleotide sequences of the cDNA codes of SEQ
ID NOs. 24-13309 and
26596-52153 or the amino acid sequences of the polypeptide codes of SEQ )D
NOS. 13310-26595.
[0547] An "identifier" refers to one or more programs which identifies certain
features within
the above-described nucleotide sequences of the cDNA codes of SEQ )D NOs. 24-
13309 and 26596-
52153 or the amino acid sequences of the polypeptide codes of SEQ ID NOS.
13310-26595. In one
embodiment, the identifier may comprise a program which identifies an open
reading frame in the cDNAs
codes of SEQ ID NOs. 24-13309 and 26596-52153.
[0548] Figure 9 is a flow diagram illustrating one embodiment of an identifier
process 300 for
detecting the presence of a feature in a sequence. The process 300 begins at a
start state 302 and then
moves to a state 304 wherein a first sequence that is to be checked for
features is stored to a memory 115
in the computer system 100. The process 300 then moves to a state 306 wherein
a database of sequence
features is opened. Such a database would include a list of each feature's
attributes along with the name
of the feature. For example, a feature name could be "Initiation Codon" and
the attribute would be
"ATG". Another example would be the feature name "TAATAA Box" and the feature
attribute would be
"TAATAA". An example of such a database is produced by the University of
Wisconsin Genetics
Computer Group (www.gcg.com j.
(05495] Once the database of features is opened at the state 306, the process
300 moves to a state
308 wherein the first feature is read from the database. A comparison of the
attribute of the first feature
with the first sequence is then made at a state 310. A determination is then
made at a decision state 316
whether the attribute of the feature was found in the first sequence. If the
amibute was found, then the
process 300 moves to a state 318 wherein the name of the found feature is
displayed to the user.
[0550] The process 300 then moves to a decision state 320 wherein a
determination is made
whether move features exist in the database. If no more features do exist,
then the process 300 terminates
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at an end state 324. However, if more features do exist in the database, then
the process 300 reads the
next sequence feature at a state 326 and loops back to the state 310 wherein
the attribute of the next
feature is compared against the first sequence.
[0551] It should be noted, that if the feature amibute is not found in the
first sequence at the
decision state 316, the process 300 moves directly to the decision state 320
in order to determine if any
more features exist in the database.
[0552] In another embodiment, the identifier may comprise a molecular modeling
program
which determines the 3-dimensional structure of the polypeptides codes of SEQ
ID NOS. 13310-26595.
In some embodiments, the molecular modeling program identifies target
sequences that are most
compatible with profiles representing the structural environments of the
residues in known three-
dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Patent No.
5,436,850 issued July 25,
1995). In another technique, the known three-dimensional structures of
proteins in a given family are
superimposed to define the structurally conserved regions in that family. This
protein modeling technique
also uses the known three-dimensional structure of a homologous protein to
approximate the structure of
the polypeptide codes of SEQ ID NOS. 13310-26595. (See e.g., Srinivasan, et
al., U.S. Patent
No. 5,557,535 issued September 17, 1996). Conventional homology modeling
techniques have been used
routinely to build models of proteases and antibodies. (Sowdhamini et al.,
Protein Engineering 10:207,
215 (1997)). Comparative approaches can also be used to develop three-
dimensional protein models
when the protein of interest has poor sequence identity to template proteins.
In some cases, proteins fold
into similar three-dimensional structures despite having very weak sequence
identities. For example, the
three-dimensional structures of a number of helical cytokines fold in similar
three-dimensional topology
in spite of weak sequence homology.
[0553] The recent development of threading methods now enables the
identification of likely
folding patterns in a number of situations where the structural relatedness
between target and templates)
is not detectable at the sequence level. Hybrid methods, in which fold
recognition is performed using
Multiple Sequence Threading (MST), structural equivalencies are deduced from
the threading output
using a distance geometry program DRAGON to construct a low resolution model,
and a full-atom
representation is constructed using a molecular modeling package such as
QUANTA.
[0554] According to this 3-step approach, candidate templates are first
identified by using the
novel fold recognition algorithm MST, which is capable of performing
simultaneous threading of
multiple aligned sequences onto one or more 3-D structures. In a second step,
the structural equivalencies
obtained from the MST output are converted into inter-residue distance
restraints and fed into the distance
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geometry program DRAGON, together with auxiliary information obtained from
secondary structure
predictions. The program combines the restraints in an unbiased manner and
rapidly generates a large
number of low resolution model confirmations. In a third step, these low
resolution model confirmations
are converted into full-atom models and subjected to energy minimization using
the molecular modeling
package QUANTA. (See e.g., Aszodi et al., Proteins:Structure, Function, and
Genetics, Supplement
1:38-42 (1997)).
[0555] The results of the molecular modeling analysis may then be used in
rational drug design
techniques to identify agents which modulate the activity of the polypeptide
codes of SEQ ID NOS.
13310-26595.
[0556] Accordingly, another aspect of the present invention is a method of
identifying a feature
within the cDNA codes of SEQ ID NOs. 24-13309 and 26596-52153 or the
polypeptide codes of SEQ ID
NOS. 13310-26595 comprising reading the nucleic acid codes) or the polypeptide
codes) through the
use of a computer program which identifies features therein and identifying
features within the nucleic
acid codes) or polypeptide codes) with the computer program. In one
embodiment, computer program
comprises a computer program which identifies open reading frames. In a
further embodiment, the
computer program identifies structural motifs in a polypeptide sequence. In
another embodiment, the
computer program comprises a molecular modeling program. The method may be
performed by reading
a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cDNA
codes of SEQ ID NOs. 24-13309
and 26596-52153 or the polypeptide codes of SEQ ID NOS. 13310-26595 through
the use of the
computer program and identifying features within the cDNA codes or polypeptide
codes with the
computer program.
[0557] The cDNA codes of SEQ ID NOs. 24-13309 and 26596-52153 or the
polypeptide codes
of SEQ ID NOS. 13310-26595 may be stored and manipulated in a variety of data
processor programs in a
variety of formats. For example, the cDNA codes of SEQ ID NOs. 24-13309 and
26596-52153 or the
polypeptide codes of SEQ ID NOS. 13310-26595 may be stored as text in a word
processing file, such as
Microsoft WORD or WORDPERFECT or as an ASCII file in a variety of database
programs familiar to
those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many
computer programs and
databases may be used as sequence comparers, identifiers, or sources of
reference nucleotide or polypeptide
sequences to be compared to the cDNA codes of SEQ ID NOs. 24-13309 and 26596-
52153 or the
polypeptide codes of SEQ ID NOS. 13310-26595. The following list is intended
not to limit the invention
but to provide guidance to programs and databases which are useful with the
cDNA codes of SEQ ID NOs.
24-13309 and 26596-52153 or the polypeptide codes of SEQ ID NOS. 13310-26595.
The programs and
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databases which may be used include, but are not limited to: MacPattern
(EMBL), DiscoveryBase (Molecular
Applications Group), GeneMine (Molecular Applications Group), Look (Molecular
Applications Group),
MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and
BLASTX
(Altschul et al, J. Mol.. Biol. 215: 403 (1990)), FASTA (Pearson and Lipman,
Proc. Natl. Acad. Sci. USA. 85:
2444 (1988)), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),
Catalyst (Molecular
Simulations Inc.), CatalysdSHAPE (Molecular Simulations Inc.),
CeriusZ.DBAccess (Molecular Simulations
Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular
Simulations Inc.), Discover (Molecular
Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular
Simulations Inc.), Delphi,
(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology
(Molecular Simulations
Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations
Inc.), Quanta/Protein Design
(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab
Diversity Explorer (Molecular
Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold
(Molecular Simulations Inc.), the
EMBL/Swissprotein database, the MDL Available Chemicals Directory database,
the MDL Drug Data
Report data base, the Comprehensive Medicinal Chemistry database, Derwents's
World Drug Index
database, the BioByteMasterFile database, the Genbank database, and the
Genseqn database. Many other
programs and data bases would be apparent to one of skill in the art given the
present disclosure.
[0558] Motifs which may be detected using the above programs include sequences
encoding
leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination
sites, alpha helices, and beta
sheets, signal sequences encoding signal peptides which direct the secretion
of the encoded proteins,
sequences implicated in transcription regulation such as homeoboxes, acidic
stretches, enzymatic active
sites, substrate binding sites, and enzymatic cleavage sites.
EXAMPLE 60
Methods of Making Nucleic Acids
[0559] The present invention also comprises methods of making the EST-related
nucleic acids,
fragments of EST-related nucleic acids, positional segments of t:he EST-
related nucleic acids, or
fragments of positional segments of the EST-related nucleic acids. The methods
comprise sequentially
linking together nucleotides to produce the nucleic acids having the preceding
sequences. A variety of
methods of synthesizing nucleic acids are known to those skilled in the art.
[0560] In many of these methods, synthesis is conducted on a solid support.
These included the
3' phosphoramidite methods in which the 3' terminal base of the desired
oligonucleotide is immobilized
on an insoluble carrier. The nucleotide base to be added is blocked at the 5'
hydroxyl and activated at the
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3' hydroxyl so as to cause coupling with the immobilized nucleotide base.
Deblocking of the new
immobilized nucleotide compound and repetition of the cycle will produce the
desired polynucleotide.
Alternatively, polynucleotides may be prepared as described in U.S. Patent No.
5,049,656, the disclosure
of which is incorporated herein by reference. In some embodiments, several
polynucleotides prepared as
described above are ligated together to generate longer polynucleotides having
a desired sequence.
EXAMPLE 6l
Methods of Making Polypeptides
[0561] The present invention also comprises methods of making the
polynucleotides encoded by
EST-related nucleic acids, fragments of EST-related nucleic acids, positional
segments of the EST-related
nucleic acids, or fragments of positional segments of the EST-related nucleic
acids and methods of
making the EST-related polypeptides, fragments of EST-related polypeptides,
positional segments of
EST-related polypeptides, or fragments of EST-related polypeptides. The
methods comprise sequentially
linking together amino acids to produce the nucleic polypeptides having the
preceding sequences. In
some embodiments, the polypeptides made by these methods are 150 amino acid or
less in length. In
other embodiments, the polypeptides made by these methods are 120 amino acids
or less in length.
[0562] A variety of methods of making polypeptides are known to those skilled
in the art,
including methods in which the carboxyl terminal amino acid is bound to
polyvinyl benzene or another
suitable resin. The amino acid to be added possesses blocking groups on its
amino moiety and any side
chain reactive groups so that only its carboxyl moiety can react. The carboxyl
group is activated with
carbodiimide or another activating agent and allowed to couple to the
immobilized amino acid. After
removal of the blocking group, the cycle is repeated to generate a polypeptide
having the desired
sequence. Alternatively, the methods described in U.S. Patent No. 5,049,656,
the disclosure of which is
incorporated herein by reference, may be used.
[0563] As discussed above, the EST-related nucleic acids, fragments of the EST-
related nucleic
acids, positional segments of the EST-related nucleic acids, or fragments of
positional segments of the
EST-related nucleic acids can be used for various purposes. The
polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use;
production of secreted polypeptides or
chimeric polypeptides, antibody production, as markers for tissues in which
the corresponding protein is
preferentially expressed (either constitutively or at a particular stage of
tissue differentiation or development
or in disease states); as molecular weight markers on Southern gels; as
chromosome markers or tags (when
labeled) to identify chromosomes or to map related gene positions; to compare
with endogenous DNA
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sequences in patients to identify potential genetic disorders; as probes to
hybridize and thus discover novel,
related DNA sequences; as a source of information to derive PCR primers for
genetic fingerprinting; for
selecting and making oligomers for attachment to a "gene chip" or other
support, including for examination
for expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an
antigen to raise anti-DNA antibodies or elicit another immune response. Where
the polynucleotide encodes a
protein or polypeptide which binds or potentially binds to another protein or
polypeptide (such as, for
example, in a receptor-ligand interaction), the polynucleotide can also be
used in interaction trap assays (such
as, for example, that described in Gyuris et al., Cell 75:791-803 (1993), the
disclosure of which is hereby
incorporated by reference) to identify polynucleotides encoding the other
protein or polypeptide with which
binding occurs or to identify inhibitors of the binding interaction.
[0564] The proteins or polypeptides provided by the present invention can
similarly be used in
assays to determine biological activity, including in a panel of multiple
proteins for high-throughput
screening; to raise antibodies or to elicit another immune response; as a
reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of the protein
(or its receptor) in biological
fluids; as markers for tissues in which the corresponding protein is
preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in a disease state); and, of
course, to isolate correlative receptors or ligands. Where the protein or
polypeptide binds or potentially binds
to another protein or polypeptide (such as, for example, in a receptor-ligand
interaction), the protein can be
used to identify the other protein with which binding occurs or to identify
inhibitors of the binding
interaction. Proteins or polypeptides involved in these binding interactions
can also be used to screen for
peptide or small molecule inhibitors or agonists of the binding interaction.
[0565] Any or all of these research utilities are capable of being developed
into reagent grade or kit
format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in the art. References
disclosing such methods include without limitation "Molecular Cloning; A
Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis
eds., 1989, and "Methods in
Enzymology; Guide to Molecular Cloning Techniques", Academic Press, Bergen
S.L. and A.R. Kimmel
eds., 1987.
[0566] Polynucleotides and proteins or polypeptides of the present invention
can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a
source of carbohydrate. In such
cases the protein or polynucleotide of the invention can be added to the feed
of a particular organism or can
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be administered as a separate solid or liquid preparation, such as in the form
of powder, pills, solutions,
suspensions or capsules. In the case of microorganisms, the protein or
polynucleotide of the invention can be
added to the medium in or on which the microorganism is cultured.
[0567] Although this invention has been described in terms of certain
preferred embodiments, other
embodiments which will be apparent to those of ordinary skill in the art in
view of the disclosure herein are
also within the scope of this invention. Accordingly, the scope of the
invention is intended to be defined only
by reference to the appended claims. All documents cited herein are
incorporated herein by reference in their
entirety.
