U.S. patent application number 10/930331 was filed with the patent office on 2005-05-19 for complementary dnas.
Invention is credited to Bougueleret, Lydie, Duclert, Aymeric, Edwards, Jean-Baptiste Dumas Milne.
Application Number | 20050106599 10/930331 |
Document ID | / |
Family ID | 27491098 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106599 |
Kind Code |
A1 |
Edwards, Jean-Baptiste Dumas Milne
; et al. |
May 19, 2005 |
Complementary DNAs
Abstract
The sequences of extended cDNAs encoding secreted proteins are
disclosed. The extended cDNAs can be used to express secreted
proteins or portions thereof or to obtain antibodies capable of
specifically binding to the secreted proteins. The extended cDNAs
may also be used in diagnostic, forensic, gene therapy, and
chromosome mapping procedures. The extended cDNAs may also be used
to design expression vectors and secretion vectors.
Inventors: |
Edwards, Jean-Baptiste Dumas
Milne; (Paris, FR) ; Duclert, Aymeric; (Saint
Maur, FR) ; Bougueleret, Lydie; (Vanves, FR) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
27491098 |
Appl. No.: |
10/930331 |
Filed: |
August 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10930331 |
Aug 30, 2004 |
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09903190 |
Jul 11, 2001 |
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09903190 |
Jul 11, 2001 |
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09247155 |
Feb 9, 1999 |
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6312922 |
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60074121 |
Feb 9, 1998 |
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60081563 |
Apr 13, 1998 |
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60096116 |
Aug 10, 1998 |
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60099273 |
Sep 4, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/183; 435/320.1; 435/325; 435/69.1; 530/350; 536/23.2 |
Current CPC
Class: |
Y02A 90/26 20180101;
C07K 14/47 20130101; C07K 14/705 20130101; Y02A 90/10 20180101;
A61K 48/00 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/183; 435/320.1; 435/325; 530/350; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C07K 014/47 |
Claims
We claim:
1. A purified or isolated nucleic acid comprising: a) at least 10
consecutive bases of one of SEQ ID NOs: 40-84, 130-154, or a
sequence complementary thereto; b) the sequence of one of SEQ ID
NOs: 40-84, 130-154, or a sequence complementary thereto; c) the
full coding sequence of one of SEQ ID NOs: 40-59, 61-73, 75, 77-82
or 130-154 wherein the full coding sequence comprises the sequence
encoding signal peptide and the sequence encoding mature protein;
d) the nucleotides of one of SEQ ID NOs: 40-59, 61-73, 75-82, 84 or
130-154 which encode a signal peptide; e) the nucleotides of one of
SEQ ID NOs: 40-59, 61-73, 75-82, 84 or 130-154 which encode a
mature peptide; f) a polynucleotide sequence encoding at least 10
amino acids of a polypeptide having the sequence of one of the
sequences of SEQ ID NOs: 85-129 or 155-179; g) a polynucleotide
sequence encoding a polypeptide having the sequence of one of the
sequences of SEQ ID NOs: 85-104, 106-120, 122-127 or 155-179; h) a
polynucleotide sequence encoding a signal peptide included in one
of the sequences of SEQ ID NOs: 85-104, 106-118, 120-127, 129 or
155-179; or i) a vector comprising a polynucleotide as set forth in
1a), 1b), 1c), 1d), 1e), 1f) 1g) or 1h).
2. A purified or isolated polypeptide comprising: a) at least 10
consecutive amino acids of one of the sequences of SEQ ID NOs:
85-129 or 155-179; b) the full length sequence of one of SEQ ID
NOs: 85-129 or 155-179; or c) a signal peptide of one of the
polypeptides of SEQ ID NOs: 85-104, 106-118, 120-127, 129 or
155-179;
3. A host cell comprising a polynucleotide or vector according to
claim 1.
4. A method of making a protein comprising one of the sequences of
SEQ ID NO: 85-129 and 155-179, comprising the steps of: a) growing
a host cell according to claim 3 under conditions whereby said
protein is expressed, and b) isolating said protein.
5. In an array of polynucleotides of at least 15 nucleotides in
length, the improvement comprising inclusion in said array of at
least one of the sequences of SEQ ID NOs: 40-84 and 130-154, or one
of the sequences complementary to the sequences of SEQ ID NOs:
40-84 and 130-154, or a fragment thereof of at least 15 consecutive
nucleotides.
6. A purified or isolated antibody capable of binding to a
polypeptide comprising at least 10 consecutive amino acids of the
sequence of one of SEQ ID NOs: 85-129 or 155-179.
7. A method of binding an antibody to an antigen comprising
contacting an antibody capable of binding to a polypeptide
comprising at least 10 consecutive amino acids of the sequence of
one of SEQ ID NOs: 85-129 or 155-179 with a polypeptide comprising
at least 10 consecutive amino acids of the sequence of one of SEQ
ID NOs: 85-129 or 155-179.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 09/247,155 filed on Feb. 9, 1999, which
claims priority from U.S. Provisional Patent Application Ser. No.
60/074,121 filed Feb. 9, 1998, U.S. Provisional Patent Application
Ser. No. 60/081,563, filed Apr. 13, 1998, U.S. Provisional Patent
Application Ser. No. 60/096,116, filed Aug. 10, 1998, and U.S.
Provisional Patent Application Ser. No. 60/099,273 filed Sep. 4,
1998, the disclosures of which are incorporated herein by reference
in their entirety.
[0002] Table I lists the SEQ ID Nos. of the extended cDNAs in the
present application, the SEQ ID Nos. of the extended cDNAs in the
provisional applications, and the identities of the provisional
applications in which the extended cDNAs were disclosed.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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. Cloning vectors such as yeast artificial chromosomes
(YACs) and bacterial artificial chromosomes (BACs) are able to
accept DNA inserts ranging from 300 to 1000 kilobases (kb) or
100-400 kb in length respectively, thereby facilitating the
manipulation and ordering of DNA sequences distributed over great
distances on the human chromosomes. Automated DNA sequencing
machines permit the rapid sequencing of human genes. Bioinformatics
software enables the comparison of nucleic acid and protein
sequences, thereby assisting in the characterization of human gene
products.
[0005] 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 using bio-informatics
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 bio-informatics
software may mischaracterize the genomic sequences obtained. Thus,
the software may produce false positives in which non-coding DNA is
mischaracterized as coding DNA or false negatives in which coding
DNA is mislabeled as non-coding DNA.
[0006] 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.
[0007] In the past, the short EST sequences which could be used to
isolate or purify extended cDNAs 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 377:174, 1996, Hillier et al., Genome
Res. 6:807-828, 1996).
[0008] 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 of the mRNA from which the cDNA is derived.
Such incomplete sequences 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 which can be
used to obtain extended cDNAs which may include the 5' sequences
contained in the 5' ESTs.
[0009] 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. Of the 50,000-100,000 protein coding genes, those
genes encoding proteins which are secreted from the cell in which
they are synthesized, 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.
[0010] In fact, several secretory proteins, including tissue
plasminogen activator, G-CSF, GM-CSF, erythropoietin, human growth
hormone, insulin, interferon-.alpha., interferon-.beta.,
interferon-.gamma., and 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 particularly valuable source of
therapeutic agents. Thus, there is a need for the identification
and characterization of secreted proteins and the nucleic acids
encoding them.
[0011] In addition to being therapeutically useful themselves,
secretory proteins include short peptides, called signal peptides,
at their arnino 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.
Because these signal peptides will direct the extracellular
secretion of any protein to which they are operably linked, the
signal sequences may be exploited to direct the efficient secretion
of any protein by operably linking the signal sequences to a gene
encoding the protein for which secretion is desired. This may prove
beneficial in gene therapy strategies in which it is desired to
deliver a particular gene product to cells other than the cell 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 5' portions of the genes for secretory proteins
which encode signal peptides.
[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, S. H. et al.,
Purification of CpG Islands using a Methylated DNA Binding Column,
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 specificity or of comprehensiveness.
[0013] 5' ESTs and extended cDNAs obtainable therefrom may be used
to efficiently identify and isolate upstream regulatory regions
which control the location, developmental stage, rate, and quantity
of protein synthesis, as well as the stability of the mRNA. (Theil
et al., BioFactors 4:87-93, (1993). 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.
[0014] In addition, ESTs containing the 5' ends of secretory
protein genes or extended cDNAs which include sequences adjacent to
the sequences of the ESTs 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 encoding secretory
proteins.
SUMMARY OF THE INVENTION
[0015] The present invention relates to purified, isolated, or
recombinant extended cDNAs which encode secreted proteins or
fragments thereof Preferably, the purified, isolated or recombinant
cDNAs contain the entire open reading frame of their corresponding
mRNAs, including a start codon and a stop codon. For example, the
extended cDNAs may include nucleic acids encoding the signal
peptide as well as the mature protein. Alternatively, the extended
cDNAs may contain a fragment of the open reading frame. In some
embodiments, the fragment may encode only the sequence of the
mature protein. Alternatively, the fragment may encode only a
portion of the mature protein. A further aspect of the present
invention is a nucleic acid which encodes the signal peptide of a
secreted protein.
[0016] The present extended cDNAs were obtained using ESTs which
include sequences derived from the authentic 5' ends of their
corresponding mRNAs. As used herein the terms "EST" or "5' EST"
refer to the short cDNAs which were used to obtain the extended
cDNAs of the present invention. As used herein, the term "extended
cDNA" refers to the cDNAs which include sequences adjacent to the
5' EST used to obtain them. The extended cDNAs may contain all or a
portion of the sequence of the EST which was used to obtain them.
The term "corresponding mRNA" refers to the mRNA which was the
template for the cDNA synthesis which produced the 5' EST. As used
herein, the term "purified" does not require absolute purity;
rather, it is intended as a relative definition. Individual
extended cDNA 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 extended 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 10.sup.4-10.sup.6 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.
[0017] 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.
[0018] As used herein, the term "recombinant" means that the
extended cDNA is adjacent to "backbone" nucleic acid to which it is
not adjacent in its natural environment. Additionally, to be
"enriched" the extended cDNAs will represent 5% 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 extended
cDNAs represent 15% or more of the number of nucleic acid inserts
in the population of recombinant backbone molecules. More
preferably, the enriched extended cDNAs 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
extended cDNAs represent 90% or more of the number of nucleic acid
inserts in the population of recombinant backbone molecules.
"Stringent", "moderate," and "low" hybridization conditions are as
defined in Example 29.
[0019] Unless otherwise indicated, a "complementary" sequence is
fully complementary. Thus, extended cDNAs encoding secreted
polypeptides or fragments thereof which are present in cDNA
libraries in which one or more extended cDNAs encoding secreted
polypeptides or fragments thereof make up 5% or more of the number
of nucleic acid inserts in the backbone molecules are "enriched
recombinant extended cDNAs" as defined herein. Likewise, extended
cDNAs encoding secreted polypeptides or fragments thereof which are
in a population of plasmids in which one or more extended cDNAs of
the present invention have been inserted such that they represent
5% or more of the number of inserts in the plasmid backbone are
"enriched recombinant extended cDNAs" as defined herein. However,
extended cDNAs encoding secreted polypeptides or fragments thereof
which are in cDNA libraries in which the extended cDNAs encoding
secreted polypeptides or fragments thereof 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
cDNA insert encoding a secreted polypeptide are extremely rare, are
not "enriched recombinant extended cDNAs."
[0020] In particular, the present invention relates to extended
cDNAs which were 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.
[0021] Extended cDNAs encoding secreted proteins may include
nucleic acid sequences, called signal sequences, which encode
signal peptides which direct the extracellular secretion of the
proteins encoded by the extended cDNAs. Generally, the signal
peptides are located at the amino termini of secreted proteins.
[0022] 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.
[0023] The extended cDNAs of the present invention have several
important applications. For example, they may be used to express
the entire secreted protein which they encode. Alternatively, they
may be used to express portions of the secreted protein. The
portions may comprise the signal peptides encoded by the extended
cDNAs or the mature proteins encoded by the extended cDNAs (i.e.
the proteins generated when the signal peptide is cleaved off). The
portions may also comprise polypeptides having at least 10
consecutive amino acids encoded by the extended cDNAs.
Alternatively, the portions may comprise at least 15 consecutive
amino acids encoded by the extended cDNAs. In some embodiments, the
portions may comprise at least 25 consecutive amino acids encoded
by the extended cDNAs. In other embodiments, the portions may
comprise at least 40 amino acids encoded by the extended cDNAs.
[0024] Antibodies which specifically recognize the entire secreted
proteins encoded by the extended cDNAs or fragments thereof having
at least 10 consecutive amino acids, at least 15 consecutive amino
acids, at least 25 consecutive amino acids, or at least 40
consecutive amino acids may also be obtained as described below.
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 encoded by the extended cDNAs may
also be obtained.
[0025] In some embodiments, the extended cDNAs include the signal
sequence. In other embodiments, the extended cDNAs may include the
full coding sequence for the mature protein (i.e. the protein
generated when the signal polypeptide is cleaved off). In addition,
the extended cDNAs 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. As discussed above, secreted proteins are
therapeutically important. Thus, the proteins expressed from the
cDNAs may be useful in treating or controlling a variety of human
conditions. The extended cDNAs may also be used to obtain the
corresponding genomic DNA. The term "corresponding genomic DNA"
refers to the genomic DNA which encodes mRNA which includes the
sequence of one of the strands of the extended cDNA in which
thymidine residues in the sequence of the extended cDNA are
replaced by uracil residues in the mRNA.
[0026] The extended cDNAs or genomic DNAs obtained therefrom may be
used in forensic procedures to identify individuals or in
diagnostic procedures to identify individuals having genetic
diseases resulting from abnormal expression of the genes
corresponding to the extended cDNAs. In addition, the present
invention is useful for constructing a high resolution map of the
human chromosomes.
[0027] The present invention also relates to secretion vectors
capable of directing the secretion of a protein of interest. Such
vectors may be used in 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.
[0028] 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 extended cDNAs such
as promoters or upstream regulatory sequences.
[0029] In addition, the present invention may also be used for gene
therapy to control or treat genetic diseases. Signal peptides may
also be fused to heterologous proteins to direct their
extracellular secretion.
[0030] One embodiment of the present invention is a purified or
isolated nucleic acid comprising the sequence of one of SEQ ID NOs:
40-84 and 130-154 or a sequence complementary thereto. In one
aspect of this embodiment, the nucleic acid is recombinant.
[0031] Another embodiment of the present invention is a purified or
isolated nucleic acid comprising at least 10 consecutive bases of
the sequence of one of SEQ ID NOs: 40-84 and 130-154 or one of the
sequences complementary thereto. In one aspect of this embodiment,
the nucleic acid comprises at least 15, 25, 30, 40, 50, 75, or 100
consecutive bases of one of the sequences of SEQ ID NOs: 40-84 and
130-154 or one of the sequences complementary thereto. The nucleic
acid may be a recombinant nucleic acid.
[0032] Another embodiment of the present invention is a purified or
isolated nucleic acid of at least 15 bases capable of hybridizing
under stringent conditions to the sequence of one of SEQ ID NOs:
40-84 and 130-154 or a sequence complementary to one of the
sequences of SEQ ID NOs: 40-84 and 130-154. In one aspect of this
embodiment, the nucleic acid is recombinant.
[0033] Another embodiment of the present invention is a purified or
isolated nucleic acid comprising the full coding sequences of one
of SEQ ID Nos: 40-84 and 130-154 wherein the full coding sequence
optionally comprises the sequence encoding signal peptide as well
as the sequence encoding mature protein. In a preferred embodiment,
the isolated or purified nucleic acid comprises the full coding
sequence of one of SEQ ID Nos. 40-59, 61-73, 75, 77-82, and 130-154
wherein the full coding sequence comprises the sequence encoding
signal peptide and the sequence encoding mature protein. In one
aspect of this embodiment, the nucleic acid is recombinant.
[0034] A further embodiment of the present invention is a purified
or isolated nucleic acid comprising the nucleotides of one of SEQ
ID NOs: 40-84 and 130-154 which encode a mature protein. In a
preferred embodiment, the purified or isolated nucleic acid
comprises the nucleotides of one of SEQ ID NOs: 40-59, 61-75,
77-82, and 130-154 which encode a mature protein. In one aspect of
this embodiment, the nucleic acid is recombinant.
[0035] Yet another embodiment of the present invention is a
purified or isolated nucleic acid comprising the nucleotides of one
of SEQ ID NOs: 40-84 and 130-154 which encode the signal peptide.
In a preferred embodiment, the purified or isolated nucleic acid
comprises the nucleotides of SEQ ID NOs: 40-59, 61-73, 75-82, 84,
and 130-154 which encode the signal peptide. In one aspect of this
embodiment, the nucleic acid is recombinant.
[0036] Another embodiment of the present invention is a purified or
isolated nucleic acid encoding a polypeptide having the sequence of
one of the sequences of SEQ ID NOs: 85-129 and 155-179.
[0037] Another embodiment of the present invention is a purified or
isolated nucleic acid encoding a polypeptide having the sequence of
a mature protein included in one of the sequences of SEQ ID NOs:
85-129 and 155-179. In a preferred embodiment, the purified or
isolated nucleic acid encodes a polypeptide having the sequence of
a mature protein included in one of the sequences of SEQ ID NOs:
85-104, 106-120, 122-127, and 155-179.
[0038] Another embodiment of the present invention is a purified or
isolated nucleic acid encoding a polypeptide having the sequence of
a signal peptide included in one of the sequences of SEQ ID NOs:
85-129 and 155-179. In a preferred embodiment, the purified or
isolated nucleic acid encodes a polypeptide having the sequence of
a signal peptide included in one of the sequences of SEQ ID NOs:
85-104, 106-118, 120-127, 129, and 155-179.
[0039] Yet another embodiment of the present invention is a
purified or isolated protein comprising the sequence of one of SEQ
ID NOs: 85-129 and 155-179.
[0040] Another embodiment of the present invention is a purified or
isolated polypeptide comprising at least 10 consecutive amino acids
of one of the sequences of SEQ ID NOs: 85-129 and 155-179. In one
aspect of this embodiment, the purified or isolated polypeptide
comprises at least 15, 20, 25, 35, 50, 75, 100, 150 or 200
consecutive amino acids of one of the sequences of SEQ ID NOs:
85-129 and 155-179. In still another aspect, the purified or
isolated polypeptide comprises at least 25 consecutive amino acids
of one of the sequences of SEQ ID NOs: 85-129 and 155-179.
[0041] Another embodiment of the present invention is an isolated
or purified polypeptide comprising a signal peptide of one of the
polypeptides of SEQ ID NOs: 85-129 and 155-179. In a preferred
embodiment, the isolated or purified polypeptide comprises a signal
peptide of one of the polypeptides of SEQ ID NOs: 85-104, 106-118,
120-127, 129, and 155-179.
[0042] Yet another embodiment of the present invention is an
isolated or purified polypeptide comprising a mature protein of one
of the polypeptides of SEQ ID NOs: 85-129 and 155-179. In a
preferred embodiment, the isolated or purified polypeptide
comprises a mature protein of one of the polypeptides of SEQ ID
NOs: 85-104, 106-120, 122-127, and 155-179. In a preferred
embodiment, the purified or isolated nucleic acid encodes a
polypeptide having the sequence of a mature protein included in one
of the sequences of SEQ ID NOs: 85-104, 106-120, 122-127, and
155-179.
[0043] A further embodiment of the present invention is a method of
making a protein comprising one of the sequences of SEQ ID NO:
85-129 and 155-179, comprising the steps of obtaining a cDNA
comprising one of the sequences of sequence of SEQ ID NO: 40-84 and
130-154, inserting the cDNA in an expression vector such that the
cDNA is operably linked to a promoter, and introducing the
expression vector into a host cell whereby the host cell produces
the protein encoded by said cDNA. In one aspect of this embodiment,
the method further comprises the step of isolating the protein.
[0044] Another embodiment of the present invention is a protein
obtainable by the method described in the preceding paragraph.
[0045] Another embodiment of the present invention is a method of
making a protein comprising the amino acid sequence of the mature
protein contained in one of the sequences of SEQ ID NOs: 85-104,
106-120, 122-127, and 155-179 comprising the steps of obtaining a
cDNA comprising one of the nucleotides sequence of sequence of SEQ
ID NOs: 40-59, 61-75, 77-82, and 130-154 which encode for the
mature protein, inserting the cDNA in an expression vector such
that the cDNA is operably linked to a promoter, and introducing the
expression vector into a host cell whereby the host cell produces
the mature protein encoded by the cDNA. In one aspect of this
embodiment, the method further comprises the step of isolating the
protein.
[0046] Another embodiment of the present invention is a mature
protein obtainable by the method described in the preceding
paragraph.
[0047] In a preferred embodiment, the above method comprises a
method of making a protein comprising the amino acid sequence of
the mature protein contained in one of the sequences of SEQ ID NOs.
85-104, 106-120, 122-127 and 155-179, comprising the steps of
obtaining a cDNA comprising one of the nucleotide sequences of SEQ
ID Nos. 40-59, 61-75, 77-82 and 130-154 which encode for the mature
protein, inserting the cDNA in an expression vector such that the
cDNA is operably linked to a promoter, and introducing the
expression vector into a host cell whereby the host cell produces
the mature protein encoded by the cDNA. In one aspect of this
embodiment, the method further comprises the step of isolating the
protein.
[0048] Another embodiment of the present invention is a host cell
containing the purified or isolated nucleic acids comprising the
sequence of one of SEQ ID NOs: 40-84 and 130-154 or a sequence
complementary thereto described herein.
[0049] Another embodiment of the present invention is a host cell
containing the purified or isolated nucleic acids comprising the
full coding sequences of one of SEQ ID NOs: 40-59, 61-73, 75,
77-82, and 130-154, wherein the full coding sequence comprises the
sequence encoding signal peptide and the sequence encoding mature
protein described herein.
[0050] Another embodiment of the present invention is a host cell
containing the purified or isolated nucleic acids comprising the
nucleotides of one of SEQ ID NOs: 40-84 and 130-154 which encode a
mature protein which are described herein. Preferably, the host
cell contains the purified or isolated nucleic acids comprising the
nucleotides of one of SEQ ID NOs: 40-59, 61-75, 77-82, and 130-154
which encode a mature protein.
[0051] Another embodiment of the present invention is a host cell
containing the purified or isolated nucleic acids comprising the
nucleotides of one of SEQ ID NOs: 40-84 and 130-154 which encode
the signal peptide which are described herein. Preferably, the host
cell contains the purified or isolated nucleic acids comprising the
nucleotides of one of SEQ ID Nos.: 40-59, 61-73, 75-82, 84, and
130-154 which encode the signal peptide.
[0052] Another embodiment of the present invention is a purified or
isolated antibody capable of specifically binding to a protein
having the sequence of one of SEQ ID NOs: 85-129 and 155-179. In
one aspect of this embodiment, the antibody is capable of binding
to a polypeptide comprising at least 10 consecutive amino acids of
the sequence of one of SEQ ID NOs: 85-129 and 155-179.
[0053] Another embodiment of the present invention is an array of
cDNAs or fragments thereof of at least 15 nucleotides in length
which includes at least one of the sequences of SEQ ID NOs: 40-84
and 130-154, or one of the sequences complementary to the sequences
of SEQ ID NOs: 40-84 and 130-154, or a fragment thereof of at least
15 consecutive nucleotides. In one aspect of this embodiment, the
array includes at least two of the sequences of SEQ ID NOs: 40-84
and 130-154, the sequences complementary to the sequences of SEQ ID
NOs: 40-84 and 130-154, or fragments thereof of at least 15
consecutive nucleotides. In another aspect of this embodiment, the
array includes at least five of the sequences of SEQ ID NOs: 40-84
and 130-154, the sequences complementary to the sequences of SEQ ID
NOs: 40-84 and 130-154, or fragments thereof of at least 15
consecutive nucleotides.
[0054] A further embodiment of the invention encompasses purified
polynucleotides comprising an insert from a clone deposited in a
deposit having an accession number selected from the group
consisting of the accession numbers listed in Table VI or a
fragment thereof comprising a contiguous span of at least 8, 10,
12, 15, 20, 25, 40, 60, 100, or 200 nucleotides of said insert. An
additional embodiment of the invention encompasses purified
polypeptides which comprise, consist of, or consist essentially of
an amino acid sequence encoded by the insert from a clone deposited
in a deposit having an accession number selected from the group
consisting of the accession numbers listed in Table VI, as well as
polypeptides which comprise a fragment of said amino acid sequence
consisting of a signal peptide, a mature protein, or a contiguous
span of at least 5, 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200
amino acids encoded by said insert.
[0055] An additional embodiment of the invention encompasses
purified polypeptides which comprise a contiguous span of at least
5, 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200 amino acids of SEQ ID
NOs: 85-129 and 155-179, wherein said contiguous span comprises at
least one of the amino acid positions which was not shown to be
identical to a public sequence in any of FIGS. 10 to 12. Also
encompassed by the invention are purified polynucleotides encoding
said polypeptides.
[0056] Another embodiment of the present invention is a computer
readable medium having stored thereon a sequence selected from the
group consisting of a cDNA code of SEQ ID NOs. 40-84 and 130-154
and a polypeptide code of SEQ ID NOs. 85-129 and 155-179.
[0057] Another embodiment of the present invention is a computer
system comprising a processor and a data storage device wherein the
data storage device has stored thereon a sequence selected from the
group consisting of a cDNA code of SEQ ID NOs. 40-84 and 130-154
and a polypeptide code of SEQ ID NOs. 85-129 and 155-179. In some
embodiments 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. In other aspects of
the computer system, the system further comprises an identifier
which identifies features in said sequence.
[0058] Another embodiment of the present invention is a method for
comparing a first sequence to a reference sequence wherein the
first sequence is selected from the group consisting of a cDNA code
of SEQ ID NOs. 40-84 and 130-154 and a polypeptide code of SEQ ID
NOs. 85-129 and 155-179 comprising the steps of reading the first
sequence and the reference sequence through use of a computer
program which compares sequences and
[0059] determining differences between the first sequence and the
reference sequence with the computer program. In some embodiments
of the method, the step of determining differences between the
first sequence and the reference sequence comprises identifying
polymorphisms.
[0060] Another embodiment of the present invention is a method for
identifying a feature in a sequence selected from the group
consisting of a cDNA code of SEQ ID NOs. 40-84 and 130-154 and a
polypeptide code of SEQ ID NOs. 85-129 and 155-179 comprising the
steps of reading the sequence through the use of a computer program
which identifies features in sequences and identifying features in
the sequence with said computer program.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 is a summary of a procedure for obtaining cDNAs which
have been selected to include the 5' ends of the mRNAs from which
they are derived.
[0062] FIG. 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.
[0063] FIG. 3 shows the distribution of von Heijne scores for 5'
ESTs in each of the categories described herein and the probability
that these 5' ESTs encode a signal peptide.
[0064] FIG. 4 shows the distribution of 5' ESTs in each category
and the number of 5' ESTs in each category having a given minimum
von Heijne's score.
[0065] FIG. 5 shows the tissues from which the mRNAs corresponding
to the 5' ESTs in each of the categories described herein were
obtained.
[0066] FIG. 6 illustrates a method for obtaining extended
cDNAs.
[0067] FIG. 7 is a map of pED6dpc2.
[0068] FIG. 8 provides a schematic description of the promoters
isolated and the way they are assembled with the corresponding 5'
tags.
[0069] FIG. 9 describes the transcription factor binding sites
present in each of these promoters.
[0070] FIG. 10 is an alignment of the proteins of SEQ ID NOs: 120
and 180 wherein the signal peptide is in italics, the predicted
transmembrane segment is underlined, the experimentally determined
transmembrane segment is double-underlined, and the ATP1G/PLMN/MAT8
signature is in bold.
[0071] FIG. 11 is an alignment of the proteins of SEQ ID NOs: 121
and 181 wherein the predicted transmembrane segment is
underlined.
[0072] FIG. 12 is an alignment of the proteins of SEQ ID NOs: 128
and 182 wherein the PPPY motif is in bold.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0073] I. Obtaining 5' ESTs
[0074] The present extended cDNAs were obtained using 5' ESTs which
were isolated as described below.
[0075] A. Chemical Methods for Obtaining mRNAs having Intact 5'
Ends
[0076] In order to obtain the 5' ESTs used to obtain the extended
cDNAs of the present invention, mRNAs having intact 5' ends must be
obtained. Currently, there are two approaches for obtaining such
mRNAs. One of these approaches is a chemical modification method
involving derivatization of the 5' ends of the mRNAs and selection
of the derivatized mRNAs. The 5' ends of eucaryotic mRNAs possess a
structure referred to as a "cap" which comprises a guanosine
methylated at the 7 position. The cap is joined to the first
transcribed base of the mRNA by a 5',5'-triphosphate bond. In some
instances, the 5' guanosine is methylated in both the 2 and 7
positions. Rarely, the 5' guanosine is trimethylated at the 2,7 and
7 positions. In the chemical method for obtaining mRNAs having
intact 5' ends, the 5' cap is specifically derivatized and coupled
to a reactive group on an immobilizing substrate. This specific
derivatization is based on the fact that only the ribose linked to
the methylated guanosine at the 5' end of the mRNA and the ribose
linked to the base at the 3' terminus of the mRNA, possess
2',3'-cis diols. Optionally, where the 3' terminal ribose has a
2',3'-cis diol, the 2',3'-cis diol at the 3' end may be chemically
modified, substituted, converted, or eliminated, leaving only the
ribose linked to the methylated guanosine at the 5' end of the mRNA
with a 2',3'-cis diol. A variety of techniques are available for
eliminating the 2',3'-cis diol on the 3' terminal ribose. For
example, controlled alkaline hydrolysis may be used to generate
mRNA fragments in which the 3' terminal ribose is a 3'-phosphate,
2'-phosphate or (2',3')-cyclophosphate. Thereafter, the fragment
which includes the original 3' ribose may be eliminated from the
mixture through chromatography on- an oligo-dT column.
Alternatively, a base which lacks the 2',3'-cis diol may be added
to the 3' end of the mRNA using an RNA ligase such as T4 RNA
ligase. Example 1 below describes a method for ligation of pCp to
the 3' end of messenger RNA.
EXAMPLE 1
Ligation of the Nucleoside Diphosphate pCp to the 3' End of
Messenger RNA
[0077] 1 .mu.g of RNA was incubated in a final reaction medium of
10 .mu.l in the presence of 5 U of T.sub.4 phage RNA ligase in the
buffer provided by the manufacturer (Gibco-BRL), 40 U of the RNase
inhibitor RNasin (Promega) and, 2 .mu.l of .sup.32pCp (Amersham #PB
10208).
[0078] The incubation was performed at 37.degree. C. for 2 hours or
overnight at 7-8.degree. C.
[0079] Following modification or elimination of the 2',3'-cis diol
at the 3' ribose, the 2',3'-cis diol present at the 5' end of the
mRNA may be oxidized using reagents such as NaBH.sub.4,
NaBH.sub.3CN, or sodium periodate, thereby converting the 2',3'-cis
diol to a dialdehyde. Example 2 describes the oxidation of the
2',3'-cis diol at the 5' end of the mRNA with sodium periodate.
EXAMPLE 2
Oxidation of 2',3'-cis diol at the 5' End of the mRNA
[0080] 0.1 OD unit of either a capped oligoribonucleotide of 47
nucleotides (including the cap) or an uncapped oligoribonucleotide
of 46 nucleotides were treated as follows. The oligoribonucleotides
were produced by in vitro transcription using the transcription kit
"AmpliScribe T7" (Epicentre Technologies). As indicated below, the
DNA template for the RNA transcript contained a single cytosine. To
synthesize the uncapped RNA, all four NTPs were included in the in
vitro transcription reaction. To obtain the capped RNA, GTP was
replaced by an analogue of the cap, m7G(5')ppp(5')G. This compound,
recognized by polymerase, was incorporated into the 5' end of the
nascent transcript during the step of initiation of transcription
but was not capable of incorporation during the extension step.
Consequently, the resulting RNA contained a cap at its 5' end. The
sequences of the oligoribonucleotides produced by the in vitro
transcription reaction were:
1 (SEQ ID NO:1) +Cap: 5'm7GpppGCAUCCUACUCCCAUCCAAUU-
CCACCCUAACUCCUCCCAUCU CCAC-3' (SEQ ID NO:2) -Cap:
5'-pppGCAUCCUACUCCCAUCCAAUUCCACCCUAACUCCUCCCAUCUCC AC-3'
[0081] The oligoribonucleotides were dissolved in 9 .mu.l of
acetate buffer (0.1 M sodium acetate, pH 5.2) and 3 .mu.l of
freshly prepared 0.1 M sodium periodate solution. The mixture was
incubated for 1 hour in the dark at 4.degree. C. or room
temperature. Thereafter, the reaction was stopped by adding 4 .mu.l
of 10% ethylene glycol. The product was ethanol precipitated,
resuspended in 10 .mu.l or more of water or appropriate buffer and
dialyzed against water.
[0082] The resulting aldehyde groups may then be coupled to
molecules having a reactive amine group, such as hydrazine,
carbazide, thiocarbazide or semicarbazide groups, in order to
facilitate enrichment of the 5' ends of the mRNAs. Molecules having
reactive amine groups which are suitable for use in selecting mRNAs
having intact 5' ends include avidin, proteins, antibodies,
vitamins, ligands capable of specifically binding to receptor
molecules, or oligonucleotides. Example 3 below describes the
coupling of the resulting dialdehyde to biotin.
EXAMPLE 3
Coupling of the Dialdehyde with Biotin
[0083] The oxidation product obtained in Example 2 was dissolved in
50 .mu.l of sodium acetate at a pH of between 5 and 5.2 and 50
.mu.l of freshly prepared 0.02 M solution of biotin hydrazide in a
methoxyethanol/water mixture (1:1) of formula: 1
[0084] In the compound used in these experiments, n=5. However, it
will be appreciated that other commercially available hydrazides
may also be used, such as molecules of the formula above in which n
varies from 0 to 5.
[0085] The mixture was then incubated for 2 hours at 37.degree. C.
Following the incubation, the mixture was precipitated with ethanol
and dialyzed against distilled water.
[0086] Example 4 demonstrates the specificity of the biotinylation
reaction.
EXAMPLE 4
Specificity of Biotinylation
[0087] The specificity of the biotinylation for capped mRNAs was
evaluated by gel electrophoresis of the following samples:
[0088] Sample 1. The 46 nucleotide uncapped in vitro transcript
prepared as in Example 2 and labeled with .sup.32pCp as described
in Example 1.
[0089] Sample 2. The 46 nucleotide uncapped in vitro transcript
prepared as in Example 2, labeled with .sup.32pCp as described in
Example 1, treated with the oxidation reaction of Example 2, and
subjected to the biotinylation conditions of Example 3.
[0090] Sample 3. The 47 nucleotide capped in vitro transcript
prepared as in Example 2 and labeled with .sup.32pCp as described
in Example 1.
[0091] Sample 4. The 47 nucleotide capped in vitro transcript
prepared as in Example 2, labeled with .sup.32pCp as described in
Example 1, treated with the oxidation reaction of Example 2, and
subjected to the biotinylation conditions of Example 3.
[0092] Samples 1 and 2 had identical migration rates, demonstrating
that the uncapped RNAs were not oxidized and biotinylated. Sample 3
migrated more slowly than Samples 1 and 2, while Sample 4 exhibited
the slowest migration. The difference in migration of the RNAs in
Samples 3 and 4 demonstrates that the capped RNAs were specifically
biotinylated.
[0093] In some cases, mRNAs having intact 5' ends may be enriched
by binding the molecule containing a reactive amine group to a
suitable solid phase substrate such as the inside of the vessel
containing the mRNAs, magnetic beads, chromatography matrices, or
nylon or nitrocellulose membranes. For example, where the molecule
having a reactive amine group is biotin, the solid phase substrate
may be coupled to avidin or streptavidin. Alternatively, where the
molecule having the reactive amine group is an antibody or receptor
ligand, the solid phase substrate may be coupled to the cognate
antigen or receptor. Finally, where the molecule having a reactive
amine group comprises an oligonucleotide, the solid phase substrate
may comprise a complementary oligonucleotide.
[0094] The mRNAs having intact 5' ends may be released from the
solid phase following the enrichment procedure. For example, where
the dialdehyde is coupled to biotin hydrazide and the solid phase
comprises streptavidin, the mRNAs may be released from the solid
phase by simply heating to 95 degrees Celsius in 2% SDS. In some
methods, the molecule having a reactive amine group may also be
cleaved from the mRNAs having intact 5' ends following enrichment.
Example 5 describes the capture of biotinylated mRNAs with
streptavidin coated beads and the release of the biotinylated mRNAs
from the beads following enrichment.
EXAMPLE 5
Canture and Release of Biotinylated mRNAs Using Strepatividin
Coated Beads
[0095] The streptavidin-coated magnetic beads were prepared
according to the manufacturer's instructions (CPG Inc., USA). The
biotinylated mRNAs were added to a hybridization buffer (1.5 M
NaCl, pH 5-6). After incubating for 30 minutes, the unbound and
nonbiotinylated material was removed. The beads were washed several
times in water with 1% SDS. The beads obtained were incubated for
15 minutes at 95.degree. C. in water containing 2% SDS.
[0096] Example 6 demonstrates the efficiency with which
biotinylated mRNAs were recovered from the streptavidin coated
beads.
EXAMPLE 6
Efficiency of Recovery of Biotinylated mRNAs
[0097] The efficiency of the recovery procedure was evaluated as
follows. RNAs were labeled with .sup.32pCp, oxidized, biotinylated
and bound to streptavidin coated beads as described above.
Subsequently, the bound RNAs were incubated for 5, 15 or 30 minutes
at 95.degree. C. in the presence of 2% SDS.
[0098] The products of the reaction were analyzed by
electrophoresis on 12% polyacrylamide gels under denaturing
conditions (7 M urea). The gels were subjected to autoradiography.
During this manipulation, the hydrazone bonds were not reduced.
[0099] Increasing amounts of nucleic acids were recovered as
incubation times in 2% SDS increased, demonstrating that
biotinylated mRNAs were efficiently recovered.
[0100] In an alternative method for obtaining mRNAs having intact
5' ends, an oligonucleotide which has been derivatized to contain a
reactive amine group is specifically coupled to mRNAs having an
intact cap. Preferably, the 3' end of the mRNA is blocked prior to
the step in which the aldehyde groups are joined to the derivatized
oligonucleotide, as described above, so as to prevent the
derivatized oligonucleotide from being joined to the 3' end of the
mRNA. For example, pCp may be attached to the 3' end of the mRNA
using T4 RNA ligase. However, as discussed above, blocking the 3'
end of the mRNA is an optional step. Derivatized oligonucleotides
may be prepared as described below in Example 7.
EXAMPLE 7
Derivatization of the Oligonucleotide
[0101] An oligonucleotide phosphorylated at its 3' end was
converted to a 3' hydrazide in 3' by treatment with an aqueous
solution of hydrazine or of dihydrazide of the formula
H.sub.2N(R1)NH.sub.2 at about 1 to 3 M, and at pH 4.5, in the
presence of a carbodiimide type agent soluble in water such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a final
concentration of 0.3 M at a temperature of 8.degree. C.
overnight.
[0102] The derivatized oligonucleotide was then separated from the
other agents and products using a standard technique for isolating
oligonucleotides.
[0103] As discussed above, the mRNAs to be enriched may be treated
to eliminate the 3' OH groups which may be present thereon. This
may be accomplished by enzymatic ligation of sequences lacking a 3'
OH, such as pCp, as described above in Example 1. Alternatively,
the 3' OH groups may be eliminated by alkaline hydrolysis as
described in Example 8 below.
EXAMPLE 8
Alkaline Hydrolysis of mRNA
[0104] The mRNAs may be treated with alkaline hydrolysis as
follows. In a total volume of 100 .mu.l of 0.1N sodium hydroxide,
1.5 .mu.g mRNA is incubated for 40 to 60 minutes at 4.degree. C.
The solution is neutralized with acetic acid and precipitated with
ethanol.
[0105] Following the optional elimination of the 3' OH groups, the
diol groups at the 5' ends of the mRNAs are oxidized as described
below in Example 9.
EXAMPLE 9
Oxidation of Diols
[0106] Up to 1 OD unit of RNA was dissolved in 9 .mu.l of buffer
(0.1 M sodium acetate, pH 6-7 or water) and 3 .mu.l of freshly
prepared 0.1 M sodium periodate solution. The reaction was
incubated for 1 h in the dark at 4.degree. C. or room temperature.
Following the incubation, the reaction was stopped by adding 4
.mu.l of 10% ethylene glycol. Thereafter the mixture was incubated
at room temperature for 15 minutes. After ethanol precipitation,
the product was resuspended in 10 .mu.l or more of water or
appropriate buffer and dialyzed against water.
[0107] Following oxidation of the diol groups at the 5' ends of the
mRNAs, the derivatized oligonucleotide was joined to the resulting
aldehydes as described in Example 10.
EXAMPLE 10
Reaction of Aldehydes with Derivatized Oligonucleotides
[0108] The oxidized mRNA was dissolved in an acidic medium such as
50 .mu.l of sodium acetate pH 4-6. 50 .mu.l of a solution of the
derivatized oligonucleotide was added such that an mRNA:derivatized
oligonucleotide ratio of 1:20 was obtained and mixture was reduced
with a borohydride. The mixture was allowed to incubate for 2 h at
37.degree. C. or overnight (14 h) at 10.degree. C. The mixture was
ethanol precipitated, resuspended in 10 .mu.l or more of water or
appropriate buffer and dialyzed against distilled water. If
desired, the resulting product may be analyzed using acrylamide gel
electrophoresis, HPLC analysis, or other conventional
techniques.
[0109] Following the attachment of the derivatized oligonucleotide
to the mRNAs, a reverse transcription reaction may be performed as
described in Example 11 below.
EXAMPLE 11
Reverse Transcription of mRNAs
[0110] An oligodeoxyribonucleotide was derivatized as follows. 3 OD
units of an oligodeoxyribonucleotide of sequence
ATCAAGAATTCGCACGAGACCATTA (SEQ ID NO:3) having 5'-OH and 3'-P ends
were dissolved in 70 .mu.l of a 1.5 M hydroxybenzotriazole
solution, pH 5.3, prepared in dimethylformamide/water (75:25)
containing 2 .mu.g of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was
incubated for 2 h 30 min at 22.degree. C. The mixture was then
precipitated twice in LiClO.sub.4/acetone. The pellet was
resuspended in 200 .mu.l of 0.25 M hydrazine and incubated at
8.degree. C. from 3 to 14 h. Following the hydrazine reaction, the
mixture was precipitated twice in LiClO.sub.4/acetone.
[0111] The messenger RNAs to be reverse transcribed were extracted
from blocks of placenta having sides of 2 cm which had been stored
at -80.degree. C. The mRNA was extracted using conventional acidic
phenol techniques. Oligo-dT chromatography was used to purify the
mRNAs. The integrity of the mRNAs was checked by
Northern-blotting.
[0112] The diol groups on 7 .mu.g of the placental mRNAs were
oxidized as described above in Example 9. The derivatized
oligonucleotide was joined to the mRNAs as described in Example 10
above except that the precipitation step was replaced by an
exclusion chromatography step to remove derivatized
oligodeoxyribonucleotides which were not joined to mRNAs. Exclusion
chromatography was performed as follows:
[0113] 10 ml of AcA34 (BioSepra#230151) gel were equilibrated in 50
ml of a solution of 10 mM Tris pH 8.0, 300 mM NaCl, 1 mM EDTA, and
0.05% SDS. The mixture was allowed to sediment. The supernatant was
eliminated and the gel was resuspended in 50 ml of buffer. This
procedure was repeated 2 or 3 times.
[0114] A glass bead (diameter 3 mm) was introduced into a 2 ml
disposable pipette (length 25 cm). The pipette was filled with the
gel suspension until the height of the gel stabilized at 1 cm from
the top of the pipette. The column was then equilibrated with 20 ml
of equilibration buffer (10 mM Tris HCl pH 7.4, 20 MM NaCl).
[0115] 10 .mu.l of the mRNA which had been reacted with the
derivatized oligonucleotide were mixed in 39 .mu.l of 10 mM urea
and 2 .mu.l of blue-glycerol buffer, which had been prepared by
dissolving 5 mg of bromophenol blue in 60% glycerol (v/v), and
passing the mixture through a filter with a filter of diameter 0.45
.mu.m.
[0116] The column was loaded. As soon as the sample had penetrated,
equilibration buffer was added. 100 .mu.l fractions were collected.
Derivatized oligonucleotide which had not been attached to mRNA
appeared in fraction 16 and later fractions. Fractions 3 to 15 were
combined and precipitated with ethanol.
[0117] The mRNAs which had been reacted with the derivatized
oligonucleotide were spotted on a nylon membrane and hybridized to
a radioactive probe using conventional techniques. The radioactive
probe used in these hybridizations was an oligodeoxyribonucleotide
of sequence TAATGGTCTCGTGCGAATTCTTGAT (SEQ ID NO:4) which was
anticomplementary to the derivatized oligonucleotide and was
labeled at its 5' end with .sup.32P. 1/10th of the mRNAs which had
been reacted with the derivatized oligonucleotide was spotted in
two spots on the membrane and the membrane was visualized by
autoradiography after hybridization of the probe. A signal was
observed, indicating that the derivatized oligonucleotide had been
joined to the mRNA.
[0118] The remaining 9/10 of the mRNAs which had been reacted with
the derivatized oligonucleotide was reverse transcribed as follows.
A reverse transcription reaction was carried out with reverse
transcriptase following the manufacturer's instructions. To prime
the reaction, 50 pmol of nonamers with random sequence were
used.
[0119] A portion of the resulting cDNA was spotted on a positively
charged nylon membrane using conventional methods. The cDNAs were
spotted on the membrane after the cDNA:RNA heteroduplexes had been
subjected to an alkaline hydrolysis in order to eliminate the RNAs.
An oligonucleotide having a sequence identical to that of the
derivatized oligonucleotide was labeled at its 5' end with .sup.32P
and hybridized to the cDNA blots using conventional techniques.
Single-stranded cDNAs resulting from the reverse transcription
reaction were spotted on the membrane. As controls, the blot
contained 1 pmol, 100 fmol, 50 fmol 10 fmol and 1 fmol respectively
of a control oligodeoxyribonucleotide of sequence identical to that
of the derivatized oligonucleotide. The signal observed in the
spots containing the cDNA indicated that approximately 15 fmol of
the derivatized oligonucleotide had been reverse transcribed.
[0120] These results demonstrate that the reverse transcription can
be performed through the cap and, in particular, that reverse
transcriptase crosses the 5'-P--P--P-5' bond of the cap of
eukaryotic messenger RNAs.
[0121] The single stranded cDNAs obtained after the above first
strand synthesis were used as template for PCR reactions. Two types
of reactions were carried out. First, specific amplification of the
mRNAs for the alpha globin, dehydrogenase, pp15 and elongation
factor E4 were carried out using the following pairs of
oligodeoxyribonucleotide primers.
2 alpha-globin (SEQ ID NO:5) GLO-S: CCG ACA AGA CCA ACG TCA AGG CCG
C (SEQ ID NO:6) GLO-As: TCA CCA GCA GGC AGT GGC TTA GGA G 3'
dehydrogenase (SEQ ID NO:7) 3 DH-S: AGT GAT TCC TGC TAC TTT GGA TGG
C (SEQ ID NO:8) 3 DH-As: GCT TGG TCT TGT TCT GGA GTT TAG A pp15
(SEQ ID NO:9) PP15-S: TCC AGA ATG GGA GAC AAG CCA ATT T (SEQ ID
NO:10) PP15-As: AGG GAG GAG GAA ACA GCG TGA GTC C Elongation factor
E4 (SEQ ID NO:11) EFA1-S: ATG GGA AAG GAA AAG ACT CAT ATC A (SEQ ID
NO:12) EF1A-As: AGC AGC AAC AAT CAG GAC AGC ACA G
[0122] Non specific amplifications were also carried out with the
antisense (_As) oligodeoxyribonucleotides of the pairs described
above and a primer chosen from the sequence of the derivatized
oligodeoxyribonucleotide (ATCAAGAATTCGCACGAGACCATTA) (SEQ ID
NO:13).
[0123] A 1.5% agarose gel containing the following samples
corresponding to the PCR products of reverse transcription was
stained with ethidium bromide. ({fraction (1/20)}th of the products
of reverse transcription were used for each PCR reaction).
[0124] Sample 1: The products of a PCR reaction using the globin
primers of SEQ ID NOs 5 and 6 in the presence of cDNA.
[0125] Sample 2: The products of a PCR reaction using the globin
primers of SEQ ID NOs 5 and 6 in the absence of added cDNA.
[0126] Sample 3: The products of a PCR reaction using the
dehydrogenase primers of SEQ ID NOs 7 and 8 in the presence of
cDNA.
[0127] Sample 4: The products of a PCR reaction using the
dehydrogenase primers of SEQ ID NOs 7 and 8 in the absence of added
cDNA.
[0128] Sample 5: The products of a PCR reaction using the pp15
primers of SEQ ID NOs 9 and 10 in the presence of cDNA.
[0129] Sample 6: The products of a PCR reaction using the pp15
primers of SEQ ID NOs 9 and 10 in the absence of added cDNA.
[0130] Sample 7: The products of a PCR reaction using the EIE4
primers of SEQ ID NOs 11 and 12 in the presence of added cDNA.
[0131] Sample 8: The products of a PCR reaction using the EIE4
primers of SEQ ID NOs 11 and 12 in the absence of added cDNA.
[0132] In Samples 1, 3, 5 and 7, a band of the size expected for
the PCR product was observed, indicating the presence of the
corresponding sequence in the cDNA population.
[0133] PCR reactions were also carried out with the antisense
oligonucleotides of the globin and dehydrogenase primers (SEQ ID
NOs 6 and 8) and an oligonucleotide whose sequence corresponds to
that of the derivatized oligonucleotide. The presence of PCR
products of the expected size in the samples corresponding to
samples 1 and 3 above indicated that the derivatized
oligonucleotide had been incorporated.
[0134] The above examples summarize the chemical procedure for
enriching mRNAs for those having intact 5' ends. Further detail
regarding the chemical approaches for obtaining mRNAs having intact
5' ends are disclosed in International Application No. W096/34981,
published Nov. 7, 1996, which is incorporated herein by reference.
Strategies based on the above chemical modifications to the 5' cap
structure may be utilized to generate cDNAs which have been
selected to include the 5' ends of the mRNAs from which they are
derived. In one version of such procedures, the 5' ends of the
mRNAs are modified as described above. Thereafter, a reverse
transcription reaction is conducted to extend a primer
complementary to the mRNA to the 5' end of the mRNA. Single
stranded RNAs are eliminated to obtain a population of cDNA/mRNA
heteroduplexes in which the mRNA includes an intact 5' end. The
resulting heteroduplexes may be captured on a solid phase coated
with a molecule capable of interacting with the molecule used to
derivatize the 5' end of the mRNA. Thereafter, the strands of the
heteroduplexes are separated to recover single stranded first cDNA
strands which include the 5' end of the mRNA. Second strand cDNA
synthesis may then proceed using conventional techniques. For
example, the procedures disclosed in WO 96/34981 or in Carninci, P.
et al. High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP
Trapper. Genomics 37:327-336 (1996), the disclosures of which are
incorporated herein by reference, may be employed to select cDNAs
which include the sequence derived from the 5' end of the coding
sequence of the mRNA.
[0135] Following ligation of the oligonucleotide tag to the 5' cap
of the mRNA, a reverse transcription reaction is conducted to
extend a primer complementary to the mRNA to the 5' end of the
mRNA. Following elimination of the RNA component of the resulting
heteroduplex using standard techniques, second strand cDNA
synthesis is conducted with a primer complementary to the
oligonucleotide tag.
[0136] FIG. 1 summarizes the above procedures for obtaining cDNAs
which have been selected to include the 5' ends of the mRNAs from
which they are derived.
[0137] B. Enzymatic Methods for Obtaining mRNAs having Intact 5'
Ends
[0138] Other techniques for selecting cDNAs extending to the 5' end
of the mRNA from which they are derived are fully enzymatic. Some
versions of these techniques 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
l'etude de la regulation de l'expression de la tryptophane
hydroxylase de rat, 20 Dec. 1993), EPO 625572 and Kato et al.
Construction of a Human Full-Length cDNA Bank. Gene 150:243-250
(1994), the disclosures of which are incorporated herein by
reference.
[0139] Briefly, in such approaches, isolated mRNA is treated with
alkaline phosphatase to remove the phosphate groups present on the
5' ends of uncapped incomplete mRNAs. Following this procedure, the
cap present on full length mRNAs is enzymatically removed with a
decapping enzyme such as T4 polynucleotide kinase or tobacco acid
pyrophosphatase. An oligonucleotide, which may be either a DNA
oligonucleotide or a DNA-RNA hybrid oligonucleotide having RNA at
its 3' end, is then ligated to the phosphate present at the 5' end
of the decapped mRNA using T4 RNA ligase. The oligonucleotide may
include a restriction site to facilitate cloning of the cDNAs
following their synthesis. Example 12 below describes one enzymatic
method based on the doctoral thesis of Dumas.
EXAMPLE 12
Enzymatic Approach for Obtaining 5' ESTs
[0140] Twenty micrograms of PolyA+ RNA were dephosphorylated using
Calf Intestinal Phosphatase (Biolabs). After a phenol chloroform
extraction, the cap structure of mRNA was hydrolyzed using the
Tobacco Acid Pyrophosphatase (purified as described by Shinshi et
al., Biochemistry 15: 2185-2190, 1976) and a hemi 5'DNA/RNA-3'
oligonucleotide having an unphosphorylated 5' end, a stretch of
adenosine ribophosphate at the 3' end, and an EcoRI site near the
5' end was ligated to the 5'P ends of mRNA using the T4 RNA ligase
(Biolabs). Oligonucleotides suitable for use in this procedure are
preferably 30-50 bases in length. Oligonucleotides having an
unphosphorylated 5' end may be synthesized by adding a fluorochrome
at the 5' end. The inclusion of a stretch of adenosine
ribophosphates at the 3' end of the oligonucleotide increases
ligation efficiency. It will be appreciated that the
oligonucleotide may contain cloning sites other than EcoRI.
[0141] Following ligation of the oligonucleotide to the phosphate
present at the 5' end of the decapped mRNA, first and second strand
cDNA synthesis may be carried out using conventional methods or
those specified in EPO 625,572 and Kato et al. Construction of a
Human Full-Length cDNA Bank. Gene 150:243-250 (1994), and Dumas
Milne Edwards, supra, the disclosures of which are incorporated
herein by reference. The resulting cDNA may then be ligated into
vectors such as those disclosed in Kato et al. Construction of a
Human Full-Length cDNA Bank. Gene 150:243-250 (1994) or other
nucleic acid vectors known to those skilled in the art using
techniques such as those described in Sambrook et al., Molecular
Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory
Press, 1989, the disclosure of which is incorporated herein by
reference.
[0142] II. Characterization of 5' ESTs
[0143] The above chemical and enzymatic approaches for enriching
mRNAs having intact 5' ends were employed to obtain 5' ESTs. First,
mRNAs were prepared as described in Example 13 below.
EXAMPLE 13
Preparation of mRNA
[0144] Total human RNAs or PolyA+ RNAs derived from 29 different
tissues were respectively purchased from LABIMO and CLONTECH and
used to generate 44 cDNA libraries as described below. The
purchased RNA had been isolated from cells or tissues using acid
guanidium thiocyanate-phenol-chloroform extraction (Chomczyniski, P
and Sacchi, N., Analytical Biochemistry 162:156-159, 1987). PolyA+
RNA was isolated from total RNA (LABIMO) by two passes of oligodT
chromatography, as described by Aviv and Leder (Aviv, H. and Leder,
P., Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972) in order to
eliminate ribosomal RNA.
[0145] The quality and the integrity of the poly A+ were checked.
Northern blots hybridized with a globin probe were used to confirm
that the mRNAs were not degraded. Contamination of the PolyA+ mRNAs
by ribosomal sequences was checked using RNAs blots and a probe
derived from the sequence of the 28S RNA. Preparations of mRNAs
with less than 5% of ribosomal RNAs 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 or of two highly
expressed mRNAs was examined using PCR.
[0146] Following preparation of the mRNAs, the above described
chemical and/or the enzymatic procedures for enriching mRNAs having
intact 5' ends discussed above were employed to obtain 5' ESTs from
various tissues. In both approaches an oligonucleotide tag was
attached to the cap at the 5' ends of the mRNAs. The
oligonucleotide tag had an EcoRI site therein to facilitate later
cloning procedures.
[0147] Following attachment of the oligonucleotide tag to the mRNA
by either the chemical or enzymatic methods, the integrity of the
mRNA was examined by performing a Northern blot with 200-500 ng of
mRNA using a probe complementary to the oligonucleotide tag.
EXAMPLE 14
cDNA Synthesis Using mRNA Templates Having Intact 5' Ends
[0148] For the mRNAs joined to oligonucleotide tags using both the
chemical and enzymatic methods, first strand cDNA synthesis was
performed using reverse transcriptase with random nonamers as
primers. 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.
[0149] For both the chemical and the enzymatic methods, the second
strand of the cDNA was synthesized with a Klenow fragment using a
primer corresponding to the 5'end of the ligated oligonucleotide
described in Example 12. 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.
[0150] Following cDNA synthesis, the cDNAs were cloned into
pBlueScript as described in Example 15 below.
EXAMPLE 15
Insertion of cDNAs into BlueScript
[0151] Following second strand synthesis, the ends of the cDNA were
blunted with T4 DNA polymerase (Biolabs) and the cDNA was digested
with EcoRI. Since methylated dCTP was used during cDNA synthesis,
the EcoRI site present in the tag was the only site which was
hemi-methylated. Consequently, only the EcoRI site in the
oligonucleotide tag was susceptible to EcoRI digestion. The cDNA
was then size fractionated using exclusion chromatography (AcA,
Biosepra). Fractions corresponding to cDNAs of more than 150 bp
were pooled and ethanol precipitated. The cDNA was directionally
cloned into the SmaI and EcoRI ends of the phagemid pBlueScript
vector (Stratagene). The ligation mixture was electroporated into
bacteria and propagated under appropriate antibiotic selection.
[0152] Clones containing the oligonucleotide tag attached were
selected as described in Example 16 below.
EXAMPLE 16
Selection of Clones Having the Oligonucleotide Tag Attached
Thereto
[0153] The plasmid DNAs containing 5' EST 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.,
Biotechniques, 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 described in Example 13. 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. Altematively, 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 estimated
to typically rank between 90 and 98% using dot blot analysis.
[0154] 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 as described below.
EXAMPLE 17
Sequencing of Inserts in Selected Clones
[0155] Plasmid inserts were first amplified by PCR on PE 9600
thermocyclers (Perkin-Elmer), using standard SETA-A and SETA-B
primers (Genset SA), AmpliTaqGold (Perkin-Elmer), dNTPs
(Boehringer), buffer and cycling conditions as recommended by the
Perkin-Elmer Corporation.
[0156] PCR products were then sequenced using automatic ABI Prism
377 sequencers (Perkin Elmer, Applied Biosystems Division, Foster
City, Calif.). Sequencing reactions were performed using PE 9600
thermocyclers (Perkin Elmer) with standard dye-primer chemistry and
ThermoSequenase (Amersham Life Science). 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.
[0157] Following the sequencing reaction, the samples were
precipitated with EtOH, 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.
[0158] The sequence data from the 44 cDNA libraries made as
described above were transferred to a proprietary database, where
quality control and validation steps were performed. A proprietary
base-caller ("Trace"), 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 EST sequences. However, the resulting 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 by case
basis.
[0159] Thereafter, the sequences were transferred to the
proprietary NETGENE.TM. Database for further analysis as described
below.
[0160] Following sequencing as described above, the sequences of
the 5' ESTs were entered in a proprietary database called
NETGENE.TM. for storage and manipulation. It will be appreciated by
those skilled in the art that the data could be stored and
manipulated on any medium which can be read and accessed by a
computer. Computer readable media include magnetically readable
media, optically readable media, or electronically readable media.
For example, the computer readable media may be a hard disc, a
floppy disc, a magnetic tape, CD-ROM, RAM, or ROM as well as other
types of other media known to those skilled in the art.
[0161] In addition, the sequence data may be stored and manipulated
in a variety of data processor programs in a variety of formats.
For example, the sequence data may be stored as text in a word
processing file, such as MicrosoftWORD 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.
[0162] The computer readable media on which the sequence
information is stored may be in a personal computer, a network, a
server or other computer systems known to those skilled in the art.
The computer or other system preferably includes the storage media
described above, and a processor for accessing and manipulating the
sequence data. Once the sequence data has been stored it may be
manipulated and searched to locate those stored sequences which
contain a desired nucleic acid sequence or which encode a protein
having a particular functional domain. For example, the stored
sequence information may be compared to other known sequences to
identify homologies, motifs implicated in biological function, or
structural motifs.
[0163] Programs which may be used to search or compare the stored
sequences include the MacPattern (EMBL), BLAST, and BLAST2 program
series (NCBI), basic local alignment search tool programs for
nucleotide (BLASTN) and peptide (BLASTX) comparisons (Altschul et
al, J. Mol. Biol. 215: 403 (1990)) and FASTA (Pearson and Lipman,
Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)). The BLAST programs
then extend the alignments on the basis of defined match and
mismatch criteria.
[0164] 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.
[0165] Before searching the cDNAs in the NETGENE.TM. database for
sequence motifs of interest, cDNAs derived from mRNAs which were
not of interest were identified and eliminated from further
consideration as described in Example 18 below.
EXAMPLE 18
Elimination of Undesired Sequences from Further Consideration
[0166] 5' ESTs in the NETGENE.TM. database which were derived from
undesired sequences such as transfer RNAs, ribosomal RNAs,
mitochondrial RNAs, procaryotic RNAs, fungal RNAs, Alu sequences,
L1 sequences, or repeat sequences were identified using the FASTA
and BLASTN programs with the parameters listed in Table II.
[0167] To eliminate 5' ESTs encoding tRNAs from further
consideration, the 5' EST sequences were compared to the sequences
of 1190 known tRNAs obtained from EMBL release 38, of which 100
were human. The comparison was performed using FASTA on both
strands of the 5' ESTs. Sequences having more than 80% homology
over more than 60 nucleotides were identified as tRNA. Of the
144,341 sequences screened, 26 were identified as tRNAs and
eliminated from further consideration.
[0168] To eliminate 5' ESTs encoding rRNAs from further
consideration, the 5' EST sequences were compared to the sequences
of 2497 known rRNAs obtained from EMBL release 38, of which 73 were
human. The comparison was performed using BLASTN on both strands of
the 5' ESTs with the parameter S=108. Sequences having more than
80% homology over stretches longer than 40 nucleotides were
identified as rRNAs. Of the 144,341 sequences screened, 3,312 were
identified as rRNAs and eliminated from further consideration.
[0169] To eliminate 5' ESTs encoding mtRNAs from further
consideration, the 5' EST sequences were compared to the sequences
of the two known mitochondrial genomes for which the entire genomic
sequences are available and all sequences transcribed from these
mitochondrial genomes including tRNAs, rRNAs, and mRNAs for a total
of 38 sequences. The comparison was performed using BLASTN on both
strands of the 5' ESTs with the parameter S=108. Sequences having
more than 80% homology over stretches longer than 40 nucleotides
were identified as mtRNAs. Of the 144,341 sequences screened, 6,110
were identified as mtRNAs and eliminated from further
consideration.
[0170] Sequences which might have resulted from exogenous
contaminants were eliminated from further consideration by
comparing the 5' EST sequences to release 46 of the EMBL bacterial
and fungal divisions using BLASTN with the parameter S=144. All
sequences having more than 90% homology over at least 40
nucleotides were identified as exogenous contaminants. Of the 42
cDNA libraries examined, the average percentages of procaryotic and
fungal sequences contained therein were 0.2% and 0.5% respectively.
Among these sequences, only one could be identified as a sequence
specific to fungi. The others were either fungal or procaryotic
sequences having homologies with vertebrate sequences or including
repeat sequences which had not been masked during the electronic
comparison.
[0171] In addition, the 5' ESTs were compared to 6093 Alu sequences
and 1115 L1 sequences to mask 5' ESTs containing such repeat
sequences from further consideration. 5' ESTs including THE and MER
repeats, SSTR sequences or satellite, micro-satellite, or telomeric
repeats were also eliminated from further consideration. On
average, 11.5% of the sequences in the libraries contained repeat
sequences. Of this 11.5%, 7% contained Alu repeats, 3.3% contained
L1 repeats and the remaining 1.2% were derived from the other types
of repetitive sequences which were screened. These percentages are
consistent with those found in cDNA libraries prepared by other
groups. For example, the cDNA libraries of Adams et al. contained
between 0% and 7.4% Alu repeats depending on the source of the RNA
which was used to prepare the cDNA library (Adams et al., Nature
377:174, 1996).
[0172] The sequences of those 5' ESTs remaining after the
elimination of undesirable sequences were compared with the
sequences of known human mRNAs to determine the accuracy of the
sequencing procedures described above.
EXAMPLE 19
Measurement of Sequencing Accuracy by Comparison to Known
Sequences
[0173] To further determine the accuracy of the sequencing
procedure described above, the sequences of 5' ESTs derived from
known sequences were identified and compared to the known
sequences. First, a FASTA analysis with overhangs shorter than 5 bp
on both ends was conducted on the 5' ESTs to identify those
matching an entry in the public human mRNA database. The 6655 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.
[0174] This analysis revealed that the sequences incorporated in
the NETGENE.TM. database had an accuracy of more than 99.5%.
[0175] To determine the efficiency with which the above selection
procedures select cDNAs which include the 5' ends of their
corresponding mRNAs, the following analysis was performed.
EXAMPLE 20
Determination of Efficiency of 5' EST Selection
[0176] To determine the efficiency at which the above selection
procedures isolated 5' ESTs which included sequences close to the
5' end of the mRNAs from which they were derived, the sequences of
the ends of the 5' ESTs which were derived from the elongation
factor 1 subunit .alpha. and ferritin heavy chain genes were
compared to the known cDNA sequences for these genes. Since the
transcription start sites for the elongation factor 1 subunit
.alpha. and ferritin heavy chain are well characterized, they may
be used to determine the percentage of 5' ESTs derived from these
genes which included the authentic transcription start sites.
[0177] For both genes, more than 95% of the cDNAs included
sequences close to or upstream of the 5' end of the corresponding
mRNAs.
[0178] To extend the analysis of the reliability of the procedures
for isolating 5' ESTs from ESTs in the NETGENE.TM. database, a
similar analysis was conducted using a database composed of human
mRNA sequences extracted from GenBank database release 97 for
comparison. For those 5' ESTs derived from mRNAs included in the
GeneBank database, more than 85% had their 5' ends close to the 5'
ends of the known sequence. As some of the mRNA sequences available
in the GenBank 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 ESTs
including the authentic 5' ends of their corresponding mRNAs.
[0179] The EST libraries made above included multiple 5' ESTs
derived from the same mRNA. The sequences of such 5' ESTs were
compared to one another and the longest 5' ESTs for each mRNA were
identified. Overlapping cDNAs were assembled into continuous
sequences (contigs). The resulting continuous sequences were then
compared to public databases to gauge their similarity to known
sequences, as described in Example 21 below.
EXAMPLE 21
[0180] Clustering of the 5' ESTs and Calculation of Novelty Indices
for cDNA Libraries
[0181] For each sequenced EST library, the sequences were clustered
by the 5' end. Each sequence in the library was compared to the
others with BLASTN2 (direct strand, parameters S=107). ESTs with
High Scoring Segment Pairs (HSPs) at least 25 bp long, having 95%
identical bases and beginning closer than 10 bp from each EST 5'
end were grouped. The longest sequence found in the cluster was
used as representative of the cluster. A global clustering between
libraries was then performed leading to the definition of
super-contigs.
[0182] To assess the yield of new sequences within the EST
libraries, a novelty rate (NR) was defined as: NR=100.times.(Number
of new unique sequences found in the library/Total number of
sequences from the library). Typically, novelty rating range
between 10% and 41% depending on the tissue from which the EST
library was obtained. For most of the libraries, the random
sequencing of 5' EST libraries was pursued until the novelty rate
reached 20%.
[0183] Following characterization as described above, the
collection of 5' ESTs in NETGENE.TM. was screened to identify those
5' ESTs bearing potential signal sequences as described in Example
22 below.
EXAMPLE 22
Identification of Potential Signal Sequences in 5' ESTs
[0184] The 5' ESTs in the NETGENE.TM. database were screened to
identify those having an uninterrupted open reading frame (ORF)
longer than 45 nucleotides beginning with an ATG codon and
extending to the end of the EST. Approximately half of the cDNA
sequences in NETGENE.TM. contained such an ORF. The ORFs of these
5' ESTs were searched to identify potential signal motifs using
slight modifications of the procedures disclosed in Von Heijne, G.
A New Method for Predicting Signal Sequence Cleavage Sites. Nucleic
Acids Res. 14:4683-4690 (1986), the disclosure of which is
incorporated herein by reference. Those 5' EST sequences encoding a
15 amino acid long stretch with a score of at least 3.5 in the Von
Heijne signal peptide identification matrix were considered to
possess a signal sequence. Those 5' ESTs which matched a known
human mRNA or EST sequence and had a 5' end more than 20
nucleotides downstream of the known 5' end were excluded from
further analysis. The remaining cDNAs having signal sequences
therein were included in a database called SIGNALTAG.TM..
[0185] To confirm the accuracy of the above method for identifying
signal sequences, the analysis of Example 23 was performed.
EXAMPLE 23
Confirmation of Accuracy of Identification of Potential Signal
Sequences in 5' ESTs
[0186] The accuracy of the above procedure for identifying signal
sequences encoding signal peptides was evaluated by applying the
method to the 43 amino terminal amino acids 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.
[0187] 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 FIGS. 2 and
3.
[0188] Using the above method of identifying secretory proteins, 5'
ESTs for human glucagon, gamma interferon induced monokine
precursor, secreted cyclophilin-like protein, human pleiotropin,
and human biotinidase precursor all of which are polypeptides which
are known to be secreted, were obtained. Thus, the above method
successfully identified those 5' ESTs which encode a signal
peptide.
[0189] To confirm that the signal peptide encoded by the 5' ESTs
actually functions as a signal peptide, the signal sequences from
the 5' ESTs may be cloned into a vector designed for the
identification of signal peptides. Some signal peptide
identification vectors are designed to confer the ability to grow
in selective medium on host cells which have a signal sequence
operably inserted into the vector. For example, to confirm that a
5' EST encodes a genuine signal peptide, the signal sequence of the
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. Pat. No. 5,536,637, the
disclosure of which is incorporated herein by reference. Growth of
host cells containing signal sequence selection vectors having the
signal sequence from the 5' EST inserted therein confirms that the
5' EST encodes a genuine signal peptide.
[0190] Alternatively, the presence of a signal peptide may be
confirmed by cloning the extended cDNAs obtained using the ESTs
into expression vectors such as pXT1 (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 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.
[0191] Those 5' ESTs which encoded a signal peptide, as determined
by the method of Example 22 above, were further grouped into four
categories based on their homology to known sequences. The
categorization of the 5' ESTs is described in Example 24 below.
EXAMPLE 24
Categorization of 5' ESTs Encoding a Signal Peptide
[0192] Those 5' ESTs having a sequence not matching any known
vertebrate sequence nor any publicly available EST sequence were
designated "new." Of the sequences in the SIGNALTAG.TM. database,
947 of the 5' ESTs having a Von Heijne's score of at least 3.5 fell
into this category.
[0193] Those 5' ESTs having a sequence not matching any vertebrate
sequence but matching a publicly known EST were designated
"EST-ext", provided that the known EST sequence was extended by at
least 40 nucleotides in the 5' direction. Of the sequences in the
SIGNALTAG.TM. database, 150 of the 5' ESTs having a Von Heijne's
score of at least 3.5 fell into this category.
[0194] Those ESTs not matching any vertebrate sequence but matching
a publicly known EST without extending the known EST by at least 40
nucleotides in the 5' direction were designated "EST." Of the
sequences in the SIGNALTAG.TM. database, 599 of the 5' ESTs having
a Von Heijne's score of at least 3.5 fell into this category.
[0195] Those 5' ESTs matching a human mRNA sequence but extending
the known sequence by at least 40 nucleotides in the 5' direction
were designated "VERT-ext." Of the sequences in the SIGNALTAG.TM.
database, 23 of the 5' ESTs having a Von Heijne's score of at least
3.5 fell into this category. Included in this category was a 5' EST
which extended the known sequence of the human translocase mRNA by
more than 200 bases in the 5' direction. A 5' EST which extended
the sequence of a human tumor suppressor gene in the 5' direction
was also identified.
[0196] FIG. 4 shows the distribution of 5' ESTs in each category
and the number of 5' ESTs in each category having a given minimum
von Heijne's score.
[0197] Each of the 5' ESTs was categorized based on the tissue from
which its corresponding mRNA was obtained, as described below in
Example 25.
EXAMPLE 25
Categorization of Expression Patterns
[0198] FIG. 5 shows the tissues from which the mRNAs corresponding
to the 5' ESTs in each of the above described categories were
obtained.
[0199] In addition to categorizing the 5' ESTs by the tissue from
which the cDNA library in which they were first identified was
obtained, the spatial and temporal expression patterns of the mRNAs
corresponding to the 5' ESTs, as well as their expression levels,
may be determined as described in Example 26 below.
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.
[0200] In addition, 5' ESTs whose corresponding mRNAs are
associated with disease states may also be identified. For example,
a particular disease may result from lack of expression, over
expression, or under expression of an mRNA corresponding to a 5'
EST. By comparing mRNA expression patterns and quantities in
samples taken from healthy individuals with those from individuals
suffering from a particular disease, 5' ESTs responsible for the
disease may be identified.
[0201] It will be appreciated that the results of the above
characterization procedures for 5' ESTs also apply to extended
cDNAs (obtainable as described below) which contain sequences
adjacent to the 5' ESTs. It will also be appreciated that if it is
desired to defer characterization until extended cDNAs have been
obtained rather than characterizing the ESTs themselves, the above
characterization procedures can be applied to characterize the
extended cDNAs after their isolation.
EXAMPLE 26
Evaluation of Expression Levels and Patterns of mRNAs Corresponding
to 5' ESTs or Extended cDNAs
[0202] Expression levels and patterns of mRNAs corresponding to 5'
ESTs or extended cDNAs (obtainable as described below) 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, a
5' EST, extended cDNA, or fragment thereof 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 5'
EST or extended cDNA has 100 or more nucleotides. 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.degree. 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, T1, Phy M, U2 or
A). The presence of the biotin-UTP modification enables capture of
the hybrid on a microtitration plate coated 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.
[0203] The 5' ESTs, extended cDNAs, or fragments thereof may also
be tagged with nucleotide sequences for the serial analysis of gene
expression (SAGE) as disclosed in UK 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 it is desired to determine
gene expression patterns. 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 cDNA 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 "tagging endonuclease" is
ligated to the digested cDNAs in the first pool. Digestion with the
second endonuclease produces short "tag" fragments from the
cDNAs.
[0204] 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 "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 5' ESTs or extended cDNAs to determine which
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 5' ESTs or
extended cDNAs in the cell, tissue, organism, or other source of
nucleic acids is obtained.
[0205] 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
full length cDNAs (i.e. extended cDNAs which include the coding
sequence for the signal peptide, the coding sequence for the mature
protein, and a stop codon), extended cDNAs, 5' ESTs or fragments of
the full length cDNAs, extended cDNAs, or 5' ESTs of sufficient
length to permit specific detection of gene expression. Preferably,
the fragments are at least 15 nucleotides in length. More
preferably, the fragments are at least 100 nucleotides in length.
More preferably, the fragments are more than 100 nucleotides in
length. In some embodiments the fragments may be more than 500
nucleotides in length.
[0206] For example, quantitative analysis of gene expression may be
performed with full length cDNAs, extended cDNAs, 5' ESTs, or
fragments thereof 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). Full length cDNAs, extended cDNAs, 5'
ESTs or fragments thereof 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.degree. C., transferred into 0.2% SDS for 1 min,
rinsed twice with water, air dried and stored in the dark at
25.degree. C.
[0207] 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 cm.sup.2 microarrays under a 14.times.14
mm glass coverslip for 6-12 hours at 60.degree. C. Arrays are
washed for 5 min at 25.degree. C. in low stringency wash buffer
(1.times.SSC/0.2% SDS), then for 10 min at room temperature in high
stringency wash buffer (0.1.times.SSC/0.2% SDS). Arrays are scanned
in 0.1.times.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.
[0208] Quantitative analysis of the expression of genes may also be
performed with full length cDNAs, extended cDNAs, 5' ESTs, or
fragments thereof in complementary DNA arrays as described by Pietu
et al. (Genome Research 6:492-503, 1996). The full length cDNAs,
extended cDNAs, 5' ESTs or fragments 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. Duplicate
experiments are performed and a quantitative analysis of
differentially expressed mRNAs is then performed.
[0209] Alternatively, expression analysis of the 5' ESTs or
extended cDNAs 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 the 5' ESTs or extended cDNAs are
synthesized directly on the chip (Lockhart et al., supra) or
synthesized and then addressed to the chip (Sosnowski et al.,
supra). Preferably, the oligonucleotides are about 20 nucleotides
in length.
[0210] 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 (Sosnowsky
et al., Proc. Natl. Acad. Sci. 94:1119-1123)., 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 or extended cDNA from which the
oligonucleotide sequence has been designed.
[0211] III. Use of 5' ESTs to Clone Extended cDNAs and to Clone the
Corresponding Genomic DNAs
[0212] Once 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. The extended cDNAs may include the entire
coding sequence of the protein encoded by the corresponding mRNA,
including the authentic translation start site, the signal
sequence, and the sequence encoding the mature protein remaining
after cleavage of the signal peptide. Such extended cDNAs are
referred to herein as "full length cDNAs." Alternatively, 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 27 below describes a general method for obtaining
extended cDNAs. Example 28 below describes the cloning and
sequencing of several extended cDNAs, 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 27, 28, and 29 can also be used to
obtain extended cDNAs which encode less than the entire coding
sequence of the secreted proteins encoded by the genes
corresponding to the 5' ESTs. In some embodiments, the extended
cDNAs isolated using these methods encode at least 10 amino acids
of one of the proteins encoded by the sequences of SEQ ID NOs:
40-84 and 130-154. In further embodiments, the extended cDNAs
encode at least 20 amino acids of the proteins encoded by the
sequences of SEQ ID NOs: 40-84 and 130-154. In further embodiments,
the extended cDNAs encode at least 30 amino amino acids of the
sequences of SEQ ID NOs: 40-84 and 130-154. In a preferred
embodiment, the extended cDNAs encode a full length protein
sequence, which includes the protein coding sequences of SEQ ID
NOs: 40-84 and 130-154.
EXAMPLE 27
General Method for Using 5' ESTs to Clone and Sequence Extended
cDNAs
[0215] The following general method has been used to quickly and
efficiently isolate extended cDNAs including sequence adjacent to
the sequences of the 5' ESTs used to obtain them. This method may
be applied to obtain extended cDNAs for any 5' EST in the
NETGENE.TM. database, including those 5' ESTs encoding secreted
proteins. The method is summarized in FIG. 6.
[0216] 1. Obtaining Extended cDNAs
[0217] a) First Strand Synthesis
[0218] The method takes advantage of the known 5' sequence of the
mRNA. A reverse transcription reaction is conducted on purified
mRNA with a poly 14dT primer containing a 49 nucleotide sequence at
its 5' end allowing the addition of a known sequence at the end of
the cDNA which corresponds to the 3' end of the mRNA. For example,
the primer may have the following sequence: 5'-ATC GTT GAG ACT CGT
ACC AGC AGA GTC ACG AGA GAG ACT ACA CGG TAC TGG TTT TTT TTT TTT
TTVN -3' (SEQ ID NO:14). Those skilled in the art will appreciate
that other sequences may also be added to the poly dT sequence and
used to prime the first strand synthesis. Using this primer arid a
reverse transcriptase such as the Superscript II (Gibco BRL) or
Rnase H Minus M-MLV (Promega) enzyme, a reverse transcript anchored
at the 3' polyA site of the RNAs is generated.
[0219] 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 are eliminated with an
exclusion column such as an AcA34 (Biosepra) matrix as explained in
Example 11.
[0220] b) Second Strand Synthesis
[0221] A pair of nested primers on each end is designed based on
the known 5' sequence from the 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).
[0222] Preferably, the nested primers at the 5' end are separated
from one another by four to nine bases. The 5' primer sequences may
be selected to have melting temperatures and specificities suitable
for use in PCR.
[0223] Preferably, the nested primers at the 3' end are separated
from one another by four to nine bases. For example, the nested 3'
primers may have the following sequences: (5'-CCA GCA GAG TCA CGA
GAG AGA CTA CAC GG-3'(SEQ ID NO:15), and 5'-CAC GAG AGA GAC TAC ACG
GTA CTG G-3' (SEQ ID NO:16). These primers were selected because
they have melting temperatures and specificities compatible with
their use in PCR. However, those skilled in the art will appreciate
that other sequences may also be used as primers.
[0224] The first PCR run of 25 cycles is performed using the
Advantage Tth Polymerase Mix (Clontech) and the outer primer from
each of the nested pairs. A second 20 cycle PCR using the same
enzyme and the inner primer from each of the nested pairs is then
performed on 1/2500 of the first PCR product. Thereafter, the
primers and nucleotides are removed.
[0225] 2. Sequencing of Full Length Extended cDNAs or Fragments
Thereof
[0226] 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 5'
primer is located upstream of the translation initiation codon thus
yielding a nested PCR product containing the whole coding sequence.
Such a fill length extended cDNA undergoes 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.
[0227] a) Nested PCR Products Containing Complete ORFs
[0228] When the resulting nested PCR product contains the complete
coding sequence, as predicted from the 5'EST sequence, it is cloned
in an appropriate vector such as pED6dpc2, as described in section
3.
[0229] b) Nested PCR Products Containing Incomplete ORFs
[0230] 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 as described in the following section.
[0231] Once the full coding sequence has been completely
determined, new primers compatible for PCR use are 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 FIG. 6. Such fill length
extended cDNAs are then cloned into an appropriate vector as
described in section 3.
[0232] c) Sequencing Extended cDNAs
[0233] Sequencing of extended cDNAs can be performed using a Die
Terminator approach with the AMPLITAQ DNA polymerase FS kit
available from Perkin Elmer.
[0234] In order to sequence 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 using minimum overlaps of 32
nucleotides. Preferably, primer walking is performed until the
sequences of full length cDNAs are obtained.
[0235] Completion of the sequencing of a given extended cDNA
fragment is assessed as follows. Since sequences located after a
polyA tract are difficult to determine precisely in the case of
uncloned products, sequencing and primer walking processes for PCR
products are interrupted when a polyA tract is identified in
extended cDNAs obtained as described in case b. The sequence length
is compared to the size of the nested PCR product obtained as
described above. Due to the limited accuracy of the determination
of the PCR product size by gel electrophoresis, a sequence is
considered complete if the size of the obtained sequence is at
least 70% the size of the first nested PCR product. If the length
of the sequence determined from the computer analysis is not at
least 70% of the length of the nested PCR product, these PCR
products are cloned and the sequence of the insertion is
determined. When Northern blot data are available, the size of the
mRNA detected for a given PCR product is used to finally assess
that the sequence is complete. Sequences which do not fulfill the
above criteria are discarded and will undergo a new isolation
procedure.
[0236] Sequence data of all extended cDNAs are then transferred to
a proprietary database, where quality controls and validation steps
are carried out as described in example 15.
[0237] 3. Cloning of Full Length Extended cDNAs
[0238] The PCR product containing the full coding sequence is then
cloned in an appropriate vector. For example, the extended cDNAs
can be cloned into the expression vector pED6dpc2 (DiscoverEase,
Genetics Institute, Cambridge, Mass.) as follows. The structure of
pED6dpc2 is shown in FIG. 7. pED6dpc2 vector DNA is prepared with
blunt ends by performing an EcoRI digestion followed by a fill in
reaction. The blunt ended vector is dephosphorylated. After removal
of PCR primers and ethanol precipitation, the PCR product
containing the full coding sequence or the extended cDNA obtained
as described above is phosphorylated with a kinase subsequently
removed by phenol-Sevag extraction and precipitation. The double
stranded extended cDNA is then ligated to the vector and the
resulting expression plasmid introduced into appropriate host
cells.
[0239] Since the PCR products obtained as described above are blunt
ended molecules that can be cloned in either direction, the
orientation of several clones for each PCR product is determined.
Then, 4 to 10 clones are ordered in microtiter plates and subjected
to a PCR reaction using a first primer located in the vector close
to the cloning site and a second primer located in the portion of
the extended cDNA corresponding to the 3' end of the mRNA. This
second primer may be the antisense primer used in anchored PCR in
the case of direct cloning (case a) or the antisense primer located
inside the 3 'UTR in the case of indirect cloning (case b). Clones
in which the start codon of the extended cDNA is operably linked to
the promoter in the vector so as to permit expression of the
protein encoded by the extended cDNA are conserved and sequenced.
In addition to the ends of cDNA inserts, approximately 50 bp of
vector DNA on each side of the cDNA insert are also sequenced.
[0240] The cloned PCR products are then entirely sequenced
according to the aforementioned procedure. In this case, contig
assembly of long fragments is then performed on walkling sequences
that have already contigated for uncloned PCR products during
primer walking. Sequencing of cloned amplicons is complete when the
resulting contigs include the whole coding region as well as
overlapping sequences with vector DNA on both ends.
[0241] 4. Computer Analysis of Full Length Extended cDNA
[0242] Sequences of all full length extended cDNAs may then be
subjected to further analysis as described below and using the
parameters found in Table II with the following modifications. For
screening of miscellaneous subdivisions of Genbank, FASTA was used
instead of BLASTN and 15 nucleotide of homology was the limit
instead of 17. For Alu detection, BLASTN was used with the
following parameters: S=72; identity=70%; and length=40
nucleotides. Polyadenylation signal and polyA tail which were not
search for the 5' ESTs were searched. For polyadenylation signal
detection the signal (AATAAA) was searched with one permissible
mismatch in the last fifty nucleotides preceding the 5' end of the
polyA. For the polyA, a stretch of 8 amino acids in the last 20
nucleotides of the sequence was searched with BLAST2N in the sense
strand with the following parameters (W=6, S=10, E=1000, and
identity=90%). Finally, patented sequences and ORF homologies were
searched using, respectively, BLASTN and BLASTP on GenSEQ
(Derwent's database of patented nucleotide sequences) and SWISSPROT
for ORFs with the following parameters (W=8 and B=10). Before
examining the extended full length cDNAs for sequences of interest,
extended cDNAs which are not of interest are searched as
follows.
[0243] a) Elimination of Undesired Sequences
[0244] Although 5'ESTs were checked to remove contaminants
sequences as described in Example 18, a last verification was
carried out to identify extended cDNAs sequences derived from
undesired sequences such as vector RNAs, transfer RNAs, ribosomal
rRNAs, mitochondrial RNAs, prokaryotic RNAs and fungal RNAs using
the FASTA and BLASTN programs on both strands of extended cDNAs as
described below.
[0245] To identify the extended cDNAs encoding vector RNAs,
extended cDNAs are compared to the known sequences of vector RNA
using the FASTA program. Sequences of extended cDNAs with more than
90% homology over stretches of 15 nucleotides are identified as
vector RNA.
[0246] To identify the extended cDNAs encoding tRNAs, extended cDNA
sequences were compared to the sequences of 1190 known tRNAs
obtained from EMBL release 38, of which 100 were human. Sequences
of extended cDNAs having more than 80% homology over 60 nucleotides
using FASTA were identified as tRNA.
[0247] To identify the extended cDNAs encoding rRNAs, extended cDNA
sequences were compared to the sequences of 2497 known rRNAs
obtained from EMBL release 38, of which 73 were human. Sequences of
extended cDNAs having more than 80% homology over stretches longer
than 40 nucleotides using BLASTN were identified as rRNAs.
[0248] To identify the extended cDNAs encoding mtRNAs, extended
cDNA sequences were compared to the sequences of the two known
mitochondrial genomes for which the entire genomic sequences are
available and all sequences transcribed from these mitochondrial
genomes including tRNAs, rRNAs, and mRNAs for a total of 38
sequences. Sequences of extended cDNAs having more than 80%
homology over stretches longer than 40 nucleotides using BLASTN
were identified as mtRNAs.
[0249] Sequences which might have resulted from other exogenous
contaminants were identified by comparing extended cDNA sequences
to release 105 of Genbank bacterial and fungal divisions. Sequences
of extended cDNAs having more than 90% homology over 40 nucleotides
using BLASTN were identified as exogenous prokaryotic or fungal
contaminants.
[0250] In addition, extended cDNAs were searched for different
repeat sequences, including Alu sequences, L1 sequences, THE and
MER repeats, SSTR sequences or satellite, micro-satellite, or
telomeric repeats. Sequences of extended cDNAs with more than 70%
homology over 40 nucleotide stretches using BLASTN were identified
as repeat sequences and masked in further identification
procedures. In addition, clones showing extensive homology to
repeats , i.e., matches of either more than 50 nucleotides if the
homology was at least 75% or more than 40 nucleotides if the
homology was at least 85% or more than 30 nucleotides if the
homology was at least 90%, were flagged.
[0251] b) Identification of Structural Features
[0252] Structural features, e.g. polyA tail and polyadenylation
signal, of the sequences of full length extended cDNAs are
subsequently determined as follows.
[0253] A polyA tail is defined as a homopolymeric stretch of at
least 11 A with at most one alternative base within it. The polyA
tail search is restricted to the last 20 nt of the sequence and
limited to stretches of 11 consecutive A's because sequencing
reactions are often not readable after such a polyA stretch.
Stretches with 100% homology over 6 nucleotides are identified as
polyA tails.
[0254] To search for a polyadenylation signal, the polyA tail is
clipped from the full-length sequence. The 50 bp preceding the
polyA tail are first searched for the canonic polyadenylation
AAUAAA signal and, if the canonic signal is not detected, for the
alternative AUUAAA signal (Sheets et al., Nuc. Acids Res. 18:
5799-5805, 1990). If neither of these consensus polyadenylation
signals is found, the canonic motif is searched again allowing one
mismatch to account for possible sequencing errors. More than 85%
of identified polyadenylation signals of either type actually ends
10 to 30 bp from the polyA tail. Alternative AUUAAA signals
represents approximately 15% of the total number of identified
polyadenylation signals.
[0255] To search for a polyadenylation signal, the polyA tail is
clipped from the full-length sequence. The 50 bp preceding the
polyA tail are searched for the canonic polyadenylation AAUAAA
signal allowing one mismatch to account for possible sequencing
errors and known variation in the canonical sequence of the
polyadenylation signal.
[0256] c) Identification of Functional Features
[0257] Functional features, e.g. ORFs and signal sequences, of the
sequences of full length extended cDNAs were subsequently
determined as follows.
[0258] 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 20 amino acids are preferred.
[0259] Each found ORF is then scanned for the presence of a signal
peptide in the first 50 amino-acids or, where appropriate, within
shorter regions down to 20 amino acids or less in the ORF, using
the matrix method of von Heijne (Nuc. Acids Res. 14: 4683-4690
(1986)), the disclosure of which is incorporated herein by
reference and the modification described in Example 22.
[0260] d) Homology to Either Nucleotidic or Proteic Sequences
[0261] Sequences of full length extended cDNAs are then compared to
known sequences on a nucleotidic or proteic basis.
[0262] Sequences of full length extended cDNAs are compared to the
following known nucleic acid sequences: vertebrate sequences, EST
sequences, patented sequences and recently identified sequences
available at the time of filing the priority documents. Full length
cDNA sequences are also compared to the sequences of a private
database (Genset internal sequences) in order to find sequences
that have already been identified by applicants. Sequences of full
length extended cDNAs with more than 90% homology over 30
nucleotides using either BLASTN or BLAST2N as indicated in Table
III are identified as sequences that have already been described.
Matching vertebrate sequences are subsequently examined using
FASTA; full length extended cDNAs with more than 70% homology over
30 nucleotides are identified as sequences that have already been
described.
[0263] ORFs encoded by full length extended cDNAs as defined in
section c) are subsequently compared to known amino acid sequences
found in public databases using Swissprot, PIR and Genptept
releases available at the time of filing the priority documents for
the present application. These analyses were performed using BLASTP
with the parameter W=8 and allowing a maximum of 10 matches.
Sequences of full length extended cDNAs showing extensive homology
to known protein sequences are recognized as already identified
proteins.
[0264] In addition, the three-frame conceptual translation products
of the top strand of full length extended cDNAs are compared to
publicly known amino acid sequences of Swissprot using BLASTX with
the parameter E=0.001. Sequences of full length extended cDNAs with
more than .sup.70% homology over 30 amino acid stretches are
detected as already identified proteins.
[0265] As used herein the term "cDNA codes of SEQ ID NOs. 40-84 and
130-154" encompasses the nucleotide sequences of SEQ ID NOs. 40-84
and 130-154, fragments of SEQ ID NOs. 40-84 and 130-154, nucleotide
sequences homologous to SEQ ID NOs. 40-84 and 130-154 or homologous
to fragments of SEQ ID NOs. 40-84 and 130-154, and sequences
complementary to all of the preceding sequences. The fragments
include portions of SEQ ID NOs. 40-84 and 130-154 comprising at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 consecutive nucleotides of SEQ ID NOs: 40-84 and 130-154.
Preferably, the fragments are novel fragments. Homologous sequences
and fragments of SEQ ID NOs. 40-84 and 130-154 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 herein, including
BLAST2N with the default parameters or with any modified
parameters. Homologous sequences also include RNA sequences in
which uridines replace the thymines in the cDNA codes of SEQ ID
NOs. 40-84 and 130-154. 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. It will be
appreciated that the cDNA codes of SEQ ID NOs. 40-84 and 130-154
can be represented in the traditional single character format (See
the inside back cover of Starrier, Lubert. Biochemistry, 3.sup.rd
edition. W. H Freeman & Co., New York.) or in any other format
which records the identity of the nucleotides in a sequence.
[0266] As used herein the term "polypeptide codes of SEQ ID NOS.
85-129 and 155-179" encompasses the polypeptide sequence of SEQ ID
NOs. 85-129 and 155-179 which are encoded by the extended cDNAs of
SEQ ID NOs. 40-84 and 130-154, polypeptide sequences homologous to
the polypeptides of SEQ ID NOS. 85-129 and 155-179, or fragments of
any of the preceding sequences. Homologous polypeptide sequences
refer to a polypeptide sequence having at least 99%, 98%, 97%, 96%,
95%, 90%, 85%, 80%, 75% homology to one of the polypeptide
sequences of SEQ ID NOS. 85-129 and 155-179. 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, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids of the polypeptides
of SEQ ID NOS. 85-129 and 155-179. Preferably, the fragments are
novel fragments. It will be appreciated that the polypeptide codes
of the SEQ ID NOS. 85-129 and 155-179 can be represented in the
traditional single character format or three letter format (See the
inside back cover of Starrier, Lubert. Biochemistry, 3.sup.rd
edition. W. H Freeman & Co., New York.) or in any other format
which relates the identity of the polypeptides in a sequence.
[0267] It will be appreciated by those skilled in the art that the
cDNA codes of SEQ ID NOs. 40-84 and 130-154 and polypeptide codes
of SEQ ID NOS. 85-129 and 155-179 can be stored, recorded, 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 cDNA codes of
SEQ ID NOs. 40-84 and 130-154, one or more of the polypeptide codes
of SEQ ID NOS. 85-129 and 155-179. 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.
40-84 and 130-154. 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. 85-129 and
155-179.
[0268] 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 disc, a floppy disc, a magnetic tape, CD-ROM, DVD,
RAM, or ROM as well as other types of other media known to those
skilled in the art.
[0269] Embodiments of the present invention include systems,
particularly computer systems which contain the sequence
information described herein. 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. 40-84 and 130-154, or the amino acid
sequences of the polypeptide codes of SEQ ID NOS. 85-129 and
155-179. The computer system preferably includes the computer
readable media described above, and a processor for accessing and
manipulating the sequence data.
[0270] Preferably, the computer is a general purpose system that
comprises a central processing unit (CPU), one or more data storage
components 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.
[0271] In one particular embodiment, the computer system includes a
processor connected to a bus which is connected to a main memory
(preferably implemented as RAM) and one or more data storage
devices, such as a hard drive and/or other computer readable media
having data recorded thereon. In some embodiments, the computer
system further includes one or more data retrieving devices for
reading the data stored on the data storage components. The data
retrieving device may represent, for example, a floppy disk drive,
a compact disk drive, a magnetic tape drive, etc. In some
embodiments, the data storage component 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 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. Software for accessing and processing the
nucleotide sequences of the cDNA codes of SEQ ID NOs. 40-84 and
130-154, or the amino acid sequences of the polypeptide codes of
SEQ ID NOS. 85-129 and 155-179 (such as search tools, compare
tools, and modeling tools etc.) may reside in main memory during
execution.
[0272] In some embodiments, the computer system may further
comprise a sequence comparer for comparing the above-described cDNA
codes of SEQ ID NOs. 40-84 and 130-154 or polypeptide codes of SEQ
ID NOS. 85-129 and 155-179 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 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 ID NOs. 40-84 and
130-154, or the amino acid sequences of the polypeptide codes of
SEQ ID NOS. 85-129 and 155-179 stored on a computer readable medium
to reference sequences stored on a computer readable medium to
identify homologies, motifs implicated in biological finction, 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.
[0273] Accordingly, one aspect of the present invention is a
computer system comprising a processor, a data storage device
having stored thereon a cDNA code of SEQ ID NOs. 40-84 and 130-154
or a polypeptide code of SEQ ID NOS. 85-129 and 155-179, a data
storage device having retrievably stored thereon reference
nucleotide sequences or polypeptide sequences to be compared to the
cDNA code of SEQ ID NOs. 40-84 and 130-154 or polypeptide code of
SEQ ID NOS. 85-129 and 155-179 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 cDNA code of SEQ ID NOs.
40-84 and 130-154 and polypeptide codes of SEQ ID NOS. 85-129 and
155-179 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 ID NOs. 40-84 and 130-154 or polypeptide codes of SEQ
ID NOS. 85-129 and 155-179.
[0274] Another aspect of the present invention is a method for
determining the level of homology between a cDNA code of SEQ ID
NOs. 40-84 and 130-154 and a reference nucleotide sequence,
comprising the steps of reading the cDNA code and the reference
nucleotide sequence through the use of a computer program which
determines homology levels and determining homology between the
cDNA 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 below, 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. 40-84 and 130-154
through use of the computer program and determining homology
between the cDNA codes and reference nucleotide sequences.
[0275] 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 cDNA code of SEQ ID NOs. 40-84 and
130-154 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 cDNA code of SEQ ID NOs. 40-84 and 130-154. In one embodiment,
the computer program may be a program which determines whether the
nucleotide sequences of the cDNA codes of SEQ ID NOs. 40-84 and
130-154 contain a single nucleotide polymorphism (SNP) with respect
to a reference nucleotide sequence. This single nucleotide
polymorphism may comprise a single base substitution, insertion, or
deletion.
[0276] Another aspect of the present invention is a method for
determining the level of homology between a polypeptide code of SEQ
ID NOS. 85-129 and 155-179 and a reference polypeptide sequence,
comprising the steps of reading the polypeptide code of SEQ ID NOS.
85-129 and 155-179 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.
[0277] Accordingly, another aspect of the present invention is a
method for determining whether a cDNA code of SEQ ID NOs. 40-84 and
130-154 differs at one or more nucleotides from a reference
nucleotide sequence comprising the steps of reading the cDNA code
and the reference nucleotide sequence through use of a computer
program which identifies differences between nucleic acid sequences
and identifying differences between the cDNA 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. 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. 40-84 and 130-154 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.
[0278] 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. 40-84 and
130-154 or the amino acid sequences of the polypeptide codes of SEQ
ID NOS. 85-129 and 155-179.
[0279] An "identifier" refers to one or more programs which
identifies certain features within the above-described nucleotide
sequences of the cDNA codes of SEQ ID NOs. 40-84 and 130-154 or the
amino acid sequences of the polypeptide codes of SEQ ID NOS. 85-129
and 155-179. In one embodiment, the identifier may comprise a
program which identifies an open reading frame in the cDNAs codes
of SEQ ID NOs. 40-84 and 130-154.
[0280] 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. 85-129 and
155-179. 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. Pat. No. 5,436,850 issued Jul. 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.
85-129 and 155-179. (See e.g., Srinivasan, et al., U.S. Pat. No.
5,557,535 issued Sep. 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.
[0281] 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 template(s) 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.
[0282] 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 interresidue distance restraints and fed into the
distance 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., Aszdi et al., Proteins:Structure, Function, and Genetics,
Supplement 1:38-42 (1997)).
[0283] 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.
85-129 and 155-179.
[0284] Accordingly, another aspect of the present invention is a
method of identifying a feature within the cDNA codes of SEQ ID
NOs. 40-84 and 130-154 or the polypeptide codes of SEQ ID NOS.
85-129 and 155-179 comprising reading the cDNA code(s) or the
polypeptide code(s) through the use of a computer program which
identifies features therein and identifying features within the
cDNA code(s) or polypeptide code(s) 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. 40-84 and 130-154 or the polypeptide
codes of SEQ ID NOS. 85-129 and 155-179 through the use of the
computer program and identifying features within the cDNA codes or
polypeptide codes with the computer program.
[0285] The cDNA codes of SEQ ID NOs. 40-84 and 130-154 or the
polypeptide codes of SEQ ID NOS. 85-129 and 155-179 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.
40-84 and 130-154 or the polypeptide codes of SEQ ID NOS. 85-129
and 155-179 may be stored as text in a word processing file, such
as MicrosoftWORD 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. 40-84 and 130-154 or the
polypeptide codes of SEQ ID NOS. 85-129 and 155-179. 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. 40-84 and 130-154 or the polypeptide codes of SEQ ID
NOS. 85-129 and 155-179. The programs and 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.), Catalyst/SHAPE (Molecular
Simulations Inc.), Cerius.sup.2.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.
[0286] 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.
[0287] 5. Selection of Cloned Full Length Sequences of the Present
Invention
[0288] Cloned full length extended cDNA sequences that have already
been characterized by the aforementioned computer analysis are then
submitted to an automatic procedure in order to preselect full
length extended cDNAs containing sequences of interest.
[0289] a) Automatic Sequence Preselection
[0290] All complete cloned full length extended cDNAs clipped for
vector on both ends are considered. First, a negative selection is
operated in order to eliminate unwanted cloned sequences resulting
from either contaminants or PCR artifacts as follows. Sequences
matching contaminant sequences such as vector RNA, tRNA, mtRNA,
rRNA sequences are discarded as well as those encoding ORF
sequences exhibiting extensive homology to repeats as defined in
section 4 a). Sequences obtained by direct cloning using nested
primers on 5' and 3' tags (section 1. case a) but lacking polyA
tail are discarded. Only ORFs containing a signal peptide and
ending either before the polyA tail (case a) or before the end of
the cloned 3'UTR (case b) are kept. Then, 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 are
eliminated.
[0291] In the selection of the ORF, priority was given to the ORF
and the frame corresponding to the polypeptides described in
SignalTag Patents (U.S. patent application Ser. Nos: 08/905,223;
08/905,135; 08/905,051; 08/905,144; 08/905,279; 08/904,468;
08/905,134; and 08/905,133). If the ORF was not found among the
ORFs described in the SignalTag Patents, the ORF encoding the
signal peptide with the highest score according to Von Heijne
method as defined in Example 22 was chosen. If the scores were
identical, then the longest ORF was chosen.
[0292] Sequences of full length extended cDNA clones are then
compared pairwise with BLAST after masking of the repeat sequences.
Sequences containing at least 90% homology over 30 nucleotides are
clustered in the same class. Each cluster is then subjected to a
cluster analysis that detects sequences resulting from internal
priming or from alternative splicing, identical sequences or
sequences with several frameshifts. This automatic analysis serves
as a basis for manual selection of the sequences.
[0293] b) Manual Sequence Selection
[0294] Manual selection can be carried out using automatically
generated reports for each sequenced full length extended cDNA
clone. During this manual procedure, a selection is operated
between clones belonging to the same class as follows. ORF
sequences encoded by clones belonging to the same class are aligned
and compared. If the homology between nucleotidic sequences of
clones belonging to the same class is more than 90% over 30
nucleotide stretches or if the homology between amino acid
sequences of clones belonging to the same class is more than 80%
over 20 amino acid stretches, than the clones are considered as
being identical. The chosen ORF is the best one according to the
criteria mentioned below. If the nucleotide and amino acid
homologies are less than 90% and 80% respectively, the clones are
said to encode distinct proteins which can be both selected if they
contain sequences of interest.
[0295] Selection of full length extended cDNA clones encoding
sequences of interest is performed using the following criteria.
Structural parameters (initial tag, polyadenylation site and
signal) are first checked. Then, homologies with known nucleic
acids and proteins are examined in order to determine whether the
clone sequence match a known nucleic/proteic 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 brings
substantial new information, such as encoding a protein resulting
from alternative slicing of an mRNA coding for an already known
protein, the sequence is kept. Examples of such cloned full length
extended cDNAs containing sequences of interest are described in
Example 28. Sequences resulting from chimera or double inserts as
assessed by homology to other sequences are discarded during this
procedure.
EXAMPLE 28
Cloning and Sequencing of Extended cDNAs
[0296] The procedure described in Example 27 above was used to
obtain the extended cDNAs of the present invention. Using this
approach, the full length cDNA of SEQ ID NO: 17 was obtained. This
cDNA falls into the "EST-ext" category described above and encodes
the signal peptide MKKVLLLITAILAVAVG (SEQ ID NO: 18) having a von
Heijne score of 8.2.
[0297] The fall length cDNA of SEQ ID NO: 19 was also obtained
using this procedure. This cDNA falls into the "EST-ext" category
described above and encodes the signal peptide
MWWFQQGLSFLPSALVIWTSA (SEQ ID NO:20) having a von Heijne score of
5.5.
[0298] Another full length cDNA obtained using the procedure
described above has the sequence of SEQ ID NO:21. This cDNA, falls
into the "EST-ext" category described above and encodes the signal
peptide MVLTTLPSANSANSPVNMPTTGPNSLSYASSALSPCLT (SEQ ID NO:22)
having a von Heijne score of 5.9.
[0299] The above procedure was also used to obtain a full length
cDNA having the sequence of SEQ ID NO:23. This cDNA falls into the
"EST-ext" category described above and encodes the signal peptide
ILSTVTALTFAXA (SEQ ID NO:24) having a von Heijne score of 5.5.
[0300] The full length cDNA of SEQ ID NO:25 was also obtained using
this procedure. This cDNA falls into the "new" category described
above and encodes a signal peptide LVLTLCTLPLAVA (SEQ ID NO:26)
having a von Heijne score of 10.1.
[0301] The full length cDNA of SEQ ID NO:27 was also obtained using
this procedure. This cDNA falls into the "new" category described
above and encodes a signal peptide LWLLFFLVTAIHA (SEQ ID NO:28)
having a von Heijne score of 10.7.
[0302] The above procedures were also used to obtain the extended
cDNAs of the present invention. 5' ESTs expressed in a variety of
tissues were obtained as described above. The appended sequence
listing provides the tissues from which the extended cDNAs were
obtained. It will be appreciated that the extended cDNAs may also
be expressed in tissues other than the tissue listed in the
sequence listing.
[0303] 5' ESTs obtained as described above were used to obtain
extended cDNAs having the sequences of SEQ ID NOs: 40-84 and
130-154. Table IV provides the sequence identification numbers of
the extended cDNAs of the present invention, the locations of the
fall coding sequences in SEQ ID NOs: 40-84 and 130-154 (i.e. the
nucleotides encoding both the signal peptide and the mature
protein, listed under the heading FCS location in Table IV), the
locations of the nucleotides in SEQ ID NOs: 40-84 and 130-154 which
encode the signal peptides (listed under the heading SigPep
Location in Table IV), the locations of the nucleotides in SEQ ID
NOs: 40-84 and 130-154 which encode the mature proteins generated
by cleavage of the signal peptides (listed under the heading Mature
Polypeptide Location in Table IV), the locations in SEQ ID NOs:
40-84 and 130-154 of stop codons (listed under the heading Stop
Codon Location in Table IV), the locations in SEQ ID NOs: 40-84 and
130-154 of polyA signals (listed under the heading Poly A Signal
Location in Table IV) and the locations of polyA sites (listed
under the heading Poly A Site Location in Table IV).
[0304] The polypeptides encoded by the extended cDNAs were 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
conserved regions have been used to derive consensus patterns or
matrices included in the PROSITE data bank, in particular in the
file prosite.dat (Release 13.0 of November 1995, located at
http://expasy.hcuge.ch/sprot/prosite.html. Prosite_convert and
prosite_scan programs (http://ulrec3.unil.ch/ftpserve-
ur/prosite_scan) were used to find signatures on the extended
cDNAs.
[0305] For each pattern obtained with the prosite_convert program
from the prosite.dat file, the accuracy of the detection on a new
protein sequence has been tested by evaluating the frequency of
irrelevant hits on the population of human secreted proteins
included in the data bank SWISSPROT. The ratio between the number
of hits on shuffled proteins (with a window size of 20 amino acids)
and the number of hits on native (unshuffled) proteins was used as
an index. Every pattern for which the ration was greater than 20%
(one hit on shuffled proteins for 5 hits on native proteins) was
skipped during the search with prosite_scan. The program used to
shuffle protein sequences (db_shuffied) and the program used to
determine the statistics for each pattern in the protein data banks
(prosite_statistics) are available on the ftp site
http://ulrec3.unil.ch/ftpserveur/prosite_scan.
[0306] Table V lists the sequence identification numbers of the
polypeptides of SEQ ID NOs: 85-129 and 155-179, the locations of
the amino acid residues of SEQ ID NOs: 85-129 and 155-179 in the
full length polypeptide (second column), the locations of the amino
acid residues of SEQ ID NOs: 85-129 and 155-179 in the signal
peptides (third column), and the locations of the amino acid
residues of SEQ ID NOs: 85-129 and 155-179 in the mature
polypeptide created by cleaving the signal peptide from the full
length polypeptide (fourth column).
[0307] The nucleotide sequences of the sequences of SEQ ID NOs:
40-84 and 130-154 and the amino acid sequences encoded by SEQ ID
NOs: 40-84 and 130-154 (i.e. amino acid sequences of SEQ ID NOs:
85-129 and 155-179) are provided in the appended sequence listing.
In some instances, the sequences are preliminary and may include
some incorrect or ambiguous sequences or amino acids. The sequences
of SEQ ID NOs: 40-84 and 130-154 can readily be screened for any
errors therein and any sequence ambiguities can be resolved by
resequencing a fragment containing such errors or ambiguities on
both strands. Sequences containing such errors will generally be at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% homologous to the sequences of SEQ ID Nos. 85-129 and 155-179
and such sequences are included in the nucleic acids and
polypeptides of the present invention. Nucleic acid fragments for
resolving sequencing errors or ambiguities may be obtained from the
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 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.
[0308] For each amino acid sequence, Applicants have identified
what they have determined to be the reading frame best identifiable
with sequence information available at the time of filing. Some of
the amino acid sequences may contain "Xaa" designators. These "Xaa"
designators 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).
[0309] Cells containing the extended cDNAs (SEQ ID NOs: 40-84 and
130-154) of the present invention in the vector pED6dpc2, are
maintained in permanent deposit by the inventors at Genset, S.A.,
24 Rue Royale, 75008 Paris, France.
[0310] Pools of cells containing the extended cDNAs (SEQ ID NOs:
40-84), from which cells containing a particular polynucleotide are
obtainable, were deposited with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va., U.S.A.,
20110-2209. Each extended cDNA clone has been transfected into
separate bacterial cells (E-coli) for this composite deposit. Table
VI lists the deposit numbers of the clones of SEQ ID Nos: 40-84. A
pool of cells designated SignalTag 28011999, which contains the
clones of SEQ ID NOs 71-84 was mailed to the European Collection of
Cell Cultures, (ECACC) Vaccine Research and Production Laboratory,
Public Health Laboratory Service, Centre for Applied Microbiology
and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United
Kingdom on Jan. 28, 1999 and was received on Jan. 29, 1999. This
pool of cells has the ECACC Accession # XXXXXX. One or more pools
of cells containing the extended cDNAs of SEQ ID Nos: 130-154, from
which the cells containing a particular polynucleotide is
obtainable, will be deposited with the European Collection of Cell
Cultures, Vaccine Research and Production Laboratory, Public Health
Laboratory Service, Centre for Applied Microbiology and Research,
Portion Down, Salisbury, Wiltshire SP4 OJG, United Kingdom and will
be assigned ECACC deposit number XXXXXXX. Table VII provides the
internal designation number assigned to each SEQ ID NO. and
indicates whether the sequence is a nucleic acid sequence or a
protein sequence.
[0311] Each extended cDNA can be removed from the pED6dpc2 vector
in which it was deposited by performing a NotI, PstI double
digestion to produce the appropriate fragment for each clone. The
proteins encoded by the extended cDNAs may also be expressed from
the promoter in pED6dpc2.
[0312] Bacterial cells containing a particular clone can be
obtained from the composite deposit as follows:
[0313] An oligonucleotide probe or probes should be designed to the
sequence that is known for that particular clone. This sequence can
be derived from the sequences provided herein, or from a
combination of those sequences. The design of the oligonucleotide
probe should preferably follow these parameters:
[0314] (a) It should be designed to an area of the sequence which
has the fewest ambiguous bases ("N's"), if any;
[0315] (b) Preferably, the probe is designed to have a T.sub.m of
approx. 80.degree. C. (assuming 2 degrees for each A or T and 4
degrees for each G or C). However, probes having melting
temperatures between 40.degree. C. and 80.degree. C. may also be
used provided that specificity is not lost.
[0316] The oligonucleotide should preferably be labeled with
(-[.sup.32P]ATP (specific activity 6000 Ci/mmole) and T4
polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be
used. Unincorporated label should preferably be removed by gel
filtration chromatography or other established methods. The amount
of radioactivity incorporated into the probe should be quantified
by measurement in a scintillation counter. Preferably, specific
activity of the resulting probe should be approximately
4.times.10.sup.6 dpm/pmole.
[0317] The bacterial culture containing the pool of full-length
clones should preferably be thawed and 100 .mu.l of the stock used
to inoculate a sterile culture flask containing 25 ml of sterile
L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be grown to saturation at 37.degree. C., and the
saturated culture should preferably be diluted in fresh L-broth.
Aliquots of these dilutions should preferably be plated to
determine the dilution and volume which will yield approximately
5000 distinct and well-separated colonies on solid bacteriological
media containing L-broth containing ampicillin at 100 .mu.g/ml and
agar at 1.5% in a 150 mm petri dish when grown overnight at
37.degree. C. Other known methods of obtaining distinct,
well-separated colonies can also be employed.
[0318] Standard colony hybridization procedures should then be used
to transfer the colonies to nitrocellulose filters and lyse,
denature and bake them.
[0319] The filter is then preferably incubated at 65.degree. C. for
1 hour with gentle agitation in 6.times.SSC (20.times. stock is
175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0
with NaOH) containing 0.5% SDS, 100 pg/ml of yeast RNA, and 10 mM
EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe
is then added to the hybridization mix at a concentration greater
than or equal to 1.times.10.sup.6 dpm/mL. The filter is then
preferably incubated at 65.degree. C. with gentle agitation
overnight. The filter is then preferably washed in 500 mL of
2.times.SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes. A third wash with 0.1.times.SSC/0.5% SDS at 65.degree. C.
for 30 minutes to 1 hour is optional. The filter is then preferably
dried and subjected to autoradiography for sufficient time to
visualize the positives on the X-ray film. Other known
hybridization methods can also be employed.
[0320] The positive colonies are picked, grown in culture, and
plasmid DNA isolated using standard procedures. The clones can then
be verified by restriction analysis, hybridization analysis, or DNA
sequencing.
[0321] 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 extended cDNA insertion. For example,
a PCR reaction may be conducted using a primer having the sequence
GGCCATACACTTGAGTGAC (SEQ ID NO:38) and a primer having the sequence
ATATAGACAAACGCACACC (SEQ. ID. NO:39). The PCR product which
corresponds to the extended cDNA can then be manipulated using
standard cloning techniques familiar to those skilled in the
art.
[0322] In addition to PCR based methods for obtaining extended
cDNAs, 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 were derived, mRNAs
corresponding to the extended cDNAs, or nucleic acids which are
homologous to extended cDNAs or 5' ESTs. Example 29 below provides
an example of such methods.
EXAMPLE 29
Methods for Obtaining Extended cDNAs or Nucleic Acids Homologous to
Extended cDNAs or 5' ESTs
[0323] 5'ESTs or extended cDNAs of the present invention may also
be used to isolate extended cDNAs or nucleic acids homologous to
extended cDNAs from a cDNA library or a genomic DNA library. Such
cDNA library or genormic DNA library may be obtained from a
commercial source or made using other techniques familiar to those
skilled in the art. One example of such cDNA library construction
is as follows.
[0324] PolyA+ RNAs are prepared and their quality checked as
described in Example 13. Then, polyA+ RNAs are ligated to an
oligonucleotide tag using either the chemical or enzymatic methods
described in above sections 1 and 2. In both cases, the
oligonucleotide tag may contain a restriction site such as Eco RI
to facilitate further subcloning procedures. Northern blotting is
then performed to check the size of ligatured mRNAs and to ensure
that the mRNAs were actually tagged.
[0325] As described in Example 14, first strand synthesis is
subsequently carried out for mRNAs joined to the oligonucleotide
tag replacing the random nonamers by an oligodT primer. For
instance, this oligodT primer may contain an internal tag of 4
nucleotides which is different from one tissue to the other.
Alternatively, the oligonucleotide of SEQ ID NO:14 may be used.
Following second strand synthesis using a primer contained in the
oligonucleotide tag attached to the 5' end of mRNA, the blunt ends
of the obtained double stranded full length DNAs are modified into
cohesive ends to allow subcloning into the Eco RI and Hind III
sites of a Bluescript vector using the addition of a Hind III
adaptor to the 3' end of full length DNAs.
[0326] The extended full length DNAs are then separated into
several fractions according to their sizes using techniques
familiar to those skilled in the art. For example, electrophoretic
separation may be applied in order to yield 3 or 6 different
fractions. Following gel extraction and purification, the DNA
fractions are subcloned into Bluescript vectors, transformed into
competent bacteria and propagated under appropriate antibiotic
conditions.
[0327] Such full length cDNA libraries may then be sequenced as
follows or used in screening procedures to obtain nucleic acids
homologous to extended cDNAs or 5' ESTs as described below.
[0328] The 5' end of extended cDNA isolated from the full length
cDNA libraries or of nucleic acid homologous thereto may then be
sequenced as described in example 27. In a first step, the sequence
corresponding to the 5' end of the mRNA is obtained. If this
sequence either corresponds to a SignalTag.TM. 5'EST or fulfills
the criteria to be one, the cloned insert is subcloned into an
appropriate vector such as pED6dpc2, double-sequenced and submitted
to the analysis and selection procedures described in Example
27.
[0329] Such cDNA or genomic DNA libraries may be used to isolate
extended cDNAs obtained from 5' EST or nucleic acids homologous to
extended cDNAs or 5' EST as follows. The cDNA library or genomic
DNA library is hybridized to a detectable probe comprising at least
10 consecutive nucleotides from the 5' EST or extended cDNA using
conventional techniques. Preferably, the probe comprises at least
12, 15, or 17 consecutive nucleotides from the 5' EST or extended
cDNA. More preferably, the probe comprises at least 20 to 30
consecutive nucleotides from the 5' EST or extended cDNA. In some
embodiments, the probe comprises at least 40, at least 50, at least
75, at least 100, at least 150, or at least 200 consecutive
nucleotides from the 5' EST or extended cDNA. [03021] Techniques
for identifying cDNA clones in a cDNA library which hybridize to a
given probe sequence are disclosed in Sambrook et al., Molecular
Cloning: 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.
[0330] Briefly, cDNA or genomic DNA clones which hybridize to the
detectable probe are identified and isolated for further
manipulation as follows. A probe comprising at least 10 consecutive
nucleotides from the 5' EST or extended cDNA is labeled with a
detectable label such as a radioisotope or a fluorescent molecule.
Preferably, the probe comprises at least 12, 15, or 17 consecutive
nucleotides from the 5' EST or extended cDNA. More preferably, the
probe comprises 20 to 30 consecutive nucleotides from the 5' EST or
extended cDNA. In some embodiments, the probe comprises at least
40, at least 50, at least 75, at least 100, at least 150, or at
least 200 consecutive nucleotides from the 5' EST or extended cDNA.
[0304] Techniques for labeling the probe are well known 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.
[0331] By varying the stringency of the hybridization conditions
used to identify extended cDNAs or genomic DNAs which hybridize to
the detectable probe, extended cDNAS having different levels of
homology to the probe can be identified and isolated as described
below.
[0332] 1. Identification of Extended cDNA or Genomic DNA Sequences
Having a High Degree of Homology to the Labeled Probe
[0333] To identify extended 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:
[0334] 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.
[0335] 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/N) where N is the length of the probe.
[0336] Prehybridization may be carried out in 6.times.SSC, 5.times.
Denhardt's reagent, 0.5% SDS, 100 .mu.g denatured fragmented salmon
sperm DNA or 6.times.SSC, 5.times. Denhardt's reagent, 0.5% SDS,
100 .mu.g denatured fragmented salmon sperm DNA, 50% formamide. The
formulas for SSC and Denhardt's solutions are listed in Sambrook et
al., supra.
[0337] 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.degree. C. below the Tm. For shorter probes, such as
oligonucleotide probes, the hybridization may be conducted at
15-25.degree. C. below the Tm. Preferably, for hybridizations in
6.times.SSC, the hybridization is conducted at approximately
68.degree. C. Preferably, for hybridizations in 50% formamide
containing solutions, the hybridization is conducted at
approximately 42.degree. C.
[0338] All of the foregoing hybridizations would be considered to
be under "stringent" conditions.
[0339] Following hybridization, the filter is washed in
2.times.SSC, 0.1% SDS at room temperature for 15 minutes. The
filter is then washed with 0.1.times.SSC, 0.5% SDS at room
temperature for 30 minutes to 1 hour. Thereafter, the solution is
washed at the hybridization temperature in 0.1.times.SSC, 0.5% SDS.
A final wash is conducted in 0.1.times.SSC at room temperature.
[0340] Extended cDNAs, nucleic acids homologous to extended cDNAs
or 5' ESTs, or genomic DNAs which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0341] 2. Obtaining Extended cDNA or Genomic DNA Sequences Having
Lower Degrees of Homology to the Labeled Probe
[0342] The above procedure may be modified to identify extended
cDNAs, nucleic acids homologous to extended cDNAs, or genomic DNAs
having decreasing levels of homology to the probe sequence. For
example, to obtain extended cDNAs, nucleic acids homologous to
extended 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.degree. C. from 68.degree. C. to 42.degree. C. in a
hybridization buffer having a sodium concentration of approximately
1M. Following hybridization, the filter may be washed with
2.times.SSC, 0.5% SDS at the temperature of hybridization. These
conditions are considered to be "moderate" conditions above
50.degree. C. and "low" conditions below 50.degree. C.
[0343] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. 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
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide.
[0344] Extended cDNAs, nucleic acids homologous to extended cDNAs,
or genomic DNAs which have hybridized to the probe are identified
by autoradiography.
[0345] 3. Determination of the Degree of Homology between the
Obtained Extended cDNAs or Genomic DNAs and the Labeled Probe
[0346] To determine the level of homology between the hybridized
nucleic acid and the extended cDNA or 5'EST from which the probe
was derived, the nucleotide sequences of the hybridized nucleic
acid and the extended cDNA or 5'EST from which the probe was
derived are compared. The sequences of the extended cDNA or 5'EST
and the homologous sequences may be stored on a computer readable
medium as described in Example 17 above and may be compared using
any of a variety of algorithms familiar to those skilled in the
art. For example, if it is desired to obtain nucleic acids
homologous to extended cDNAs, such as allelic variants thereof or
nucleic acids encoding proteins related to the proteins encoded by
the extended cDNAs, the level of homology between the hybridized
nucleic acid and the extended cDNA or 5' EST used as the probe may
be determined using algorithms such as BLAST2N; parameters may be
adapted depending on the sequence length and degree of homology
studied. For example, the default parameters or the parameters in
Table I and II may be used to determine homology levels.
[0347] Alternatively, the level of homology between the hybridized
nucleic acid and the extended cDNA or 5'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 or 5'EST
from which the probe was derived the homology level is manually
adjusted by calculating the number of nucleotides of the extended
cDNA or 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 sequence is 1000
nucleotides in length wherein the first 300 bases at the 5' end of
the extended cDNA 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. 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.
[0348] 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 homology, at least 99% nucleic acid homology, or more than 99%
nucleic acid homology to the extended cDNA or 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 or 5'EST from which the probe was
derived.
[0349] To determine whether a clone encodes a protein having a
given amount of homology to the protein encoded by the extended
cDNA or 5' EST, the amino acid sequence encoded by the extended
cDNA or 5' EST is compared to the amino acid sequence encoded by
the hybridizing nucleic acid. The sequences encoded by the extended
cDNA or 5'EST and the sequences encoded by the homologous sequences
may be stored on a computer readable medium as described in Example
17 above and may be compared using any of a variety of algorithms
familiar to those skilled in the art. Homology is determined to
exist when an amino acid sequence in the extended cDNA or 5' EST is
closely related to an amino acid sequence in the hybridizing
nucleic acid. A sequence is closely related when it is identical to
that of the extended cDNA or 5' EST or when it contains one or more
amino acid substitutions therein in which amino acids having
similar characteristics have been substituted for one another.
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 or the parameters in
Table I and II, 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 proteins encoded by the extended cDNA or 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)).
[0350] Alternatively, the level of 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=1, 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 or 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 or
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 or 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 or 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 or 5'EST. This represents 20% of the length of the
amino acid sequence encoded by the extended cDNA or 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.
[0351] In addition to the above described methods, other protocols
are available to obtain extended cDNAs using 5' ESTs as outlined in
the following paragraphs.
[0352] 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
known 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.
[0353] The first cDNA strand is hybridized to a second primer
containing at least 10 consecutive nucleotides of the sequences of
the 5' EST for which an extended cDNA is desired. Preferably, the
primer comprises at least 12, 15, or 17 consecutive nucleotides
from the sequences of the 5' EST. More preferably, the primer
comprises 20 to 30 consecutive nucleotides from the sequences of
the 5' EST. In some embodiments, the primer comprises more than 30
nucleotides from the sequences of the 5' EST. If it is desired to
obtain extended cDNAs containing the fall 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.
[0354] Extended cDNAs containing 5' fragments of the mRNA may be
prepared by hybridizing an mRNA comprising the sequence of the 5'
EST for which an extended cDNA is desired with a primer comprising
at least 10 consecutive nucleotides of the sequences complementary
to the 5' EST and reverse transcribing the hybridized primer to
make a first cDNA strand from the mRNAs. Preferably, the primer
comprises at least 12, 15, or 17 consecutive nucleotides from the
5' EST. More preferably, the primer comprises 20 to 30 consecutive
nucleotides from the 5' EST.
[0355] 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.
[0356] 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.
[0357] 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 503
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.
[0358] 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 5' EST,
or a fragment containing at least 10 nucleotides thereof, is
hybridized to the single stranded phagemids. Preferably, the
fragment comprises at least 12, 15, or 17 consecutive nucleotides
from the 5' EST. More preferably, the fragment comprises 20-30
consecutive nucleotides from the 5' EST. In some procedures, the
fragment may comprise at least 40, at least 50, at least 75, at
least 100, at least 150, or at least 200 consecutive nucleotides
from the 5' EST.
[0359] Hybrids between the biotinylated oligonucleotide and
phagemids having inserts containing the 5' EST sequence 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 containing the 5' EST sequence are released from the
beads and converted into double stranded DNA using a primer
specific for the 5' EST sequence. Alternatively, protocols such as
the Gene Trapper kit (Gibco BRL) may be used. The resulting double
stranded DNA is transformed into bacteria. Extended cDNAs
containing the 5' EST sequence are identified by colony PCR or
colony hybridization.
[0360] Using any of the above described methods in section III, a
plurality of extended cDNAs containing full length protein coding
sequences or sequences encoding only the mature protein remaining
after the signal peptide is cleaved off may be provided as cDNA
libraries for subsequent evaluation of the encoded proteins or use
in diagnostic assays as described below.
[0361] IV. Expression of Proteins Encoded by Extended cDNAs
Isolated Using 5' ESTs
[0362] Extended cDNAs containing the full protein coding sequences
of their corresponding mRNAs or portions thereof, such as cDNAs
encoding the mature protein, may be used to express the secreted
proteins or portions thereof which they encode as described in
Example 30 below. If desired, the extended cDNAs may contain the
sequences encoding the signal peptide to facilitate secretion of
the expressed protein. It will be appreciated that a plurality of
extended cDNAs containing the full protein coding sequences or
portions thereof may be simultaneously cloned into expression
vectors to create an expression library for analysis of the encoded
proteins as described below.
EXAMPLE 30
Expression of the Proteins Encoded by Extended cDNAs or Portions
Thereof
[0363] To express the proteins encoded by the extended cDNAs or
portions thereof, nucleic acids containing the coding sequence for
the proteins or portions thereof to be expressed are obtained as
described in Examples 27-29 and cloned into a suitable expression
vector. If desired, the nucleic acids may contain the sequences
encoding the signal peptide to facilitate secretion of the
expressed protein. For example, the nucleic acid may comprise the
sequence of one of SEQ ID NOs: 40-84 and 130-154 listed in Table IV
and in the accompanying sequence listing. Alternatively, the
nucleic acid may comprise those nucleotides which make up the full
coding sequence of one of the sequences of SEQ ID NOs: 40-84 and
130-154 as defined in Table IV above.
[0364] It will be appreciated that should the extent of the full
coding sequence (i.e. the sequence encoding the signal peptide and
the mature protein resulting from cleavage of the signal peptide)
differ from that listed in Table IV as a result of a sequencing
error, reverse transcription or amplification error, mRNA splicing,
post-translational modification of the encoded protein, enzymatic
cleavage of the encoded protein, or other biological factors, one
skilled in the art would be readily able to identify the extent of
the full coding sequences in the sequences of SEQ ID NOs. 40-84 and
130-154. Accordingly, the scope of any claims herein relating to
nucleic acids containing the full coding sequence of one of SEQ ID
NOs. 40-84 and 130-154 is not to be construed as excluding any
readily identifiable variations from or equivalents to the full
coding sequences listed in Table IV Similarly, should the extent of
the full length polypeptides differ from those indicated in Table V
as a result of any of the preceding factors, the scope of claims
relating to polypeptides comprising the amino acid sequence of the
full length polypeptides is not to be construed as excluding any
readily identifiable variations from or equivalents to the
sequences listed in Table V.
[0365] Alternatively, the nucleic acid used to express the protein
or portion thereof may comprise those nucleotides which encode the
mature protein (i.e. the protein created by cleaving the signal
peptide off) encoded by one of the sequences of SEQ ID NOs: 40-84
and 130-154 as defined in Table IV above.
[0366] It will be appreciated that should the extent of the
sequence encoding the mature protein differ from that listed in
Table IV as a result of a sequencing error, reverse transcription
or amplification error, mRNA splicing, post-translational
modification of the encoded protein, enzymatic cleavage of the
encoded protein, or other biological factors, one skilled in the
art would be readily able to identify the extent of the sequence
encoding the mature protein in the sequences of SEQ ID NOs. 40-84
and 130-154. Accordingly, the scope of any claims herein relating
to nucleic acids containing the sequence encoding the mature
protein encoded by one of SEQ ID Nos. 40-84 and 130-154 is not to
be construed as excluding any readily identifiable variations from
or equivalents to the sequences listed in Table IV. Thus, claims
relating to nucleic acids containing the sequence encoding the
mature protein encompass equivalents to the sequences listed in
Table IV, such as sequences encoding biologically active proteins
resulting from post-translational modification, enzymatic cleavage,
or other readily identifiable variations from or equivalents to the
secreted proteins in addition to cleavage of the signal peptide.
Similarly, should the extent of the mature polypeptides differ from
those indicated in Table V as a result of any of the preceding
factors, the scope of claims relating to polypeptides comprising
the sequence of a mature protein included in the sequence of one of
SEQ ID NOs. 85-129 and 155-179 is not to be construed as excluding
any readily identifiable variations from or equivalents to the
sequences listed in Table V. Thus, claims relating to polypeptides
comprising the sequence of the mature protein encompass equivalents
to the sequences listed in Table IV, such as biologically active
proteins resulting from post-translational modification, enzymatic
cleavage, or other readily identifiable variations from or
equivalents to the secreted proteins in addition to cleavage of the
signal peptide. It will also be appreciated that should the
biologically active form of the polypeptides included in the
sequence of one of SEQ ID NOs. 85-129 and 155-179 or the nucleic
acids encoding the biologically active form of the polypeptides
differ from those identified as the mature polypeptide in Table V
or the nucleotides encoding the mature polypeptide in Table IV as a
result of a sequencing error, reverse transcription or
amplification error, mRNA splicing, post-translational modification
of the encoded protein, enzymatic cleavage of the encoded protein,
or other biological factors, one skilled in the art would be
readily able to identify the amino acids in the biologically active
form of the polypeptides and the nucleic acids encoding the
biologically active form of the polypeptides. In such instances,
the claims relating to polypetides comprising the mature protein
included in one of SEQ ID NOs. 85-129 and 155-179 or nucleic acids
comprising the nucleotides of one of SEQ ID NOs. 40-84 and 130-154
encoding the mature protein shall not be construed to exclude any
readily identifiable variations from the sequences listed in Table
IV and Table V.
[0367] In some embodiments, the nucleic acid used to express the
protein or portion thereof may comprise those nucleotides which
encode the signal peptide encoded by one of the sequences of SEQ ID
NOs: 40-84 and 130-154 as defined in Table IV above.
[0368] It will be appreciated that should the extent of the
sequence encoding the signal peptide differ from that listed in
Table IV as a result of a sequencing error, reverse transcription
or amplification error, mRNA splicing, post-translational
modification of the encoded protein, enzymatic cleavage of the
encoded protein, or other biological factors, one skilled in the
art would be readily able to identify the extent of the sequence
encoding the signal peptide in the sequences of SEQ ID NOs. 40-84
and 130-154. Accordingly, the scope of any claims herein relating
to nucleic acids containing the sequence encoding the signal
peptide encoded by one of SEQ ID Nos. 40-84 and 130-154 is not to
be construed as excluding any readily identifiable variations from
the sequences listed in Table IV. Similarly, should the extent of
the signal peptides differ from those indicated in Table V as a
result of any of the preceding factors, the scope of claims
relating to polypeptides comprising the sequence of a signal
peptide included in the sequence of one of SEQ ID NOs. 85-129 and
155-179 is not to be construed as excluding any readily
identifiable variations from the sequences listed in Table V.
[0369] Alternatively, the nucleic acid may encode a polypeptide
comprising at least 10 consecutive amino acids of one of the
sequences of SEQ ID NOs: 85-129 and 155-179. In some embodiments,
the nucleic acid may encode a polypeptide comprising at least 15
consecutive amino acids of one of the sequences of SEQ ID NOs:
85-129 and 155-179. In other embodiments, the nucleic acid may
encode a polypeptide comprising at least 25 consecutive amino acids
of one of the sequences of SEQ ID NOs: 85-129 and 155-179. In other
embodiments, the nucleic acid may encode a polypeptide comprising
at least 60, at least 75, at least 100 or more than 100 consecutive
amino acids of one of the sequences of SEQ ID Nos: 85-129 and
155-179.
[0370] 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.
[0371] 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 vectors and expression
systems are available from a variety of suppliers including
Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla,
Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego,
Calif.). 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. Pat. No. 5,082,767, incorporated herein by
this reference.
[0372] The following is provided as one exemplary method to express
the proteins encoded by the extended cDNAs corresponding to the 5'
ESTs or the nucleic acids described above. First, the methionine
initiation codon for the gene and the poly A signal of the gene are
identified. 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
extended cDNA lacks a poly A signal, this sequence can be added to
the construct by, for example, splicing out the Poly A signal from
pSG5 (Stratagene) using BglI and SalI restriction endonuclease
enzymes and incorporating it into the mammalian expression vector
pXT1 (Stratagene). pXT1 contains the LTRs and a portion of the gag
gene from Moloney Murine Leukemia Virus. The position of the LTRs
in the construct allow efficient stable transfection. The vector
includes the Herpes Simplex Thymidine Kinase promoter and the
selectable neomycin gene. The extended cDNA or portion thereof
encoding the polypeptide to be expressed is obtained by PCR from
the bacterial vector using oligonucleotide primers complementary to
the extended cDNA or portion thereof and containing restriction
endonuclease sequences for Pst I incorporated into the 5'primer and
BglII at the 5' end of the corresponding cDNA 3' primer, taking
care to ensure that the extended cDNA is positioned in frame with
the poly A signal. The purified fragment obtained from the
resulting PCR reaction is digested with PstI, blunt ended with an
exonuclease, digested with Bgl II, purified and ligated to pXT1,
now containing a poly A signal and digested with BglII.
[0373] The ligated product is transfected into mouse NIH 3T3 cells
using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.)
under conditions outlined in the product specification. Positive
transfectants are selected after growing the transfected cells in
600ug/ml G418 (Sigma, St. Louis, Mo.). Preferably the expressed
protein is released into the culture medium, thereby facilitating
purification.
[0374] Alternatively, the extended cDNAs 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. Preferably,
the protein expressed from the extended cDNA is released into the
culture medium thereby facilitating purification.
[0375] Proteins in the culture medium are separated by gel
electrophoresis. If desired, the proteins may be ammonium sulfate
precipitated or separated based on size or charge prior to
electrophoresis.
[0376] As a control, the expression vector lacking a cDNA insert is
introduced into host cells or organisms and the proteins in the
medium are harvested. The secreted proteins present in the medium
are detected using techniques such as Coomassie or silver staining
or using antibodies against the protein encoded by the extended
cDNA. Coomassie and silver staining techniques are familiar to
those skilled in the art.
[0377] Antibodies capable of specifically recognizing the protein
of interest may be generated using synthetic 15-mer peptides having
a sequence encoded by the appropriate 5' EST, extended cDNA, or
portion thereof. The synthetic peptides are injected into mice to
generate antibody to the polypeptide encoded by the 5' EST,
extended cDNA, or portion thereof.
[0378] Secreted proteins from the host cells or organisms
containing an expression vector which contains the extended cDNA
derived from a 5' EST or a portion thereof are compared to those
from the control cells or organism. The presence of a band in the
medium from the cells containing the expression vector which is
absent in the medium from the control cells indicates that the
extended cDNA encodes a secreted protein. Generally, the band
corresponding to the protein encoded by the extended cDNA 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.
[0379] 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 containing an insert encoding a secreted protein
or portion thereof can be compared to the proteins expressed in
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.
[0380] The protein encoded by the extended cDNA may be purified
using standard immunochromatography techniques. In such procedures,
a solution containing the secreted protein, such as the culture
medium or a cell extract, is applied to a column having antibodies
against the secreted protein attached to the chromatography matrix.
The secreted protein is allowed to bind the immunochromatography
column. Thereafter, the column is washed to remove non-specifically
bound proteins. The specifically bound secreted protein is then
released from the column and recovered using standard
techniques.
[0381] If antibody production is not possible, the extended cDNA
sequence or portion thereof may be incorporated into expression
vectors designed for use in purification schemes employing chimeric
polypeptides. In such strategies the coding sequence of the
extended cDNA or portion thereof is inserted in frame with the gene
encoding the other half of the chimera. The other half of the
chimera may be .beta.-globin or a nickel binding polypeptide
encoding sequence. A chromatography matrix having antibody to
.beta.-globin or nickel attached thereto is then used to purify the
chimeric protein. Protease cleavage sites may be engineered between
the .beta.-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.
[0382] One useful expression vector for generating .beta.-globin
chimerics is pSG5 (Stratagene), which encodes rabbit .beta.-globin.
Intron II of the rabbit .beta.-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 Express.TM. Translation
Kit (Stratagene).
[0383] Following expression and purification of the secreted
proteins encoded by the 5' ESTs, extended cDNAs, or fragments
thereof, the purified proteins may be tested for the ability to
bind to the surface of various cell types as described in Example
31 below. It will be appreciated that a plurality of proteins
expressed from these cDNAs 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 31
Analysis of Secreted Proteins to Determine Whether they Bind to the
Cell Surface
[0384] The proteins encoded by the 5' ESTs, extended cDNAs, or
fragments thereof are cloned into expression vectors such as those
described in Example 30. The proteins are purified by size, charge,
immunochromatography or other techniques familiar to those skilled
in the art. Following purification, the proteins are labeled using
techniques known to those skilled in the art. The labeled proteins
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 protein. The labeled
proteins are detected by autoradiography. Alternatively, unlabeled
proteins may be incubated with the cells and detected with
antibodies having a detectable label, such as a fluorescent
molecule, attached thereto.
[0385] Specificity of cell surface binding may be analyzed by
conducting a competition analysis in which various amounts of
unlabeled protein are incubated along with the labeled protein. The
amount of labeled protein bound to the cell surface decreases as
the amount of competitive unlabeled protein increases. As a
control, various amounts of an unlabeled protein unrelated to the
labeled protein is included in some binding reactions. The amount
of labeled protein bound to the cell surface does not decrease in
binding reactions containing increasing amounts of unrelated
unlabeled protein, indicating that the protein encoded by the cDNA
binds specifically to the cell surface.
[0386] As discussed above, secreted proteins have been shown to
have a number of important physiological effects and, consequently,
represent a valuable therapeutic resource. The secreted proteins
encoded by the extended cDNAs or portions thereof made according to
Examples 27-29 may be evaluated to determine their physiological
activities as described below.
EXAMPLE 32
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Cytokine, Cell Proliferation or Cell Differentiation
Activity
[0387] As discussed above, secreted proteins may 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 of the
present invention is evidenced by any one of a number of routine
factor dependent cell proliferation assays for cell lines
including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2,
TF-1, Mo7c and CMK. The proteins encoded by the above extended
cDNAs or portions thereof 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.
Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol.
152:1756-1761, 1994.
[0388] 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., Vol 1 pp.
3.12.1-3.12.14 John Wiley and Sons, Toronto. 1994; and Schreiber,
R. D. Current Protocols in Immunology., supra Vol 1 pp.
6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
[0389] The proteins encoded by the cDNAs 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, K., Davis, L. S. and Lipsky, P. E.,
Measurement of Human and Murine Interleukin 2 and Interleukin 4,
Current Protocols in Immunology., J. E. Coligan et al. Eds. Vol 1
pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; 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. U.S.A.
80:2931-2938, 1983; Nordan, R., Measurement of Mouse and Human
Interleukin 6 Current Protocols in Immunology. J. E. Coligan et al.
Eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986;
Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.,
Measurement of Human Interleukin 11 Current Protocols in
Immunology. J. E. Coligan et al. Eds. Vol 1 pp. 6.15.1 John Wiley
and Sons, Toronto. 1991; Ciarletta, A., Giannotti, J., Clark, S. C.
and Turner, K. J., Measurement of Mouse and Human Interleukin 9
Current Protocols in Immunology. J. E. Coligan et al., Eds. Vol 1
pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
[0390] The proteins encoded by the cDNAs 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, J. E. Coligan et al. Eds.
Greene Publishing Associates and Wiley-Interscience; 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.
[0391] Those proteins 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 below, genes encoding
these proteins or nucleic acids regulating the expression of these
proteins may be introduced into appropriate host cells to increase
or decrease the expression of the proteins as desired.
EXAMPLE 33
Assaving the Proteins Expressed from Extended cDNAs or Portions
Thereof for Activity as Imune System Regulators
[0392] The proteins encoded by the cDNAs may also be evaluated for
their effects as immune regulators. For example, the proteins 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; Herrmann et al.,
Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann 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; Herrmann et al., Proc.
Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann 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;
Bowman et al., J. Virology 61:1992-1998; Takai et al., J. Immunol.
140:508-512, 1988; Bertagnolli et al., Cellular Immunology
133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,
1994.
[0393] The proteins encoded by the cDNAs 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, J. J. and
Brunswick, M Assays for B Cell Function: In vitro Antibody
Production, Vol 1 pp. 3.8.1-3.8.16 in Current Protocols in
Immunology. J. E. Coligan et al Eds., John Wiley and Sons, Toronto.
1994.
[0394] The proteins encoded by the cDNAs may also be evaluated for
their effect on immune effector cells, including their effect on
Th1 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, J. E.
Coligan et al. Eds., Greene Publishing Associates and
Wiley-Interscience; 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.
[0395] The proteins encoded by the cDNAs 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 herein by reference: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0396] The proteins encoded by the cDNAs 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 Research 53:1945-1951, 1993; Itoh et al.,
Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology
145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993;
Gorczyca et al., International Journal of Oncology 1:639-648,
1992.
[0397] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
88:7548-7551, 1991.
[0398] Those proteins 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 may be useful in
the treatment of various immune deficiencies and disorders
(including severe combined immunodeficiency (SCID)), 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
fingal infections, or may result from autoinunune disorders. More
specifically, infectious diseases caused by viral, bacterial,
fungal or other infection may be treatable using a protein of the
present invention, including infections by HIV, hepatitis viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and
various fungal infections such as candidiasis. Of course, in this
regard, a protein of the present invention may also be useful where
a boost to the immune system generally may be desirable, i.e., in
the treatment of cancer.
[0399] Autoimmune disorders which may be treated using a protein of
the present invention include, for example, connective tissue
disease, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis, myasthenia gravis, graft-versus-host disease and
autoimmune inflammatory eye disease. Such a protein of the present
invention may also to be usefuil 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 a protein of the
present invention.
[0400] Using the proteins of the invention it may also be possible
to regulate immune responses, in a number of ways. Down regulation
may be in the form of inhibiting or blocking an immune response
already in progress or may involve preventing the induction of an
immune response. The finctions 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 exposure to the tolerizing agent has ceased.
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.
[0401] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen finctions (such
as, for example, B7)), 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., B7-1,
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 block the function of a combination of B lymphocyte
antigens.
[0402] 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, murine models of
GVHD (see Paul ed., Fundamental Immunology, 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.
[0403] Blocking antigen finction may also be therapeutically useful
for treating autoimmune diseases. Many autoinmmune 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 may be 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 murine experimental
autoimmune encephalitis, systemic lupus erythmatosis in MRL/pr/pr
mice or NZB hybrid mice, murine autoimmuno collagen arthritis,
diabetes mellitus in OD mice and BB rats, and murine experimental
myasthenia gravis (see Paul ed., Fundamental Immunology, Raven
Press, New York, 1989, pp. 840-856).
[0404] Upregulation of an antigen function (preferably a B
lymphocyte antigen fu8nction), as a means of up regulating immune
responses, may also be useful in therapy. Upregulation of immune
responses may be in the form of enhancing an existing immune
response or eliciting an initial immune response. For example,
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.
[0405] Alternatively, anti-viral 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 a peptide of the present invention or together
with a stimulatory form of a soluble peptide of the present
invention and reintroducing the in vitro activated 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.
[0406] In another application, up regulation or enhancement of
antigen function (preferably B lymphocyte antigen fumction) may be
useful in the induction of tumor immunity. Tumor cells (e.g.,
sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
transfected with a nucleic acid encoding at least one peptide of
the present invention 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 B7-1-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.
[0407] The presence of the peptide of the present invention having
the activity of a B lymphocyte antigen(s) 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 MHC class I or MHC
class II molecules, 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 .alpha.
chain protein and .beta..sub.2 macroglobulin protein or an MHC
class II .alpha. chain protein and an MHC class II .beta. chain
protein to thereby express MHC class I or MHC class II proteins on
the cell surface. Expression of the appropriate class II or class
II MHC in conjunction with a peptide having the activity of a B
lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell
mediated immune response against the transfected tumor cell.
Optionally, a gene 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
peptide 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, genes encoding these proteins or nucleic acids
regulating the expression of these proteins may be introduced into
appropriate host cells to increase or decrease the expression of
the proteins as desired.
EXAMPLE 34
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Hematopoiesis Regulating Activity
[0408] The proteins encoded by the extended cDNAs or portions
thereof may also be evaluated for their hematopoiesis regulating
activity. For example, the effect of the proteins 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. Cellular Biology
15:141-151, 1995; Keller et al., Molecular and Cellular Biology
13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
[0409] The proteins encoded by the extended cDNAs or portions
thereof 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 Hematopoietic Cells. R. I.
Freshney, et al. Eds. pp. 265-268, Wiley-Liss, Inc., New York, N.Y.
1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911,
1992; McNiece, I. K. 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, N.Y. 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. 1-21, Wiley-Liss, Inc., New York, N.Y.
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, N.Y. 1994.
[0410] Those proteins which exhibit hematopoiesis regulatory
activity may then be formulated as pharmaceuticals and used to
treat clinical conditions in which regulation of hematopoeisis is
beneficial. For example, a protein 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 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 transfuisions; 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 irradiation/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, genes encoding these proteins or nucleic acids
regulating the expression of these proteins may be introduced into
appropriate host cells to increase or decrease the expression of
the proteins as desired.
EXAMPLE 35
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Regulation of Tissue Growth
[0411] The proteins encoded by the extended cDNAs or portions
thereof 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 intemational Patent
Publication No. WO95/16035, International Patent Publication No.
WO95/05846 and International Patent Publication No. WO91/07491,
which are incorporated herein by reference.
[0412] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H1 and Rovee, D T, 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.
[0413] Those proteins 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 of the present invention also
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.
[0414] A protein of the present invention, which induces cartilage
and/or bone growth in circumstances where bone is not normally
formed, has application in the healing of bone fractures and
cartilage damage or defects in humans and other animals. Such a
preparation employing a protein 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
formation 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.
[0415] A protein 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.
[0416] Another category of tissue regeneration activity that may be
attributable to the protein of the present invention is
tendon/ligament formation. A protein of the present invention,
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 composition of the present invention
contributes to the repair of congenital, trauma induced, or other
tendon or ligament defects of other origin, and is also useful in
cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The compositions 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 compositions of the invention
may also be useful in the treatment of tendinitis, carpal tunnel
syndrome and other tendon or ligament defects. The compositions may
also include an appropriate matrix and/or sequestering agent as a
carrier as is well known in the art.
[0417] The protein 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 may 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 may be 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 of
the invention.
[0418] Proteins 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.
[0419] It is expected that a protein 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 of the
invention may also exhibit angiogenic activity.
[0420] A protein 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 cytokinc damage.
[0421] A protein 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.
[0422] Alternatively, as described in more detail below, genes
encoding these proteins or nucleic acids regulating the expression
of these proteins may be introduced into appropriate host cells to
increase or decrease the expression of the proteins as desired.
EXAMPLE 36
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Regulation of Reproductive Hormones or Cell
Movement
[0423] The proteins encoded by the extended cDNAs or portions
thereof 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 incorporated herein by reference: Vale et
al., Endocrinology 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 (Measurement of Alpha and
Beta Chemokines) 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. of Immunol. 152:5860-5867, 1994; Johnston et al.
J. of Immunol. 153:1762-1768, 1994.
[0424] Those proteins 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 or cell movement are
beneficial. For example, a protein of the present invention may
also 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 folic stimulating hormone
(FSH). Thus, a protein of the present invention, alone or in
heterodimers with a member of the inhibin .alpha. 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 of the invention, as a homodimer or as a heterodimer
with other protein subunits of the inhibin-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, U.S. Pat. No. 4,798,885, the
disclosure of which is incorporated herein by reference. A protein
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.
[0425] Alternatively, as described in more detail below, genes
encoding these proteins or nucleic acids regulating the expression
of these proteins may be introduced into appropriate host cells to
increase or decrease the expression of the proteins as desired.
EXAMPLE 36A
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Chemotactic/Chemokinetic Activity
[0426] The proteins encoded by the extended cDNAs or portions
thereof may also be evaluated for chemotactic/chemokinetic
activity. For example, a protein 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, cosinophils, epithelial and/or
endothelial cells. Chemotactic and chmokinetic proteins can be used
to mobilize or attract a desired cell population to a desired site
of action. Chemotactic or chemokinetic proteins 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.
[0427] A protein or peptide 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 peptide has the ability to
directly stimulate directed movement of cells. Whether a particular
protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any
known assay for cell chemotaxis.
[0428] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0429] Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhension 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. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 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. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol, 153:1762-1768,
1994.
EXAMPLE 37
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Regulation of Blood Clotting
[0430] The proteins encoded by the extended cDNAs or portions
thereof 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.
[0431] Those proteins 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 of the invention may also
exhibit hemostatic or thrombolytic activity. As a result, such a
protein is expected to be useful in treatment of various
coagulations disorders (including hereditary disorders, such as
hemophilias) or to enhance coagulation and other hemostatic events
in treating wounds resulting from trauma, surgery or other causes.
A protein 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, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
Alternatively, as described in more detail below, genes encoding
these proteins or nucleic acids regulating the expression of these
proteins may be introduced into appropriate host cells to increase
or decrease the expression of the proteins as desired.
EXAMPLE 38
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Involvement in Receptor/Ligand Interactions
[0432] The proteins encoded by the extended cDNAs or a portion
thereof 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 7.28 (Measurement of Cellular Adhesion
under Static Conditions 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., J. 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., Cell 75:791-803,
1993.
[0433] For example, the proteins 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 respones). Receptors and ligands are
also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein
of the present invention (including, without limitation, fragments
of receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand interactions.
EXAMPLE 38A
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Anti-Inflammatory Activity
[0434] The proteins encoded by the extended cDNAs or a portion
thereof may also be evaluated for anti-inflammatory activity. The
anti-inflarnmatory 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 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 (SIRS)), 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 of the invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic
substance or material.
EXAMPLE 38B
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Tumor Inhibition Activity
[0435] The proteins encoded by the extended cDNAs or a portion
thereof may also be evaluated for tumor inhibition activity. In
addition to the activities described above for immunological
treatment or prevention of tumors, a protein of the invention may
exhibit other anti-tumor activities. A protein may inhibit tumor
growth directly or indirectly (such as, for example, via ADCC). A
protein 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 suppressing,
climinating or inhibiting factors, agents or cell types which
promote tumor growth.
[0436] A protein 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 limitation, 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 clirnination 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.
EXAMPLE 39
Identification of Proteins which Interact with Polypeptides Encoded
by Extended cDNAs
[0437] Proteins which interact with the polypeptides encoded by
extended cDNAs or portions thereof, 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 Matchmaker Two Hybrid System 2
(Catalog No. K1604-1, Clontech), which is incorporated herein by
reference, the extended cDNAs or portions thereof, 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 which
might interact with the polypeptides encoded by the extended cDNAs
or portions thereof are inserted into a second expression vector
such that they are in frame with DNA encoding the activation domain
of GAL4. 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 which interact with the polypeptide
encoded by the extended cDNAs or portions thereof.
[0438] Alternatively, the system described in Lustig et al.,
Methods in Enzymology 283: 83-99 (1997), the disclosure of which is
incorporated herein by reference, may be used for identifying
molecules which interact with the polypeptides encoded by extended
cDNAs. In such systems, in vitro transcription reactions are
performed on a pool of vectors containing extended cDNA inserts
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
acitivity.
[0439] 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 polypeptide.
[0440] Proteins or other molecules interacting with polypeptides
encoded by extended cDNAs can be found by a variety of additional
techniques. In one method, affinity columns containing the
polypeptide encoded by the extended cDNA or a portion thereof can
be constructed. In some versions, of this method the affinity
column contains chimeric proteins in which the protein encoded by
the extended cDNA or a portion thereof 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.
Proteins interacting with the 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 proteins 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.
[0441] Proteins interacting with polypeptides encoded by extended
cDNAs or portions thereof 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 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 manometers from the sensor
surface). In these screening assays, the target molecule can be one
of the polypeptides encoded by extended cDNAs or a portion thereof
and the test sample can be a collection of proteins extracted from
tissues or cells, a pool of expressed proteins, combinatorial
peptide and/or chemical libraries,or phage displayed peptides. The
tissues or cells from which the test proteins are extracted can
originate from any species.
[0442] In other methods, a target protein is immobilized and the
test population is a collection of unique polypeptides encoded by
the extended cDNAs or portions thereof.
[0443] To study the interaction of the proteins encoded by the
extended cDNAs or portions thereof 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 referenc can be
used.
[0444] The system described in U.S. Pat. No. 5,654,150, the
disclosure of which is incorporated herein by reference, may also
be used to identify molecules which interact with the polypeptides
encoded by the extended cDNAs. In this system, pools of extended
cDNAs are transcribed and translated in vitro and the reaction
products are assayed for interaction with a known polypeptide or
antibody.
[0445] It will be appreciated by those skilled in the art that the
proteins expressed from the extended cDNAs or portions may be
assayed for numerous activities in addition to those specifically
enumerated above. For example, the expressed proteins may be
evaluated for applications involving control and regulation of
inflammation, tumor proliferation or metastasis, infection, or
other clinical conditions. In addition, the proteins expressed from
the extended cDNAs or portions thereof may be useful as nutritional
agents or cosmetic agents.
[0446] The proteins expressed from the extended cDNAs or portions
thereof may be used to generate antibodies capable of specifically
binding to the expressed protein or fragments thereof as described
in Example 40 below. The antibodies may be capable of binding a
full length protein encoded by one of the sequences of SEQ ID NOs:
40-59, 61-73, 75, 77-82, and 130-154, a mature protein encoded by
one of the sequences of SEQ ID NOs. 40-59, 61-75, 77-82, and
130-154, or a signal peptide encoded by one of the sequences of SEQ
ID Nos. 40-59, 61-73, 75-82, 84 and 130-154. Alternatively, the
antibodies may be capable of binding fragments of the proteins
expressed from the extended cDNAs which comprise at least 10 amino
acids of the sequences of SEQ ID NOs: 85-129 and 155-179. In some
embodiments, the antibodies may be capable of binding fragments of
the proteins expressed from the extended cDNAs which comprise at
least 15 amino acids of the sequences of SEQ ID NOs: 85-129 and
155-179. In other embodiments, the antibodies may be capable of
binding fragments of the proteins expressed from the extended cDNAs
which comprise at least 25 amino acids of the sequences of SEQ ID
NOs: 85-129 and 155-179. In further embodiments, the antibodies may
be capable of binding fragments of the proteins expressed from the
extended cDNAs which comprise at least 40 amino acids of the
sequences of SEQ ID NOs: 85-129 and 155-179.
EXAMPLE 40
Production of an Antibody to a Human Protein
[0447] Substantially pure protein or polypeptide is isolated from
the transfected or transformed cells as described in Example 30.
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 micrograms/ml. Monoclonal or polyclonal antibody to
the protein can then be prepared as follows:
[0448] A. Monoclonal Antibody Production by Hybridoma Fusion
[0449] Monoclonal antibody to epitopes of any of the peptides
identified and isolated as described can be prepared from murine
hybridomas according to the classical method of Kohler, G. and
Milstein, C., 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 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 supematant fluid of the wells by immunoassay procedures,
such as Elisa, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), 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. Basic Methods
in Molecular Biology Elsevier, New York. Section 21-2.
[0450] B. Polyclonal Antibody Production by Immunization
[0451] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein or peptides derived therefrom
described above, which can be unmodified or modified to enhance
imrnmunogenicity. 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 vary in response to site of inoculations and dose,
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, J. et al. J. Clin. Endocrinol. Metab. 33:988-991
(1971).
[0452] 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, O. et al., Chap. 19 in: Handbook of
Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Afinity 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).
[0453] Antibody preparations prepared according to either protocol
are useful in quantitative immunoassays which determine
concentrations of antigen-bearing substances in biological samples;
they are also used semi-quantitatively or qualitatively to identify
the presence of antigen 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.
[0454] V. Use of Extended cDNAs or Portions Thereof as Reagents
[0455] The extended cDNAs of the present invention may be used as
reagents in isolation procedures, diagnostic assays, and forensic
procedures. For example, sequences from the extended cDNAs (or
genomic DNAs obtainable therefrom) may be detectably labeled and
used as probes to isolate other sequences capable of hybridizing to
them. In addition, sequences from the extended cDNAs (or genomic
DNAs obtainable therefrom) may be used to design PCR primers to be
used in isolation, diagnostic, or forensic procedures.
EXAMPLE 41
Preparation of PCR Primers and Amplification of DNA
[0456] The extended cDNAs (or genomic DNAs obtainable therefrom)
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. The PCR primers are at least 10 bases, and preferably
at least 12, 15, or 17 bases in length. More preferably, the PCR
primers are at least 20-30 bases 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. 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 polymerase such as Taq
polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in
the sample is denatured and the PCR primers are specifically
hybridized to complementary nucleic acid 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 42
Use of Extended cDNAs as Probes
[0457] Probes derived from extended cDNAs or portions thereof (or
genomic DNAs obtainable therefrom) 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. The nucleic acid sample may
comprise nucleic acids obtained from a variety of sources,
including genomic DNA, cDNA libraries, RNA, or tissue samples.
[0458] 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 cDNA
library which are capable of hybridizing to the detectable probe as
described in Example 30 above.
[0459] PCR primers made as described in Example 41 above may be
used in forensic analyses, such as the DNA fingerprinting
techniques described in Examples 43-47 below. Such analyses may
utilize detectable probes or primers based on the sequences of the
extended cDNAs isolated using the 5' ESTs (or genomic DNAs
obtainable therefrom).
EXAMPLE 43
Forensic Matching by DNA Sequencing
[0460] 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 extended cDNAs (or genomic DNAs obtainable
therefrom), is then utilized in accordance with Example 41 to
amplify DNA of approximately 100-200 bases in length from the
forensic specimen. Corresponding sequences are obtained from a 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 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 44
Positive Identification by DNA Sequencing
[0461] 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 sequences from Table IV and the
appended sequence listing. 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 41. Each of these DNA segments is
sequenced, using the methods set forth in Example 43. 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 45
Southern Blot Forensic Identification
[0462] The procedure of Example 44 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 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).
[0463] A panel of probes based on the sequences of the extended
cDNAs (or genomic DNAs obtainable therefrom), or fragments thereof
of at least 10 bases, are radioactively or calorimetrically 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
comprises at least 12, 15, or 17 consecutive nucleotides from the
extended cDNA (or genomic DNAs obtainable therefrom). More
preferably, the probe comprises at least 20-30 consecutive
nucleotides from the extended cDNA (or genomic DNAs obtainable
therefrom). In some embodiments, the probe comprises more than 30
nucleotides from the extended cDNA (or genomic DNAs obtainable
therefrom). In other embodiments, the probe comprises at least 40,
at least 50, at least 75, at least 100, at least 150, or at least
200 consecutive nucleotides from the extended cDNA (or genomic DNAs
obtainable therefrom).
[0464] 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 extended cDNAs (or genomic DNAs
obtainable therefrom) will be a unique identifier. Since the
restriction enzyme cleavage will be different for every individual,
the band pattern on the Southem blot will also be unique.
Increasing the number of extended cDNA 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 46
Dot Blot Identification Procedure
[0465] Another technique for identifying individuals using the
extended cDNA sequences disclosed herein utilizes a dot blot
hybridization technique.
[0466] Genomic DNA is isolated from nuclei of subject to be
identified. Oligonucleotide probes of approximately 30 bp in length
are synthesized that correspond to at least 10, preferably 50
sequences from the extended cDNAs or genomic DNAs obtainable
therefrom. The probes are used to hybridize to the genomic DNA
through conditions known to those in the art. The oligonucleotides
are end labeled with P.sup.32 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 Calif.). The nitrocellulose filter containing the
genomic sequences is baked or UV linked to the filter,
prehybridized and hybridized with labeled probe using techniques
known in the art (Davis et al. supra). The .sup.32P labeled DNA
fragments are sequentially hybridized with successively stringent
conditions to detect minimal differences between the 30 bp 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.
[0467] Extended cDNAs or oligonucleotides containing at least 10
consecutive bases from these sequences can be used as probes in the
following alternative fingerprinting technique. Preferably, the
probe comprises at least 12, 15, or 17 consecutive nucleotides from
the extended cDNA (or genomic DNAs obtainable therefrom). More
preferably, the probe comprises at least 20-30 consecutive
nucleotides from the extended cDNA (or genomic DNAs obtainable
therefrom). In some embodiments, the probe comprises more than 30
nucleotides from the extended cDNA (or genomic DNAs obtainable
therefrom). In other embodiments, the probe comprises at least 40,
at least 50, at least 75, at least 100, at least 150, or at least
200 consecutive nucleotides from the extended cDNA (or genomic DNAs
obtainable therefrom).
[0468] Preferably, a plurality of probes having sequences from
different genes are used in the alternative fingerprinting
technique. Example 47 below provides a representative alternative
fingerprinting procedure in which the probes are derived from
extended cDNAs.
EXAMPLE 47
Alternative "Fingerprint" Identification Technique
[0469] 20-mer oligonucleotides are prepared from a large number,
e.g. 50, 100, or 200, of extended cDNA sequences (or genomic DNAs
obtainable therefrom) using commercially available oligonucleotide
services such as Genset, Paris, France. 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 containing 5 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.
[0470] 10 ng of each of the oligonucleotides are pooled and
end-labeled with P.sup.32. 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.
[0471] It is additionally contemplated within this example that the
number of probe sequences used can be varied for additional
accuracy or clarity.
[0472] The antibodies generated in Examples 30 and 40 above may be
used to identify the tissue type or cell species from which a
sample is derived as described above.
EXAMPLE 48
Identification of Tissue Types or Cell Species by Means of Labeled
Tissue Specific Antibodies
[0473] Identification of specific tissues is accomplished by the
visualization of tissue specific antigens by means of antibody
preparations according to Examples 30 and 40 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.
[0474] 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.
[0475] A. Immunohistochemical Techniques
[0476] 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, 3rd Ed. Lange, Los Altos, Calif. (1980) or Rose, N. et
al., Chap. 12 in: Methods in Immunodiagnosis, 2d Ed. John Wiley 503
Sons, New York (1980).
[0477] 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 .sup.125I, and detected by
overlaying the antibody treated preparation with photographic
emulsion.
[0478] 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.
[0479] Tissue sections and cell suspensions are prepared for
immunohistochemical examination according to common histological
techniques. Multiple cryostat sections (about 4 .mu.m, unfixed) of
the unknown 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.
[0480] 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.
[0481] 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.
[0482] 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.
[0483] B. Identification of Tissue Specific Soluble Proteins
[0484] 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.
[0485] 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.
[0486] 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), 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 .mu.l, and containing from about
1 to 100 .mu.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., (above) 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 30 and
40. In this procedure, as in procedure A above, appropriate
positive and negative sample and reagent controls are run.
[0487] In either procedure A or B, 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.
[0488] The visualization of tissue specific antigen binding at
levels above those seen in control tissues to one or more tissue
specific antibodies, prepared from the gene sequences identified
from extended cDNA sequences, can identify tissues of unknown
origin, for example, forensic samples, or differentiated tumor
tissue that has metastasized to foreign bodily sites.
[0489] In addition to their applications in forensics and
identification, extended cDNAs (or genomic DNAs obtainable
therefrom) may be mapped to their chromosomal locations. Example 49
below describes radiation hybrid (RH) mapping of human chromosomal
regions using extended cDNAs. Example 50 below describes a
representative procedure for mapping an extended cDNA (or a genomic
DNA obtainable therefrom) to its location on a human chromosome.
Example 51 below describes mapping of extended cDNAs (or genomic
DNAs obtainable therefrom) on metaphase chromosomes by Fluorescence
In Situ Hybridization (FISH).
EXAMPLE 49
Radiation Hybrid Mapping of Extended cDNAs to the Human Genome
[0490] 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
any human genorme marker. Human DNA isolated from a panel of 80-100
cell lines provides a mapping reagent for ordering extended cDNAs
(or genomic DNAs obtainable therefrom). 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).
[0491] 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 (Warrington et al., Genomics
11:701-708, 1991).
EXAMPLE 50
Mapping of Extended cDNAs to Human Chromosomes using PCR
Techniques
[0492] Extended cDNAs (or genomic DNAs obtainable therefrom) may be
assigned to human chromosomes using PCR based methodologies. In
such approaches, oligonucleotide primer pairs are designed from the
extended cDNA sequence (or the sequence of a genomic DNA obtainable
therefrom) to minimize the chance of amplifying through an intron.
Preferably, the oligonucleotide primers are 18-23 bp in length and
are designed for PCR amplification. The creation of PCR primers
from known sequences is well known to those with skill in the art.
For a review of PCR technology see Erlich, H. A., PCR Technology;
Principles and Applications for DNA Amplification, 1992. W.H.
Freeman and Co., New York.
[0493] 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 .mu.Cu of a .sup.32P-labeled deoxycytidine triphosphate. The
PCR is performed in a microplate thennocycler (Techne) under the
following conditions: 30 cycles of 94.degree. C., 1.4 min;
55.degree. C., 2 min; and 72.degree. C., 2 min; with a final
extension at 72.degree. 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 the ends of the primer sequences
in the extended cDNA 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.J.).
[0494] 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 extended cDNA (or genomic DNA obtainable therefrom). DNA
is isolated from the somatic hybrids and used as starting templates
for PCR reactions using the primer pairs from the extended cDNAs
(or genomic DNAs obtainable therefrom). Only those somatic cell
hybrids with chromosomes containing the human gene corresponding to
the extended cDNA (or genomic DNA obtainable therefrom) will yield
an amplified fragment. The extended cDNAs (or genomic DNAs
obtainable therefrom) 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 extended cDNA (or genomic DNA obtainable
therefrom). For a review of techniques and analysis of results from
somatic cell gene mapping experiments. (See Ledbetter et al.,
Genomics 6:475-481 (1990).)
[0495] Alternatively, the extended cDNAs (or genomic DNAs
obtainable therefrom) may be mapped to individual chromosomes using
FISH as described in Example 51 below.
EXAMPLE 51
Mapping of Extended 5' ESTs to Chromosomes Using Fluorescence in
situ Hybridization
[0496] Fluorescence in situ hybridization allows the extended cDNA
(or genomic DNA obtainable therefrom) 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.
[0497] In a preferred embodiment, chromosomal localization of an
extended cDNA (or genomic DNA obtainable therefrom) is obtained by
FISH as described by Cherif et al. (Proc. Natl. Acad. Sci. U.S.A.,
87:6639-6643, 1990). 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 .mu.M)
is added for 17 h, followed by addition of 5-bromodeoxyuridine
(5-BudR, 0.1 mM) for 6 h. Colcemid (1 .mu.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.degree. 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 extended cDNA (or genomic DNA
obtainable therefrom) 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, Upssala, Sweden) and precipitated. Just
prior to hybridization, the DNA pellet is dissolved in
hybridization buffer (50% formamide, 2.times.SSC, 10% dextran
sulfate, 1 mg/ml sonicated salmon sperm DNA, pH 7) and the probe is
denatured at 70.degree. C. for 5-10 mm.
[0498] Slides kept at -20.degree. C. are treated for 1 h at
37.degree. C. with RNase A (100 .mu.g/ml), rinsed three times in
2.times.SSC and dehydrated in an ethanol series. Chromosome
preparations are denatured in 70% formamide, 2.times.SSC for 2 min
at 70.degree. C., then dehydrated at 4.degree. C. The slides are
treated with proteinase K (10 .mu.g/100 ml in 20 mM Tris-HCl, 2 mM
CaCl.sub.2) at 37.degree. 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.degree. C. After hybridization
and post-hybridization washes, the biotinylated probe is detected
by avidin-FITC and amplified with additional layers of biotinylated
goat anti-avidin and avidin-FITC. 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 extended
cDNA (or genomic DNA obtainable therefrom) may be localized to a
particular cytogenetic R-band on a given chromosome.
[0499] Once the extended cDNAs (or genomic DNAs obtainable
therefrom) have been assigned to particular chromosomes using the
techniques described in Examples 49-51 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.
EXAMPLE 52
Use of Extended cDNAs to Construct or Expand Chromosome Maps
[0500] 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
extended cDNAs (or genomic DNAs obtainable therefrom) are obtained.
This approach is described in Ramaiah Nagaraja et al. Genome
Research 7:210-222, March 1997. 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 extended cDNA
(or genomic DNA obtainable therefrom) whose position is to be
determined. Once an insert has been found which includes the
extended cDNA (or genomic DNA obtainable therefrom), the insert can
be analyzed by PCR or other methods to determine whether the insert
also contains other sequences known to be on the chromosome or in
the region from which the extended cDNA (or genomic DNA obtainable
therefrom) was derived. This process can be repeated for each
insert in the YAC library to determine the location of each of the
extended cDNAs (or genomic DNAs obtainable therefrom) 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 organisms chromosomes may be obtained.
[0501] As described in Example 53 below extended cDNAs (or genomic
DNAs obtainable therefrom) may also be used to identify genes
associated with a particular phenotype, such as hereditary disease
or drug response.
EXAMPLE 53
Identification of Genes Associated with Hereditary Diseases or Drug
Response
[0502] This example illustrates an approach useful for the
association of extended cDNAs (or genomic DNAs obtainable
therefrom) with particular phenotypic characteristics. In this
example, a particular extended cDNA (or genomic DNA obtainable
therefrom) is used as a test probe to associate that extended cDNA
(or genomic DNA obtainable therefrom) with a particular phenotypic
characteristic.
[0503] Extended cDNAs (or genomic DNAs obtainable therefrom) are
mapped to a particular location on a human chromosome using
techniques such as those described in Examples 49 and 50 or other
techniques known in 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 extended cDNA (or
genomic DNA obtainable therefrom) 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 extended cDNA (or genomic DNA obtainable therefrom) thus
becomes an immediate candidate for each of these genetic
diseases.
[0504] Cells from patients with these diseases or phenotypes are
isolated and expanded in culture. PCR primers from the extended
cDNA (or genomic DNA obtainable therefrom) are used to screen
genomic DNA, mRNA or cDNA obtained from the patients. Extended
cDNAs (or genomic DNAs obtainable therefrom) that are not amplified
in the patients can be positively associated with a particular
disease by further analysis. Alternatively, the PCR analysis may
yield fragments of different lengths when the samples are derived
from an individual having the phenotype associated with the disease
than when the sample is derived from a healthy individual,
indicating that the gene containing the extended cDNA may be
responsible for the genetic disease.
[0505] VI. Use of Extended cDNAs (or Genomic DNAs Obtainable
Therefrom) to Construct Vectors
[0506] The present extended cDNAs (or genomic DNAs obtainable
therefrom) may also be used to construct secretion vectors capable
of directing the secretion of the proteins encoded by genes
inserted in the vectors. 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 54 below.
EXAMPLE 54
Construction of Secretion Vectors
[0507] 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.
[0508] A signal sequence from an extended cDNA (or genomic DNA
obtainable therefrom), such as one of the signal sequences in SEQ
ID NOs: 40-59, 61-73, 75-82, 84, and 130-154 as defined in Table IV
above, is operably linked to the promoter such that the mRNA
transcribed from the promoter will direct the translation of the
signal peptide. 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 extended cDNA (or genomic DNA obtainable
therefrom). Suitable hosts include mammalian cells, tissues or
organisms, avian cells, tissues, or organisms, insect cells,
tissues or organisms, or yeast.
[0509] 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 inserted gene
is expressed from the mRNA transcribed from the promoter. The
signal peptide directs the extracellular secretion of the fusion
protein.
[0510] 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. 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, baculovirus vectors, or bacterial
plasmids capable of being transiently introduced into the host.
[0511] 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.
[0512] 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, DEAE-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,
immunochromatography, 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.
[0513] 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.
[0514] The extended cDNAs or 5' ESTs may also be used to clone
sequences located upstream of the extended cDNAs or 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 55 describes a method for cloning sequences
upstream of the extended cDNAs or 5' ESTs.
EXAMPLE 55
Use of Extended cDNAs or 5' ESTs to Clone Upstream Sequences from
Genomic DNA
[0515] Sequences derived from extended cDNAs or 5' ESTs may be used
to isolate the promoters of the corresponding genes using
chromosome walking techniques. In one chromosome walking technique,
which utilizes the GenomeWalkerm kit available from Clontech, five
cornplete 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.
[0516] 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 adaptor
primer provided in the kit and an outer gene specific primer. The
gene specific primer should be selected to be specific for the
extended cDNA or 5' EST of interest and should have a melting
temperature, length, and location in the extended cDNA or ' EST
which is consistent with its use in PCR reactions. Each first PCR
reaction contains 5 ng of genomic DNA, 5 .mu.l of 10.times. Tth
reaction buffer, 0.2 mM of each dNTP, 0.2 .mu.M each of outer
adaptor primer and outer gene specific primer, 1.1 mM of
Mg(OAc).sub.2, and 1 .mu.l of the Tth polymerase 50.times. mix in a
total volume of 50 .mu.l. The reaction cycle for the first PCR
reaction is as follows: 1 min @ 94.degree. C./2 sec @ 94.degree.
C., 3 min @ 72.degree. C. (7 cycles)/2 sec @ 94.degree. C., 3 min @
67.degree. C. (32 cycles)/5 min @ 67.degree. C.
[0517] 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 .mu.l of the reaction product of the first
PCR reaction mixture may be diluted 180 times. Reactions are made
in a 50 .mu.l 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 adaptor, and is provided with the
GenomeWalker.TM. kit. The second nested primer is specific for the
particular extended cDNA or 5' EST for which the promoter is to be
cloned and should have a melting temperature, length, and location
in the extended cDNA or 5' EST which is consistent with its use in
PCR reactions. The reaction parameters of the second PCR reaction
are as follows: 1 min @ 94.degree. C./2 sec @ 94.degree. C., 3 min
@ 72.degree. C. (6 cycles)/2 sec @ 94.degree. C., 3 min @
67.degree. C. (25 cycles)/5 min @ 67.degree. C.
[0518] The product of the second PCR reaction is purified, cloned,
and sequenced using standard techniques. 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 extended cDNA or 5' EST sequence is hybridized
to the single stranded DNA. Hybrids between the biotinylated
oligonucleotide and the single stranded DNA containing the extended
cDNA or EST sequence are isolated as described in Example 29 above.
Thereafter, the single stranded DNA containing the extended cDNA or
EST sequence is released from the beads and converted into double
stranded DNA using a primer specific for the extended cDNA or 5'
EST sequence or a primer corresponding to a sequence included in
the cloning vector. The resulting double stranded DNA is
transformed into bacteria. DNAs containing the 5' EST or extended
cDNA sequences are identified by colony PCR or colony
hybridization.
[0519] 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 extended
cDNAs or 5' ESTs with databases containing known transcription
start sites, transcription factor binding sites, or promoter
sequences.
[0520] In addition, promoters in the upstream sequences may be
identified using promoter reporter vectors as described in Example
56.
EXAMPLE 56
Identification of Promoters in Cloned Upstream Sequences
[0521] The genomic sequences upstream of the extended cDNAs or 5'
ESTs are cloned into a suitable promoter reporter vector, such as
the pSEAP-Basic, pSEAP-Enhancer, p.beta.gal-Basic,
p.beta.gal-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, .beta. galactosidase, or green fluorescent protein.
The sequences upstream of the extended cDNAs or 5' ESTs 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.
[0522] 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 extended cDNAs and ESTs. For example,
if the expression pattern analysis indicates that the mRNA
corresponding to a particular extended cDNA or 5' EST is expressed
in fibroblasts, the promoter reporter vector may be introduced into
a human fibroblast cell line.
[0523] 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. The effects of these mutations on
transcription levels may be determined by inserting the mutations
into the cloning sites in the promoter reporter vectors.
EXAMPLE 57
Cloning and Identification of Promoters
[0524] Using the method described in Example 55 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:29) and CTG CCA
TGT ACA TGA TAG AGA GAT TC (SEQ ID NO:30), the promoter having the
internal designation P13H2 (SEQ ID NO:31) was obtained.
[0525] Using the primer pairs GTA CCA GGGG ACT GTG ACC ATT GC (SEQ
ID NO:32) and CTG TGA CCA TTG CTC CCA AGA GAG (SEQ ID NO:33), the
promoter having the internal designation P15B4 (SEQ ID NO:34) was
obtained.
[0526] Using the primer pairs CTG GGA TGG AAG GCA CGG TA (SEQ ID
NO:35) and GAG ACC ACA CAG CTA GAC AA (SEQ ID NO:36), the promoter
having the internal designation P29B6 (SEQ ID NO:37) was
obtained.
[0527] FIG. 8 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 Matlnspector
release 2.0, August 1996.
[0528] FIG. 9 describes the transcription factor binding sites
present in each of these promoters. The columns labeled matrice
provides the name of the MatInspector 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 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.
[0529] The promoters and other regulatory sequences located
upstream of the extended cDNAs or 5' ESTs 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 in
Example 26 above. For example, if a promoter which confers a high
level of expression in muscle is desired, the promoter sequence
upstream of an extended cDNA or 5' EST derived from an mRNA which
is expressed at a high level in muscle, as determined by the method
of Example 26, may be used in the expression vector.
[0530] 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 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.
[0531] 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.
[0532] Following the identification of promoter sequences using the
procedures of Examples 55-57, proteins which interact with the
promoter may be identified as described in Example 58 below.
EXAMPLE 58
Identification of Proteins which Interact with Promoter Sequences,
Upstream Regulatory Sequences, or mRNA
[0533] 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. 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.
[0534] 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 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.
[0535] VII. Use of Extended cDNAs (or Genomic DNAs Obtainable
Therefrom) in Gene Therapy
[0536] The present invention also comprises the use of extended
cDNAs (or genomic DNAs obtainable therefrom) in gene therapy
strategies, including antisense and triple helix strategies as
described in Examples 57 and 58 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 59
Preparation and Use of Antisense Oligonucleotides
[0537] 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 extended cDNA (or
genomic DNA obtainable therefrom). The antisense nucleic acids
should have a length and melting temperature sufficient to permit
formation of an intracellular duplex having 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.
[0538] 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 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.
[0539] 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).
[0540] Various types of antisense oligonucleotides complementary to
the sequence of the extended cDNA (or genomic DNA obtainable
therefrom) may be used. In one preferred embodiment, stable and
semi-stable antisense oligonucleotides described in International
Application No. PCT WO94/23026, 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.
[0541] 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.
[0542] 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.
[0543] 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 proteins and
are effective as decoys therefor. These molecules may contain
"hairpin" structures, "dumbbell" structures, "modified dumbbell"
structures, "cross-linked" decoy structures and "loop"
structures.
[0544] 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.
[0545] 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.
[0546] 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
encapsulated 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.
[0547] The antisense molecules are introduced onto cell samples at
a number of different concentrations preferably between
1.times.10.sup.-10M to 1.times.10.sup.-4M. 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 1.times.10.sup.-7 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.
[0548] 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.
[0549] 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 that include but are not limited to
antibody-mediated tests such as RIAs and ELISA, functional assays,
or radiolabeling.
[0550] The extended cDNAs of the present invention (or genomic DNAs
obtainable therefrom) 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 extended
cDNAs (or genomic DNAs obtainable therefrom) 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, a
portion of the extended cDNA (or genomic DNA obtainable therefrom)
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 extended cDNA or from the gene corresponding to the
extended cDNA are contemplated within the scope of this
invention.
EXAMPLE 60
Preparation and Use of Triple Helix Probes
[0551] The sequences of the extended cDNAs (or genomic DNAs
obtainable therefrom) 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.
[0552] 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.
[0553] Treated cells are monitored for altered cell finction 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 finctions to be
monitored are predicted based upon the homologies of the target
gene corresponding to the extended cDNA from which the
oligonucleotide was derived with known gene sequences that have
been associated with a particular function. The cell finctions can
also be predicted based on the presence of abnormal physiologies
within cells derived from individuals with a particular inherited
disease, particularly when the extended cDNA is associated with the
disease using techniques described in Example 53.
[0554] 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 59 at a dosage
calculated based on the in vitro results, as described in Example
59.
[0555] 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 Griffm et
al. (Science 245:967-971 (1989), which is hereby incorporated by
this reference).
EXAMPLE 61
Use of Extended cDNAs to Express an Encoded Protein in a Host
Organism
[0556] The extended cDNAs of the present invention may also be used
to express an encoded protein in a host organism to produce a
beneficial effect. In such procedures, the encoded protein may be
transiently expressed in the host organism or stably expressed in
the host organism. The encoded protein may have any of the
activities described above. The encoded protein may be a protein
which the host organism lacks or, alternatively, the encoded
protein may augment the existing levels of the protein in the host
organism.
[0557] A full length extended cDNA encoding the signal peptide and
the mature protein, or an extended cDNA encoding only the mature
protein is introduced into the host organism. The extended cDNA 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.
[0558] Alternatively, the extended cDNA 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.
[0559] 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. Cells
containing the expression vector are thereafter selected and
introduced into the host organism, where they express the encoded
protein to produce a beneficial effect.
EXAMPLE 62
Use of Signal Peptides Encoded By 5' Ests or Sequences Obtained
Therefrom to Import Proteins into Cells
[0560] The short core hydrophobic region (h) of signal peptides
encoded by the 5'ESTS or extended cDNAs derived from the 5'ESTs of
the present invention may also be used as a carrier 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)).
[0561] 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 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.
[0562] This method may be applied to study diverse intracellular
functions and cellular processes. For instance, it has been used to
probe finctionally relevant domains of intracellular proteins and
to examine protein-protein interactions involved in signal
transduction pathways (Lin 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., Bioch. Biophys. Res. Commun.,
234: 675-680 (1997)).
[0563] 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.
[0564] 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 in
examples 59 and 60 respectively, in order to inhibit processing and
maturation of a target cellular RNA.
EXAMPLE 63
Reassembling & Resequencing of Clones
[0565] Full length cDNA clones obtained by the procedure described
in Example 27 were double-sequenced. These sequences were assembled
and the resulting consensus sequences were then reanalyzed. Open
reading frames were reassigned following essentially the same
process as the one described in Example 27.
[0566] After this reanalysis process a few abnormalities were
revealed. The sequence presented in SEQ ID NO: 84 is apparently
unlikely to be genuine full length cDNAs. This clone is more
probably a 3' truncated cDNA sequence based on homology studies
with existing protein sequences. Similarly, the sequences presented
in SEQ ID NOs: 60, 76, 83 and 84 may also not be genuine full
length cDNAs based on homology studies with existing protein
sequences. Although these sequences encode a potential start
methionine, except for SEQ ID NO:60, they could represent a 5'
truncated cDNA.
[0567] Finally, after the reassignment of open reading frames for
the clones, new open reading frames were chosen in some instances.
For example, in the case of SEQ ID NOs: 60, 74 and 83 the new open
reading frames were no longer predicted to contain a signal
peptide.
[0568] As discussed above, Table IV provides the sequence
identification numbers of the extended cDNAs of the present
invention, the locations of the full coding sequences in SEQ ID
NOs: 40-84 and 130-154 (i.e. the nucleotides encoding both the
signal peptide and the mature protein, listed under the heading FCS
location in Table IV), the locations of the nucleotides in SEQ ID
NOs: 40-84 and 130-154 which encode the signal peptides (listed
under the heading SigPep Location in Table IV), the locations of
the nucleotides in SEQ ID NOs: 40-84 and 130-154 which encode the
mature proteins generated by cleavage of the signal peptides
(listed under the heading Mature Polypeptide Location in Table IV),
the locations in SEQ ID NOs: 40-84 and 130-154 of stop codons
(listed under the heading Stop Codon Location in Table IV) the
locations in SEQ ID NOs: 40-84 and 130-154 of polyA signals (listed
under the heading g PolyA Signal Location in Table IV) and the
locations of polyA sites (listed under the heading PolyA Site
Location in Table IV).
[0569] As discussed above, Table V lists the sequence
identification numbers of the polypeptides of SEQ ID NOs: 85-129
and 155-179, the locations of the amino acid residues of SEQ ID
NOs: 85-129 and 155-179 in the full length polypeptide (second
column), the locations of the amino acid residues of SEQ ID NOs:
85-129 and 155-179 in the signal peptides (third column), and the
locations of the amino acid residues of SEQ ID NOs: 85-129 and
155-179 in the mature polypeptide created by cleaving the signal
peptide from the full length polypeptide (fourth column). In Table
V, and in the appended sequence listing, the first amino acid of
the mature protein resulting from cleavage of the signal peptide is
designated as amino acid number 1 and the first amino acid of the
signal peptide is designated with the appropriate negative number,
in accordance with the regulations governing sequence listings.
EXAMPLE 64
Functional Analysis of Predicted Protein Sequences
[0570] Following double-sequencing, new contigs were assembled for
each of the extended cDNAs of the present invention and each was
compared to known sequences available at the time of filing. These
sequences originate from the following databases: Genbank (release
108 and daily releases up to October, 15, 1998), Genseq (release
32) PIR (release 53) and Swissprot (release 35). The predicted
proteins of the present invention matching known proteins were
further classified into 3 categories depending on the level of
homology.
[0571] The first category contains proteins of the present
invention exhibiting more than 80% identical amino acid residues on
the whole length of the matched protein. They are clearly close
homologues which most probably have the same function or a very
similar function as the matched protein.
[0572] The second category contains proteins of the present
invention exhibiting more remote homologies (30 to 80% over the
whole protein) indicating that the protein of the present invention
is susceptible to have a function similar to the one of the matched
protein.
[0573] The third category contains proteins exhibiting either high
homology (90 to 100%) to a short domain or more remote homology (40
to 60%) to a larger domain of a known protein indicating that the
matched protein and the protein of the invention may share similar
features.
[0574] It should be noted that the numbering of amino acids in the
protein sequences discussed in FIGS. 10 to 12, and Table VIII, the
first methionine encountered is designated as amino acid number 1.
In the appended sequence listing, the first amino acid of the
mature protein resulting from cleavage of the signal peptide is
designated as amino acid number 1 and the first amino acid of the
signal peptide is designated with the appropriate negative number,
in accordance with the regulations governing sequence listings.
[0575] In addition, all of the corrected amino acid sequences (SEQ
ID NOs: 85-129 and 155-179) were scanned for the presence of known
protein signatures and motifs. This search was performed against
the Prosite 15.0 database, using the Proscan software from the GCG
package. Functional signatures and their locations are indicated in
Table VIII.
[0576] A) Proteins which are Closely Related to Known Proteins
[0577] Protein of SEQ ID NO: 120 (Internal Designation
26-44-1-B5-CL3 1)
[0578] The protein of SEQ ID NO: 120 encoded by the extended cDNA
SEQ ID NO: 75 isolated from ovary shows extensive homology to a
human protein called phospholemman or PLM and its homologues in
rodent and canine species. PLM is encoded by the nucleic acid
sequence of Genbank accession number U72245 and has the amino acid
sequence of SEQ ID NO: 180. Phospholemman is a prominent plasma
membrane protein whose phosphorylation correlates with an increase
in contractility of myocardium and skeletal muscle. Initially
described as a simple chloride channel, it has recently been shown
to be a channel for taurine that acts as an osmolyte in the
regulation of cell volume (Moorman et al, Adv Exp. Med. Biol.,
442:219-228 (1998)).
[0579] As shown by the alignment in FIG. 10 between the protein of
SEQ ID NO:120 and PLM, the amino acid residues are identical except
for positions 3 and 5 in the 92 amino acid long matched protein.
The substitution of a proline residue at position 3 par another
neutral residue, serine, is conservative. In addition, the protein
of the invention also exhibits the typical ATP1G/PLM/MAT8 PROSITE
signature (position 27 to 40 in bold in FIG. 10) for a family
containing mostly proteins known to be either chloride channels or
chloride channel regulators. In addition, the protein of invention
contains 2 short transmembrane segments from positions 1 to 21 and
from 37 to 57 as predicted by the software TopPred II (Claros and
von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)). The first
segment (in italic) corresponds to the signal peptide of PLM and
the second transmembrane domains (underlined) matches the
transmembrane region (double-underlined) shown to be the chloride
channel itself (Chen et al., Circ. Res., 82:367-374 (1998)).
[0580] Taken together, these data suggest that the protein of SEQ
ID NO 120 may be involved in the regulation of cell volume and in
tissue contractility. Thus, this protein may be useful in
diagnosing and/or treating several types of disorders including,
but not limited to, cancer, diarrhea, fertility disorders, and in
contractility disorders including muscle disorders, pulmonary
disorders and myocardial disorders.
[0581] Proteins of SEQ ID NOs: 121 (Internal Designation
47-4-4-C6-CL2 3)
[0582] The protein of SEQ ID NO: 121 encoded by the extended cDNA
SEQ ID NO: 76 found in substantia nigra shows extensive homology
with the human E25 protein. The E25 protein is encoded by the
nucleic acid sequence of Genbank accession number AF038953 and has
the amino acid sequence of SEQ ID NO: 181. The matched protein
might be involved in the development and differentiation of
haematopoietic stem/progenitor cells. In addition, it is the human
homologue of a munne protein thought to be involved in
chondro-osteogenic differentiation and belonging to a novel
multigene family of integral membrane proteins (Deleersnijder et
al. J. Biol. Chem., 271 :19475-19482 (1996)).
[0583] As shown by the alignments in FIG. 11 between the protein of
SEQ ID NO:121 and E25, the amino acid residues are identical except
for positions 9, 24 and 121 in the 263 amino acid long matched
sequence. All these substitutions are conservative. In addition,
the protein of invention contains one short transmembrane segment
from positions 1 to 21 (underlined in FIG. 11) matching the one
predicted for the murine E25 protein as predicted by the software
TopPred II (Claros and von Heijne, CABIOS applic. Notes, 10
:685-686 (1994)).
[0584] Taken together, these data suggest that the protein of SEQ
ID NO: 121 may be involved in cellular proliferation and
differentiation, and/or in haematopoiesis. Thus, this protein may
be useful in diagnosing and/or treating several types of disorders
including, but not limited to, cancer, hematological,
chondro-osteogenic and embryogenetic disorders.
[0585] Proteins of SEQ ID NO: 128 (Internal Designation
58-34-2-H8-CL1 3)
[0586] The protein of SEQ ID NO: 128 encoded by the extended cDNA
SEQ ID NO: 83 isolated from kidney shows extensive homology to the
murine WW-domain binding protein 1 or WWBP-1. WWBP-1 is encoded by
the nucleic acid sequence of Genbank accession number U40825 and
has the amino acid sequence of SEQ ID NO: 182. This protein is
expressed in placenta, lung, liver and kidney is thought to play a
role in intracellular signaling by binding to the WW domain of the
Yes protooncogene-associated protein via its so-called PY domain
(Chen and Sudol, Proc. Natl. Acad. Sci., 92 :7819-7823 (1995)). The
WW--PY domains are thought to represent a new set of modular
protein-binding sequences just like the SH3--PXXP domains (Sudol et
al., FEBS Lett., 369 :67-71 (1995)).
[0587] As shown by the alignments of FIG. 12 between the protein of
SEQ ID NO: 128 and WWBP-1, the amino acid residues are identical to
those of the 305 amino acid long matched protein except for
positions 53, 66, 78, 89, 92, 94, 96, 100, 102, 106, 110, 113, 124,
128, 136, 139, 140, 142-144, 166, 168, 173, 176, 178, 181, 182,
188, 196, 199, 201, 202, 207 and 210 of the matched protein. 68% of
these substitutions are conservative. Indeed the histidine-rich PY
domain is present in the protein of the invention (positions 82-86
in bold in FIG. 12).
[0588] Taken together, these data suggest that the protein of SEQ
ID NO: 128 may play a role in intracellular signaling. 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.
[0589] B) Proteins which are Remotely Related to Proteins with
Known Functions
[0590] Protein of SEO ID NO: 97 (Internal Designation
108-004-5-0-G6-FL)
[0591] The protein SEQ ID NO: 97 found in liver encoded by the
extended cDNA SEQ ID NO: 52 shows homology to a lectin-like
oxidized LDL receptor (LOX-1) found in human, bovine and murine
species. Such type II proteins with a C-lectin-like domain,
expressed in vascular endothelium and vascular-rich organs, bind
and internalize oxidatively modified low-density lipoproteins
(Sawamura et al, Nature, 386:73-77, (1997)). The oxidized
lipoproteins have been implicated in the pathogenesis of
atherosclerosis, a leading cause of death in industrialized
countries (see review by Parthasarathy et al, Biochem. Pharmacol.
56:279-284 (1998)). In addition, type II membrane proteins with a
C-terminus C-type lectin domain, also known as
carbohydrate-recognition domains, also include proteins involved in
target-cell recognition and cell activation.
[0592] The protein of invention has the typical structure of a type
II protein belonging to the C-type lectin family. Indeed, it
contains a short 31-amino-acid-long N-terminal tail, a
transmembrane segment from positions 32 to 52 matching the one
predicted for human LOX-1 and a large 177-amino-acid-long
C-terminal tail as predicted by the software TopPred II (Claros and
von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)). All six
cysteines of LOX-1 C-type lectin domain are also conserved in the
protein of the invention (positions 102, 113, 130, 195, 208 and
216) although the characteristic PROSITE signature of this family
is not. The LOX-1 protein is encoded by the nucleic acid sequence
of Genbank accession number: AB010710.
[0593] Taken together, these data suggest that the protein of SEQ
ID NO: 97 may be involved in the metabolism of lipids and/or in
cell-cell or cell-matrix interactions and/or in cell activation.
Thus, this protein or part therein, may be useful in diagnosing and
treating several disorders including, but not limited to, cancer,
hyperlipidaemia, cardiovascular disorders and neurodegenerative
disorders.
[0594] Protein of SEQ ID NO: 111 (Internal Designation
108-008-5-0-G12-FL)
[0595] The protein SEQ ID NO: 111 encoded by the extended cDNA SEQ
ID NO:66 shows homology to a mitochondrial protein found in
Saccharomyces Cerevisiae (PIR:S72254) which is similar to E. Coli
ribosomal protein L36. The typical PROSITE signature for ribosomal
L36 is present in the protein of the invention (positions 76-102)
except for a substitution of a tryptophane residue instead of a
valine, leucine, isoleucine, methionine or asparagine residue.
[0596] Taken together, these data suggest that the protein of SEQ
ID NO: 111 may be involved in protein biosynthesis. Thus, this
protein may be useful in diagnosing and/or treating several types
of disorders including, but not limited to, cancer.
[0597] Protein of SEQ ID NO: 94 (Internal Designation
108-004-5-0-D10-FL)
[0598] The protein SEQ ID NO: 94 encoded by the extended cDNA SEQ
ID NO: 49 shows remote homology to a subfamily of
beta4-galactosyltransferases widely conserved in animals (human,
rodents, cow and chicken). Such enzymes, usually type II membrane
proteins located in the endoplasmic reticulum or in the Golgi
apparatus, catalyzes the biosynthesis of glycoproteins, glycolipid
glycans and lactose. Their characteristic features defined as those
of subfamily A in Breton et al, J. Biochem., 123:1000-1009 (1998)
are pretty well conserved in the protein of the invention,
especially the region I containing the DVD motif (positions
163-165) thought to be involved either in UDP binding or in the
catalytic process itself.
[0599] In addition, the protein of invention has the typical
structure of a type II protein. Indeed, it contains a short
28-amino-acid-long N-terminal tail, a transmembrane segment from
positions 29 to 49 and a large 278-amino-acid-long C-terminal tail
as predicted by the software TopPred II (Claros and von Heijne,
CABIOS applic. Notes, 10 :685-686 (1994)).
[0600] Taken together, these data suggest that the protein of SEQ
ID NO: 94 may play a role in the biosynthesis of polysaccharides,
and of the carbohydrate moieties of glycoproteins and glycolipids
and/or in cell-cell recognition. Thus, this protein may be useful
in diagnosing and/or treating several types of disorders including,
but not limited to, cancer, atherosclerosis, cardiovascular
disorders, autoimmune disorders and rheumatic diseases including
rheumatoid arthritis.
[0601] Protein of SEQ ID NO: 104 (Internal Designation
108-006-5-0-G2-FL)
[0602] The protein of SEQ ID NO: 104 encoded by the extended cDNA
SEQ ID NO: 59 shows homology to a neuronal murine protein NP15.6
whose expression is developmentally regulated. NP15.6 protein is
encoded by the nucleic acid sequence of Genbank accession number
Y08702.
[0603] Taken together, these data suggest that the protein of SEQ
ID NO: 104 may be involved in cellular proliferation and
differentiation. Thus, this protein may be useful in diagnosing
and/or treating several types of disorders including, but not
limited to, cancer, neurodegenerative disorders and embryogenetic
disorders.
[0604] C) Proteins Homologous to a Domain of a Protein with Known
Function
[0605] Protein of SEQ ID NO: 113 (Internal Designation
108-009-5-0-A2-FL)
[0606] The protein of SEQ ID NO: 113 encoded by the extended cDNA
SEQ ID NO: 68 shows extensive homology to the bZIP family of
transcription factors, and especially to the human luman protein.
(Lu et al., Mol. Cell. Biol., 17 :5117-5126 (1997)). The human
luman protein is encoded by the nucleic acid sequence of Genbank
accession number: AF009368. The match include the whole bZIP domain
composed of a basic DNA-binding domain and of a leucine zipper
allowing protein dimerization. The basic domain is conserved in the
protein of the invention as shown by the characteristic PROSITE
signature (positions 224-237) except for a conservative
substitution of a glutamic acid with an aspartic acid in position
233. The typical PROSITE signature for leucine zipper is also
present (positions 259 to 280). Secreted proteins may have nucleic
acid binding domain as shown by a nematode protein thought to
regulate gene expression which exhibits zinc fingers as well as a
functional signal peptide (Holst and Zipfel, J. Biol. Chem., 271
:16275-16733, 1996).
[0607] Taken together, these data suggest that the protein of SEQ
ID NO: 113 may bind to DNA, hence regulating gene expression as a
transcription factor. Thus, this protein may be useful in
diagnosing and/or treating several types of disorders including,
but not limited to, cancer.
[0608] Proteins of SEO ID NO: 129 (Internal Designation
76-13-3-A9-CL1 1)
[0609] The protein of SEQ ID NO: 129 encoded by the extended cDNA
SEQ ID NO: 84 shows homology with part of a human seven
transmembrane protein. The human seven transmembrane protein is
encoded by the nucleic acid sequence of Genbank accession number
Y11395. The matched protein potentially associated to stomatin may
act as a G-protein coupled receptor and is likely to be important
for the signal transduction in neurons and haematopoietic cells
(Mayer et al, Biochem. Biophys. Acta., 1395 :301-308 (1998)).
[0610] Taken together, these data suggest that the protein of SEQ
ID NO: 129 may be involved in signal transduction. 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.
[0611] Proteins of SEQ ID NO: 95 (Internal Designation
108-004-5-0-E8-FL)
[0612] The protein of SEQ ID NO: 95 encoded by the extended cDNA
SEQ ID NO: 50 exhibit the typical PROSITE signature for amino acid
perneases (positions 5 to 66) which are integral membrane proteins
involved in the transport of amino acids into the cell. In
addition, the protein of invention has a transmembrane segment from
positions 9 to 29 as predicted by the software TopPred II (Claros
and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)).
[0613] Taken together, these data suggest that the protein of SEQ
ID NO: 95 may be involved in amino acid transport. Thus, this
protein may be useful in diagnosing and/or treating several types
of disorders including, but not limited to, cancer, aminoacidurias,
neurodegenerative diseases, anorexia, chronic fatigue, coronary
vascular disease, diphtheria, hypoglycemia, male infertility,
muscular and myopathies.
[0614] As discussed above, the extended cDNAs of the present
invention or portions thereof can be used for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; 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 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 pattems; 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 which
binds or potentially binds to another protein (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)) to identify
polynucleotides encoding the other protein with which binding
occurs or to identify inhibitors of the binding interaction.
[0615] 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 binds or
potentially binds to another protein (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 involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0616] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0617] 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., Cole 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,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0618] Polynucleotides and proteins 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 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.
[0619] 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.
3TABLE I SEQ ID NO. in SEQ ID NO. in Provisional Present
Application Provisional Application Disclosing Sequence Application
40 U.S. Application No. 60/096,116, filed on Aug. 10, 1998 40 41
U.S. Application No. 60/096,116, filed on Aug. 10, 1998 41 42 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 62 43 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 47 44 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 43 45 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 42 46 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 43 47 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 45 48 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 44 49 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 50 50 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 49 51 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 44 52 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 45 53 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 46 54 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 51 55 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 59 56 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 61 57 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 53 58 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 52 59 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 54 60 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 47 61 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 63 62 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 46 63 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 48 64 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 58 65 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 56 66 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 49 67 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 57 68 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 55 69 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 42 70 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 41 71 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 48 72 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 60 73 U.S.
Application No. 60/096,116, filed on Aug. 10, 1998 50 74 U.S.
Application No. 60/099,273, filed on Sep. 4, 1998 40 75 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 42 76 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 56 77 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 57 78 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 84 79 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 69 80 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 62 81 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 79 82 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 64 83 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 51 84 U.S.
Application No. 60/074,121, filed on Feb. 9, 1998 71 130 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 40 131 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 41 132 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 42 133 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 43 134 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 44 135 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 45 136 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 46 137 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 47 138 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 48 139 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 49 140 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 50 141 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 53 142 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 54 143 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 55 144 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 56 145 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 57 146 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 58 147 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 59 148 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 60 149 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 61 150 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 62 151 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 63 152 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 64 153 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 65 154 U.S.
Application No. 60/081,563, filed on Apr. 13, 1998 66
[0620]
4TABLE II Parameters used for each step of EST analysis Selection
Characteristics Search Characteristics Identity Length Step Program
Strand Parameters (%)) (bp) Miscellaneous Blastn both S = 61 X = 16
90 17 tRNA Fasta both -- 80 60 rRNA Blastn both S = 108 80 40 mtRNA
Blastn both S = 108 80 40 Procaryotic Blastn both S = 144 90 40
Fungal Blastn both S = 144 90 40 Alu fasta* both -- 70 40 L1 Blastn
both S = 72 70 40 Repeats Blastn both S = 72 70 40 Promoters Blastn
top S = 54 X = 16 90 15.perp. Vertebrate fasta* both S = 108 90 30
ESTs Blatsn both S = 108 X = 16 90 30 Proteins blastx.eta. top E =
0.001 -- -- *use "Quick Fast" Database Scanner .perp.alignment
further constrained to begin closer than 10 bp to EST.backslash.5'
end .eta.using BLOSUM62 substitution matrix
[0621]
5TABLE III Parameters used for each step of extended cDNA analysis
Search Selection characteristics characteristics Identity Length
Step Program Strand Parameters (%) (bp) Comments miscellaneous*
FASTA both -- 90 15 tRNA.sup.$ FASTA both -- 80 90 rRNA.sup.$
BLASTN both S = 108 80 40 mtRNA.sup.$ BLASTN both S = 108 80 40
Procaryotic.sup.$ BLASTN both S = 144 90 40 Fungal* BLASTN both S =
144 90 40 Alu* BLASTN both S = 72 70 40 max 5 matches, masking
L1.sup.$ BLASTN both S = 72 70 40 max 5 matches, masking
Repeats.sup.$ BLASTN both S = 72 70 40 masking PolyA BLAST2N top W
= 6, S = 10, E = 1000 90 8 in the last 20 nucleotides
Polyadenylation -- top AATAAA allowing in the 50 signal 1 mismatch
nucleotides preceding the 5' end of the polA Vertibrate* BLASTN
both -- 90 then 70 30 first BLASTN and then then FASTA on FASTA
matching sequences ESTs* BLAST2N both -- 90 30 Geneseq BLASTN both
W = 8, B = 10 90 30 ORF BLASTP top W = 8, B = 10 -- -- on ORF
proteins, max 10 matches Proteins* BLASTX top E = 0.001 70 30
.sup.$steps common to EST analysis and using the same algorithms
and parameters *steps also used in EST analysis but with different
algorithms and/or parameters
[0622]
6TABLE IV Mature Stop FCS SigPep Polypeptide Codon PolyA Signal
PolyA Site Id Location Location Location Location Location Location
40 35 through 568 35 through 100 101 through 568 569 667 through
672 685 through 699 41 68 through 337 68 through 124 125 through
337 338 462 through 467 482 through 497 42 39 through 413 39
through 83 84 through 413 414 566 through 571 583 through 598 43
235 through 642 235 through 336 337 through 642 643 1540 through
1545 1564 through 1579 44 42 through 755 42 through 200 201 through
755 756 860 through 865 878 through 893 45 23 through 340 23
through 235 236 through 340 341 611 through 616 629 through 644 46
12 through 380 12 through 263 264 through 380 381 -- 523 through
538 47 8 through 232 8 through 154 155 through 232 233 -- 737
through 752 48 183 through 422 183 through 302 303 through 422 423
505 through 510 523 through 537 49 24 through 1004 24 through 170
171 through 1004 1005 -- 1586 through 1602 50 80 through 784 80
through 139 140 through 784 785 910 through 915 933 through 948 51
67 through 222 67 through 159 160 through 222 223 -- 673 through
687 52 46 through 732 46 through 186 187 through 732 733 781
through 786 806 through 821 53 81 through 356 81 through 152 153
through 356 357 406 through 411 429 through 445 54 72 through 1346
72 through 140 141 through 1346 1347 1482 through 1487 1502 through
1517 55 194 through 454 194 through 379 380 through 454 455 -- 1545
through 1560 56 48 through 494 48 through 347 348 through 494 495
1031 through 1036 1051 through 1066 57 111 through 671 111 through
215 216 through 671 672 990 through 995 1045 through 1061 58 5
through 373 5 through 82 83 through 373 374 1986 through 1991 2010
through 2025 59 14 through 472 14 through 319 320 through 472 473
555 through 560 576 through 591 60 2 through 217 -- 2 through 217
218 489 through 494 529 through 544 61 51 through 575 51 through
110 111 through 575 576 1653 through 1658 1674 through 1689 62 69
through 977 69 through 128 129 through 977 978 1076 through 1081
1096 through 1111 63 44 through 238 44 through 160 161 through 238
239 443 through 448 540 through 554 64 114 through 524 114 through
164 165 through 524 525 1739 through 1744 1758 through 1773 65 26
through 487 26 through 64 65 through 487 488 883 through 888 901
through 917 66 80 through 388 80 through 187 188 through 388 389
609 through 614 627 through 641 67 186 through 443 186 through 407
408 through 443 444 827 through 832 839 through 854 68 75 through
1259 75 through 1004 1005 through 1259 1260 1536 through 1541 1553
through 1568 69 98 through 376 98 through 151 152 through 376 377
471 through 476 491 through 506 70 72 through 254 72 through 134
135 through 254 255 506 through 511 528 through 542 71 148 through
1140 148 through 240 241 through 1140 1141 1590 through 1595 1614
through 1629 72 109 through 738 109 through 405 406 through 738 739
1633 through 1638 1650 through 1665 73 55 through 291 55 through
255 256 through 291 292 390 through 395 410 through 425 74 25
through 276 -- 25 through 276 277 508 through 513 533 through 546
75 32 through 307 32 through 91 92 through 307 308 452 through 457
472 through 485 76 46 through 675 46 through 87 88 through 675 676
1363 through 1368 1382 through 1394 77 329 through 943 329 through
745 746 through 943 944 -- 1322 through 1333 78 27 through 281 27
through 77 78 through 281 282 -- -- 79 61 through 405 61 through
213 214 through 405 406 675 through 680 692 through 703 80 137
through 379 137 through 229 230 through 379 380 728 through 733 755
through 768 81 37 through 741 37 through 153 154 through 741 742
969 through 974 994 through 1007 82 80 through 265 80 through 142
143 through 265 266 491 through 496 517 through 527 83 612 through
644 -- 612 through 644 645 829 through 834 850 through 861 84 61
through 228 61 through 162 163 through 228 229 208 through 213 --
130 15 through 311 15 through 110 111 through 311 312 507 through
512 531 through 542 131 50 through 529 50 through 130 131 through
529 530 877 through 882 899 through 909 132 240 through 416 240
through 305 306 through 416 417 1117 through 1122 1139 through 1149
133 111 through 446 111 through 254 255 through 446 447 890 through
895 909 through 921 134 123 through 455 123 through 290 291 through
455 456 886 through 891 904 through 916 135 2 through 433 2 through
232 233 through 433 434 488 through 493 510 through 520 136 34
through 363 34 through 87 88 through 363 364 536 through 541 558
through 568 137 50 through 286 50 through 157 158 through 286 287
385 through 390 405 through 416 138 50 through 637 50 through 151
152 through 637 638 -- 1277 through 1289 139 72 through 602 72
through 125 126 through 602 603 -- 704 through 715 140 120 through
434 120 through 185 186 through 434 435 899 through 904 918 through
931 141 4 through 447 4 through 147 148 through 447 448 858 through
863 880 through 891 142 28 through 804 28 through 96 97 through 804
805 -- 806 through 817 143 27 through 359 27 through 212 213
through 359 360 988 through 993 1009 through 1020 144 25 through
957 25 through 93 94 through 957 958 1368 through 1373 1388 through
1399 145 47 through 319 47 through 226 227 through 319 320 -- 656
through 666 146 80 through 940 80 through 130 131 through 940 941
1101 through 1106 1119 through 1130 147 146 through 457 146 through
292 293 through 457 458 442 through 447 465 through 475 148 100
through 351 100 through 207 208 through 351 352 -- 940 through 949
149 177 through 569 177 through 236 237 through 569 570 -- 931
through 939 150 67 through 459 67 through 135 136 through 459 460
856 through 861 875 through 887 151 65 through 1069 65 through 112
113 through 1069 1070 1978 through 1983 1999 through 2010 152 70
through 321 70 through 234 235 through 321 322 364 through 369 375
through 387 153 38 through 877 38 through 91 92 through 877 878 947
through 952 974 through 983 154 51 through 470 51 through 203 204
through 470 471 1585 through 1590 1604 through 1614
[0623]
7TABLE V Full Length Polypeptide Signal Peptide Mature Polypeptide
Id Location Location Location 85 -22 through 156 -22 through -1 1
through 156 86 -19 through 71 -19 through -1 1 through 71 87 -15
through 110 -15 through -1 1 through 110 88 -34 through 102 -34
through -1 1 through 102 89 -53 through 185 -53 through -1 1
through 185 90 -71 through 35 -71 through -1 1 through 35 91 -84
through 39 -84 through -1 1 through 39 92 -49 through 26 -49
through -1 1 through 26 93 -40 through 40 -40 through -1 1 through
40 94 -49 through 278 -49 through -1 1 through 278 95 -20 through
215 -20 through -1 1 through 215 96 -31 through 21 -31 through -1 1
through 21 97 -47 through 182 -47 through -1 1 through 182 98 -24
through 68 -24 through -1 1 through 68 99 -23 through 402 -23
through -1 1 through 402 100 -62 through 25 -62 through -1 1
through 25 101 -100 through 49 -100 through -1 1 through 49 102 -35
through 152 -35 through -1 1 through 152 103 -26 through 97 -26
through -1 1 through 97 104 -102 through 51 -102 through -1 1
through 51 105 1 through 72 -- 1 through 72 106 -20 through 155 -20
through -1 1 through 155 107 -20 through 283 -20 through -1 1
through 283 108 -39 through 26 -39 through -1 1 through 26 109 -17
through 120 -17 through -1 1 through 120 110 -13 through 141 -13
through -1 1 through 141 111 -36 through 67 -36 through -1 1
through 67 112 -74 through 12 -74 through -1 1 through 12 113 -310
through 85 -310 through -1 1 through 85 114 -18 through 75 -18
through -1 1 through 75 115 -21 through 40 -21 through -1 1 through
40 116 -31 through 300 -31 through -1 1 through 300 117 -99 through
111 -99 through -1 1 through 111 118 -67 through 12 -67 through -1
1 through 12 119 1 through 84 -- 1 through 84 120 -20 through 72
-20 through -1 1 through 72 121 -14 through 196 -14 through -1 1
through 196 122 -139 through 66 -139 through -1 1 through 66 123
-17 through 68 -17 through -1 1 through 68 124 -51 through 64 -51
through -1 1 through 64 125 -31 through 50 -31 through -1 1 through
50 126 -39 through 196 -39 through -1 1 through 196 127 -21 through
41 -21 through -1 1 through 41 128 1 through 11 -- 1 through 11 129
-34 through 22 -34 through -1 1 through 22 155 -32 through 67 -32
through -1 1 through 67 156 -27 through 133 -27 through -1 1
through 133 157 -22 through 37 -22 through -1 1 through 37 158 -48
through 64 -48 through -1 1 through 64 159 -56 through 55 -56
through -1 1 through 55 160 -77 through 67 -77 through -1 1 through
67 161 -18 through 92 -18 through -1 1 through 92 162 -36 through
43 -36 through -1 1 through 43 163 -34 through 162 -34 through -1 1
through 162 164 -18 through 159 -18 through -1 1 through 159 165
-22 through 83 -22 through -1 1 through 83 166 -48 through 100 -48
through -1 1 through 100 167 -23 through 236 -23 through -1 1
through 236 168 -62 through 49 -62 through -1 1 through 49 169 -23
through 288 -23 through -1 1 through 288 170 -60 through 31 -60
through -1 1 through 31 171 -17 through 270 -17 through -1 1
through 270 172 -49 through 55 -49 through -1 1 through 55 173 -36
through 48 -36 through -1 1 through 48 174 -20 through 111 -20
through -1 1 through 111 175 -23 through 108 -23 through -1 1
through 108 176 -16 through 319 -16 through -1 1 through 319 177
-55 through 29 -55 through -1 1 through 29 178 -18 through 262 -18
through -1 1 through 262 179 -51 through 89 -51 through -1 1
through 89
[0624]
8TABLE VI Id Collection refs Deposit Name 40 ATCC# 98921 SignalTag
121-144 41 ATCC# 98921 SignalTag 121-144 42 ATCC# 98919 SignalTag
145-165 43 ATCC# 98919 SignalTag 145-165 44 ATCC# 98919 SignalTag
145-165 45 ATCC# 98921 SignalTag 121-144 46 ATCC# 98921 SignalTag
121-144 47 ATCC# 98919 SignalTag 145-165 48 ATCC# 98919 SignalTag
145-165 49 ATCC# 98919 SignalTag 145-165 50 ATCC# 98919 SignalTag
145-165 51 ATCC# 98921 SignalTag 121-144 52 ATCC# 98921 SignalTag
121-144 53 ATCC# 98921 SignalTag 121-144 54 ATCC# 98919 SignalTag
145-165 55 ATCC# 98919 SignalTag 145-165 56 ATCC# 98919 SignalTag
145-165 57 ATCC# 98919 SignalTag 145-165 58 ATCC# 98919 SignalTag
145-165 59 ATCC# 98919 SignalTag 145-165 60 ATCC# 98921 SignalTag
121-144 61 ATCC# 98919 SignalTag 145-165 62 ATCC# 98919 SignalTag
145-165 63 ATCC# 98921 SignalTag 121-144 64 ATCC# 98919 SignalTag
145-165 65 ATCC# 98919 SignalTag 145-165 66 ATCC# 98921 SignalTag
121-144 67 ATCC# 98919 SignalTag 145-165 68 ATCC# 98919 SignalTag
145-165 69 ATCC# 98919 SignalTag 145-165 70 ATCC# 98919 SignalTag
145-165 71 ECACC# XXXX Signal Tag 28011 999 72 ECACC# XXXX Signal
Tag 28011 999 73 ECACC# XXXX Signal Tag 28011 999 74 ECACC# XXXX
Signal Tag 28011 999 75 ECACC# XXXX Signal Tag 28011 999 76 ECACC#
XXXX Signal Tag 28011 999 77 ECACC# XXXX Signal Tag 28011 999 78
ECACC# XXXX Signal Tag 28011 999 79 ECACC# XXXX Signal Tag 28011
999 80 ECACC# XXXX Signal Tag 28011 999 81 ECACC# XXXX Signal Tag
28011 999 82 ECACC# XXXX Signal Tag 28011 999 83 ECACC# XXXX Signal
Tag 28011 999 84 ECACC# XXXX Signal Tag 28011 999
[0625]
9 TABLE VII Internal designation Id Type of sequence
108-002-5-0-B1-FL 40 DNA 108-002-5-0-F3-FL 41 DNA 108-002-5-0-F4-FL
42 DNA 108-003-5-0-A8-FL 43 DNA 108-003-5-0-D2-FL 44 DNA
108-003-5-0-E5-FL 45 DNA 108-003-5-0-H2-FL 46 DNA 108-004-5-0-B7-FL
47 DNA 108-004-5-0-C8-FL 48 DNA 108-004-5-0-D10-FL 49 DNA
108-004-5-0-E8-FL 50 DNA 108-004-5-0-F5-FL 51 DNA 108-004-5-0-G6-FL
52 DNA 108-005-5-0-B11-FL 53 DNA 108-005-5-0-C1-FL 54 DNA
108-005-5-0-F11-FL 55 DNA 108-005-5-0-F6-FL 56 DNA
108-006-5-0-C2-FL 57 DNA 108-006-5-0-E6-FL 58 DNA 108-006-5-0-G2-FL
59 DNA 108-006-5-0-G4-FL 60 DNA 108-008-5-0-A6-FL 61 DNA
108-008-5-0-A8-FL 62 DNA 108-008-5-0-C10-FL 63 DNA
108-008-5-0-E6-FL 64 DNA 108-008-5-0-F6-FL 65 DNA
108-008-5-0-G12-FL 66 DNA 108-008-5-0-G4-FL 67 DNA
108-009-5-0-A2-FL 68 DNA 108-013-5-0-C12-FL 69 DNA
108-013-5-0-G11-FL 70 DNA 108-003-5-0-E4-FL 71 DNA
108-005-5-0-D6-FL 72 DNA 108-008-5-0-G3-FL 73 DNA 108-013-5-0-B5-FL
74 DNA 26-44-1-B5-CL3_1 75 DNA 47-4-4-C6-CL2_3 76 DNA
47-40-4-G9-CL1_1 77 DNA 48-25-4-D8-CL1_7 78 DNA 48-28-3-A9-CL0_1 79
DNA 51-25-1-A2-CL3_1 80 DNA 55-10-3-F5-CL0_3 81 DNA
57-19-2-G8-CL1_3 82 DNA 58-34-2-H8-CL1_3 83 DNA 76-13-3-A9-CL1_1 84
DNA 78-7-2-B8-FL1 130 DNA 77-8-4-F9-FL1 131 DNA 58-8-1-F2-FL2 132
DNA 77-13-1-A7-FL2 133 DNA 47-2-3-G9-FL1 134 DNA 33-75-4-H7-FL1 135
DNA 51-41-1-F10-FL1 136 DNA 48-51-4-C11-FL1 137 DNA 33-58-3-C8-FL1
138 DNA 76-20-4-C11-FL1 139 DNA 76-28-3-A12-FL1 140 DNA
76-25-4-F11-FL1 141 DNA 58-20-4-G7-FL1 142 DNA 33-54-1-B9-FL1 143
DNA 76-20-3-H1-FL1 144 DNA 47-20-2-G3-FL1 145 DNA 78-25-1-H11-FL1
146 DNA 78-6-2-B10-FL1 147 DNA 58-49-3-G10-FL1 148 DNA
78-21-1-B7-FL1 149 DNA 57-28-4-B12-FL1 150 DNA 33-77-4-E2-FL1 151
DNA 58-19-3-D3-FL2 152 DNA 37-7-4-E7-FL1 153 DNA 60-14-2-H10-FL1
154 DNA 108-002-5-0-B1-FL 85 PRT 108-002-5-0-F3-FL 86 PRT
108-002-5-0-F4-FL 87 PRT 108-003-5-0-A8-FL 88 PRT 108-003-5-0-D2-FL
89 PRT 108-003-5-0-E5-FL 90 PRT 108-003-5-0-H2-FL 91 PRT
108-004-5-0-B7-FL 92 PRT 108-004-5-0-C8-FL 93 PRT
108-004-5-0-D10-FL 94 PRT 108-004-5-0-E8-FL 95 PRT
108-004-5-0-F5-FL 96 PRT 108-004-5-0-G6-FL 97 PRT
108-005-5-0-B11-FL 98 PRT 108-005-5-0-C1-FL 99 PRT
108-005-5-0-F11-FL 100 PRT 108-005-5-0-F6-FL 101 PRT
108-006-5-0-C2-FL 102 PRT 108-006-5-0-E6-FL 103 PRT
108-006-5-0-G2-FL 104 PRT 108-006-5-0-G4-FL 105 PRT
108-008-5-0-A6-FL 106 PRT 108-008-5-0-A8-FL 107 PRT
108-008-5-0-C10-FL 108 PRT 108-008-5-0-E6-FL 109 PRT
108-008-5-0-F6-FL 110 PRT 108-008-5-0-G12-FL 111 PRT
108-008-5-0-G4-FL 112 PRT 108-009-5-0-A2-FL 113 PRT
108-013-5-0-C12-FL 114 PRT 108-013-5-0-G11-FL 115 PRT
108-003-5-0-E4-FL 116 PRT 108-005-5-0-D6-FL 117 PRT
108-008-5-0-G3-FL 118 PRT 108-013-5-0-B5-FL 119 PRT
26-44-1-B5-CL3_1 120 PRT 47-4-4-C6-CL2_3 121 PRT 47-40-4-G9-CL1_1
122 PRT 48-25-4-D8-CL1_7 123 PRT 48-28-3-A9-CL0_1 124 PRT
51-25-1-A2-CL3_1 125 PRT 55-10-3-F5-CL0_3 126 PRT 57-19-2-G8-CL1_3
127 PRT 58-34-2-H8-CL1_3 128 PRT 76-13-3-A9-CL1_1 129 PRT
78-7-2-B8-FL1 155 PRT 77-8-4-F9-FL1 156 PRT 58-8-1-F2-FL2 157 PRT
77-13-1-A7-FL2 158 PRT 47-2-3-G9-FL1 159 PRT 33-75-4-H7-FL1 160 PRT
51-41-1-F10-FL1 161 PRT 48-51-4-C11-FL1 162 PRT 33-58-3-C8-FL1 163
PRT 76-20-4-C11-FL1 164 PRT 76-28-3-A12-FL1 165 PRT 76-25-4-F11-FL1
166 PRT 58-20-4-G7-FL1 167 PRT 33-54-1-B9-FL1 168 PRT
76-20-3-H1-FL1 169 PRT 47-20-2-G3-FL1 170 PRT 78-25-1-H11-FL1 171
PRT 78-6-2-B10-FL1 172 PRT 58-49-3-G10-FL1 173 PRT 78-21-1-B7-FL1
174 PRT 57-28-4-B12-FL1 175 PRT 33-77-4-E2-FL1 176 PRT
58-19-3-D3-FL2 177 PRT 37-7-4-E7-FL1 178 PRT 60-14-2-H10-FL1 179
PRT
[0626]
10 TABLE VIII PROSITE signature Id Locations Name 89 205-226
Leucine zipper 95 5-66 Amino acid permease 103 46-67 Leucine zipper
113 259-280 Leucine zipper 120 27-40 MAT8 family 122 123-125 Cell
attachment sequence
[0627]
Sequence CWU 0
0
* * * * *
References