U.S. patent application number 11/455201 was filed with the patent office on 2007-01-11 for complementary dnas.
This patent application is currently assigned to Serono Genetics Institute S.A.. Invention is credited to Lydie Bougueleret, Aymeric Duclert, Jean-Baptiste Dumas Milne Edwards.
Application Number | 20070009941 11/455201 |
Document ID | / |
Family ID | 27491098 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009941 |
Kind Code |
A1 |
Dumas Milne Edwards; Jean-Baptiste
; et al. |
January 11, 2007 |
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: |
Dumas Milne Edwards;
Jean-Baptiste; (Paris, FR) ; Duclert; Aymeric;
(Saint-Maur, FR) ; Bougueleret; Lydie;
(Petit-Lancy, CH) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Assignee: |
Serono Genetics Institute
S.A.
Evry
FR
|
Family ID: |
27491098 |
Appl. No.: |
11/455201 |
Filed: |
June 16, 2006 |
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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11455201 |
Jun 16, 2006 |
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09903190 |
Jul 11, 2001 |
6936692 |
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10930331 |
Aug 30, 2004 |
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09247155 |
Feb 9, 1999 |
6312922 |
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09903190 |
Jul 11, 2001 |
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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 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101;
Y02A 90/26 20180101; A61K 48/00 20130101; Y02A 90/10 20180101; C07K
14/705 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/705 20060101 C07K014/705 |
Claims
1. An isolated or purified polypeptide: a) comprising at least 50,
75, 100 or 150 consecutive amino acids of SEQ ID NO: 104; b)
comprising amino acids 1 to 51 of SEQ ID NO: 104; c) consisting of
amino acids 1 to 51 of SEQ ID NO: 104; d) comprising SEQ ID NO:
104; e) consisting of SEQ ID NO: 104; f) comprising the amino acid
sequence encoded by the insert from deposited clone
108-006-5-0-G2-FL in ATCC accession number 98921; or g) comprising
an amino acid sequence has at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identity to the polypeptide of SEQ ID NO:104.
2. The polypeptide according to claim 1, wherein said polypeptide
comprises amino acids 1 to 51 of SEQ ID NO:104.
3. The polypeptide according to claim 1, wherein said polypeptide
consists of amino acids 1 to 51 of SEQ ID NO:104.
4. The polypeptide according to claim 1, wherein said polypeptide
comprises the sequence of SEQ ID NO:104.
5. The polypeptide according to claim 4, wherein said polypeptide
consists of an amino acid sequence of SEQ ID NO:104.
6. The polypeptide according to claim 1, wherein said polypeptide
comprises an amino acid sequence encoded by the insert from
deposited clone 108-006-5-0-G2-FL in ATCC accession number
98921.
7. The polypeptide of claim 1, wherein said polypeptide has at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the
polypeptide of SEQ ID NO:104.
8. The polypeptide of claim 1, wherein said polypeptide is a
neuronal protein involved in cellular proliferation and
differentiation.
9. A method of making a polypeptide comprising the steps of: a)
obtaining a cDNA encoding a polypeptide: i) comprising at least 50,
75, 100 or 150 consecutive amino acids of SEQ ID NO: 104; ii)
comprising amino acids 1 to 51 of SEQ ID NO: 104; iii) consisting
of amino acids 1 to 51 of SEQ ID NO: 104; iv) comprising SEQ ID NO.
104; v) consisting of SEQ ID NO: 104; vi) comprising the amino acid
sequence encoded by the insert from deposited clone
108-006-5-O-G2-FL in ATCC accession number 98921; or vii).
comprising an amino acid sequence has at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identity to the polypeptide of SEQ ID NO:104;
b) inserting said cDNA in an expression vector such that said cDNA
is operably linked to a promoter; and c) introducing said
expression vector into a host cell whereby said host cell produces
the protein encoded by said cDNA.
10. The method of claim 9, further comprising the step of isolating
said polypeptide.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/930,331, filed Aug. 30, 2004, which is a
divisional application of U.S. patent application Ser. No.
09/903,190, filed Jul. 11, 2001, now U.S. Pat. No. 6,936,692, which
is a divisional application of U.S. patent application Ser. No.
09/247,155, filed Feb. 9, 1999, now U.S. Pat. No. 6,312,922, which
claims the benefit of 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.
[0003] The Sequence Listing for this application is on duplicate
compact discs labeled "Copy 1" and "Copy 2." Copy 1 and Copy 2 each
contain only one file named "SEQ LIST-36US3.txt" which was created
on Dec. 10, 2002, and is 330 KB. The entire contents of each of the
computer discs are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0004] The estimated 50,000 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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).
[0009] 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.
[0010] 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 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.
[0011] 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.
[0012] In addition to being therapeutically useful themselves,
secretory proteins include short peptides, called signal peptides,
at their amino termini which direct their secretion. These signal
peptides are encoded by the signal sequences located at the 5' ends
of the coding sequences of genes encoding secreted proteins.
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.
[0013] 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.
[0014] 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.
[0015] 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
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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."
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] Another embodiment of the present invention is a protein
obtainable by the method described in the preceding paragraph.
[0046] 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.
[0047] Another embodiment of the present invention is a mature
protein obtainable by the method described in the preceding
paragraph.
[0048] 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.
[0049] 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.
[0050] 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.
[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 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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
[0060] 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.
[0061] 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
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] FIG. 5 shows the tissues from which the mRNAs corresponding
to the 5' ESTs in each of the categories described herein were
obtained.
[0067] FIG. 6 illustrates a method for obtaining extended
cDNAs.
[0068] FIG. 7 is a map of pED6dpc2.
[0069] FIG. 8 provides a schematic description of the promoters
isolated and the way they are assembled with the corresponding 5'
tags.
[0070] FIG. 9 describes the transcription factor binding sites
present in each of these promoters.
[0071] 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.
[0072] FIG. 11 is an alignment of the proteins of SEQ ID NOs: 121
and 181 wherein the predicted transmembrane segment is
underlined.
[0073] 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
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.32 pCp (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: TABLE-US-00001 +Cap: (SEQ ID NO:1)
5'm7GpppGCAUCCUACUCCCAUCCAAUUCCACCCUAACUCCUCCCAUCU CCAC-3' -Cap:
(SEQ ID NO:2) 5'-pppGCAUCCUACUCCCAUCCAAUUCCACCCUAACUCCUCCCAUCUC-
CAC-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: ##STR1##
[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.32 pCp as described
in Example 1.
[0089] Sample 2. The 46 nucleotide uncapped in vitro transcript
prepared as in Example 2, labeled with .sup.32 pCp 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.32 pCp as described
in Example 1.
[0091] Sample 4. The 47 nucleotide capped in vitro transcript
prepared as in Example 2, labeled with .sup.32 pCp 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
Capture 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.32 pCp, 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 GEL (BioSepra#230151) 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. TABLE-US-00002 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. ( 1/20th 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. WO96/34981,
published Nov. 7, 1996, which is incorporated herein by
reference.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] B. Enzymatic Methods for Obtaining mRNAs having Intact 5'
Ends
[0139] 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), EP0 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.
[0140] 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
[0141] 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.
[0142] 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 EP0 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.
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
II1 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. Alternatively, protocols
such as the Gene Trapper kit (Gibco BRL) may be used. The double
stranded DNA was then electroporated into bacteria. The percentage
of positive clones having the 5' tag oligonucleotide was 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 a 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
GENBANK 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
Clustering of the 5' ESTs and Calculation of Novelty Indices for
cDNA Libraries
[0180] 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.
[0181] 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%.
[0182] 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
[0183] 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..
[0184] 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
[0185] 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.
[0186] 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.
[0187] 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 biotimidase 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.
[0188] 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.
[0189] 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.
[0190] 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
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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
[0197] FIG. 5 shows the tissues from which the mRNAs corresponding
to the 5' ESTs in each of the above described categories were
obtained.
[0198] 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.
[0199] 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.
[0200] 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
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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 cm2 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.
[0207] 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.
[0208] 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.
[0209] 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.
III. Use of 5' ESTs to Clone Extended cDNAs and to Clone the
Corresponding Genomic DNAs
[0210] 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.
[0211] 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.
[0212] 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
[0213] 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.
1. Obtaining Extended cDNAs
a) First Strand Synthesis
[0214] 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 and 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.
[0215] 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.
b) Second Strand Synthesis
[0216] 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.weizrnann.ac.il/software/PC-Rare/doc/manuel.html).
[0217] 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.
[0218] 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.
[0219] 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.
2. Sequencing of Full Length Extended cDNAs or Fragments
Thereof
[0220] 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 full 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.
a) Nested PCR Products Containing Complete ORFs
[0221] 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.
b) Nested PCR Products Containing Incomplete ORFs
[0222] 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.
[0223] 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 full length
extended cDNAs are then cloned into an appropriate vector as
described in section 3.
c) Sequencing Extended cDNAs
[0224] Sequencing of extended cDNAs can be performed using a Die
Terminator approach with the AMPLITAQ DNA polymerase FS kit
available from Perkin Elmer.
[0225] 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.
[0226] 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.
[0227] 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.
3. Cloning of Full Length Extended cDNAs
[0228] The PCR product containing the full coding sequence is then
cloned in an appropriate vector. For example, the extended cDNAs
can be cloned into 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.
[0229] 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.
[0230] 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 walking 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.
4. Computer Analysis of Full Length Extended cDNA
[0231] 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.
a) Elimination of Undesired Sequences
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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.
b) Identification of Structural Features
[0239] Structural features, e.g. polyA tail and polyadenylation
signal, of the sequences of full length extended cDNAs are
subsequently determined as follows.
[0240] 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.
[0241] 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.
[0242] 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.
c) Identification of Functional Features
[0243] Functional features, e.g. ORFs and signal sequences, of the
sequences of full length extended cDNAs were subsequently
determined as follows.
[0244] 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.
[0245] 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.
d) Homology to either Nucleotidic or Proteic Sequences
[0246] Sequences of full length extended cDNAs are then compared to
known sequences on a nucleotidic or proteic basis.
[0247] 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.
[0248] 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.
[0249] 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 70% homology over 30 amino acid stretches are detected as
already identified proteins.
[0250] 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.
[0251] 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.
[0252] 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.
[0253] 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.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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 function, or
structural motifs. The various sequence comparer programs
identified elsewhere in this patent specification are particularly
contemplated for use in this aspect of the invention.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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., Aszodi et al., Proteins:Structure, Function, and Genetics,
Supplement 1:38-42 (1997)).
[0268] 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.
[0269] 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 programidentifies 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.
[0270] 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.
[0271] 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.
5. Selection of Cloned Full Length Sequences of the Present
Invention
[0272] 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.
a) Automatic Sequence Preselection
[0273] 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 4a). 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.
[0274] 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.
[0275] 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.
b) Manual Sequence Selection
[0276] 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.
[0277] 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
[0278] 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.
[0279] The full 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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
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 Poly A Signal
Location in Table IV) and the locations of polyA sites (listed
under the heading Poly A Site Location in Table IV).
[0286] 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/ftpserveur/prosite_scan) were used to find
signatures on the extended cDNAs.
[0287] 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_shuffled) 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.
[0288] 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).
[0289] 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.
[0290] 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).
[0291] 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.
[0292] 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,
Porton 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.
[0293] 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.
[0294] Bacterial cells containing a particular clone can be
obtained from the composite deposit as follows:
[0295] 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: [0296] (a) It
should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any; [0297] (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.
[0298] The oligonucleotide should preferably be labeled with
.gamma.-[.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.
[0299] 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.
[0300] Standard colony hybridization procedures should then be used
to transfer the colonies to nitrocellulose filters and lyse,
denature and bake them.
[0301] 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 NaC1/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.
[0302] 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.
[0303] 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.
[0304] 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
[0305] 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 genomic 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] 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.
1. Identification of Extended cDNA or Genomic DNA Sequences Having
a High Degree of Homology to the Labeled Probe
[0316] 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:
[0317] For probes between 14 and 70 nucleotides in length the
melting temperature (T.sub.m) 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.
[0318] 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.
[0319] 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.
[0320] 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.
[0321] All of the foregoing hybridizations would be considered to
be under "stringent" conditions.
[0322] 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.
[0323] 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.
2. Obtaining Extended cDNA or Genomic DNA Sequences Having Lower
Degrees of Homology to the Labeled Probe
[0324] 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.
[0325] 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.
[0326] Extended cDNAs, nucleic acids homologous to extended cDNAs,
or genomic DNAs which have hybridized to the probe are identified
by autoradiography.
3. Determination of the Degree of Homology between the Obtained
Extended cDNAs or Genomic DNAs and the Labeled Probe
[0327] 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.
[0328] 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.
[0329] 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.
[0330] 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)).
[0331] 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.
[0332] In addition to the above described methods, other protocols
are available to obtain extended cDNAs using 5' ESTs as outlined in
the following paragraphs.
[0333] 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.
[0334] 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 full protein coding sequence,
including the authentic translation initiation site, the second
primer used contains sequences located upstream of the translation
initiation site. The second primer is extended to generate a second
cDNA strand complementary to the first cDNA strand. Alternatively,
RT-PCR may be performed as described above using primers from both
ends of the cDNA to be obtained.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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 conscutive nucleotides
from the 5' EST.
[0340] 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.
[0341] 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.
IV. Expression of Proteins Encoded by Extended cDNAs Isolated Using
5' ESTs
[0342] 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
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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
600 ug/ml G418 (Sigma, St. Louis, Mo.). Preferably the expressed
protein is released into the culture medium, thereby facilitating
purification.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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).
[0363] 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
[0364] 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.
[0365] 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.
[0366] 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
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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
Assaying the Proteins Expressed from Extended cDNAs or Portions
Thereof for Activity as Immune System Regulators
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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 67:40624069, 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.
[0376] 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.
[0377] 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.
[0378] 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
fungal infections, or may result from autoimmune 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.
[0379] 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 useful in the treatment of allergic
reactions and conditions, such as asthma (particularly allergic
asthma) or other respiratory problems. Other conditions, in which
immune suppression is desired (including, for example, organ
transplantation), may also be treatable using a protein of the
present invention.
[0380] 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 functions of activated T-cells may be
inhibited by suppressing T cell responses or by inducing specific
tolerance in T cells, or both. Immunosuppression of T cell
responses is generally an active, non-antigen-specific, process
which requires continuous exposure of the T cells to the
suppressive agent. Tolerance, which involves inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and
persists after 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.
[0381] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions (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.
[0382] 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.
[0383] Blocking antigen function may also be therapeutically useful
for treating autoimmune diseases. Many autoimmune disorders are the
result of inappropriate activation of T cells that are reactive
against self tissue and which promote the production of cytokines
and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or
eliminate disease symptoms. Administration of reagents which block
costimulation of T cells by disrupting receptor ligand interactions
of B lymphocyte antigens can be used to inhibit T cell activation
and prevent production of autoantibodies or T cell-derived
cytokines which 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).
[0384] Upregulation of an antigen function (preferably a B
lymphocyte antigen function), as a means of up regulating immune
responses, may also be useful in therapy. Upregulation of immune
responses may 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.
[0385] 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.
[0386] In another application, up regulation or enhancement of
antigen function (preferably B lymphocyte antigen function) may be
useful in the induction of tumor immunity. Tumor cells (e.g.,
sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
transfected with 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.
[0387] 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
[0388] 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.
[0389] 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, N.Y. 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.
[0390] 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 transfusions; and/or in supporting
the growth and proliferation of hematopoietic stem cells which are
capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantion, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post 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
[0391] 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 International Patent
Publication No. WO95/16035, International Patent Publication No.
WO95/05846 and International Patent Publication No. WO91/07491,
which are incorporated herein by reference.
[0392] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H I 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.
[0393] 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.
[0394] 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.
[0395] 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.
[0396] 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.
[0397] 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.
[0398] 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.
[0399] 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.
[0400] 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.
[0401] 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.
[0402] 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
[0403] 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.
[0404] 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.
[0405] 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
[0406] 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.
[0407] 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.
[0408] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0409] 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
Chemokincs 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
[0410] 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.
[0411] 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
[0412] 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.
[0413] 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 selecting,
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
[0414] The proteins encoded by the extended cDNAs or a portion
thereof may also be evaluated for anti-inflammatory activity. The
anti-inflammatory activity may be achieved by providing a stimulus
to cells involved in the inflammatory response, by inhibiting or
promoting cell-cell interactions (such as, for example, cell
adhesion), by inhibiting or promoting chemotaxis of cells involved
in the inflammatory process, inhibiting or promoting cell
extravasation, or by stimulating or suppressing production of other
factors which more directly inhibit or promote an inflammatory
response. Proteins 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
[0415] 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.
[0416] 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 climination 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
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] 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.
[0422] 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.
[0423] 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.
[0424] 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.
[0425] 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.
[0426] 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
[0427] 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:
A. Monoclonal Antibody Production by Hybridoma Fusion
[0428] 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 supernatant 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.
B. Polyclonal Antibody Production by Immunization
[0429] 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
immunogenicity. Effective polyclonal antibody production is
affected by many factors related both to the antigen and the host
species. For example, small molecules tend to be less immunogenic
than others and may require the use of carriers and adjuvant. Also,
host animals 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).
[0430] 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). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc.
For Microbiol., Washington, D.C. (1980).
[0431] 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.
V. Use of Extended cDNAs or Portions Thereof as Reagents
[0432] 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
[0433] 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
[0434] 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.
[0435] 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.
[0436] 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
[0437] 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
[0438] 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
[0439] 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).
[0440] 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).
[0441] 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 Southern 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
[0442] Another technique for identifying individuals using the
extended cDNA sequences disclosed herein utilizes a dot blot
hybridization technique.
[0443] 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.
[0444] 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).
[0445] 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
[0446] 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.
[0447] 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.
[0448] It is additionally contemplated within this example that the
number of probe sequences used can be varied for additional
accuracy or clarity.
[0449] 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
[0450] 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.
[0451] 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.
A. Immunohistochemical Techniques
[0452] 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).
[0453] 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.
[0454] 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.
[0455] 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.
[0456] 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.
[0457] 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.
[0458] 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.
B. Identification of Tissue Specific Soluble Proteins
[0459] 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.
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] 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
[0465] 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 genome 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).
[0466] 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
[0467] 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.
[0468] 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 thermocycler (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.).
[0469] 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).)
[0470] 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
[0471] 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.
[0472] 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 min.
[0473] 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.
[0474] 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
[0475] 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.
[0476] 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
[0477] 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.
[0478] 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.
[0479] 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.
VI. Use of Extended cDNAs (or Genomic DNAs Obtainable Therefrom) to
Construct Vectors
[0480] 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
[0481] 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.
[0482] 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.
[0483] 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.
[0484] 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.
[0485] 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.
[0486] 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.
[0487] 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.
[0488] 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
[0489] 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 GenomeWalker.TM. kit available from Clontech,
five complete genomic DNA samples are each digested with a
different restriction enzyme which has a 6 base recognition site
and leaves a blunt end. Following digestion, oligonucleotide
adapters are ligated to each end of the resulting genomic DNA
fragments.
[0490] 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 ' EST of interest 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. 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.
[0491] 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.
[0492] 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.
[0493] 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.
[0494] 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
[0495] 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, P 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.
[0496] 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.
[0497] 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
[0498] 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.
[0499] 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.
[0500] 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.
[0501] 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 MatInspector
release 2.0, August 1996.
[0502] 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.
[0503] 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.
[0504] 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.
[0505] 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.
[0506] 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
[0507] 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.
[0508] 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.
VII. Use of Extended cDNAs (or Genomic DNAs Obtainable Therefrom)
in Gene Therapy
[0509] 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
[0510] 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.
[0511] 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.
[0512] 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).
[0513] 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.
[0514] 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.
[0515] 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.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] 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.
[0520] The antisense molecules are introduced onto cell samples at
a number of different concentrations preferably between
1.times.10.sup.-10 M to 1.times.10.sup.-4 M. Once the minimum
concentration that can adequately control gene expression is
identified, the optimized dose is translated into a dosage suitable
for use in vivo. For example, an inhibiting concentration in
culture of 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.
[0521] 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.
[0522] 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.
[0523] 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
[0524] 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.
[0525] 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.
[0526] Treated cells are monitored for altered cell function or
reduced gene expression using techniques such as Northern blotting,
RNase protection assays, or PCR based strategies to monitor the
transcription levels of the target gene in cells which have been
treated with the oligonucleotide. The cell functions to be
monitored are predicted based upon the homologies of the target
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 functions can
also be predicted based on the presence of abnormal physiologies
within cells derived from individuals with a particular inherited
disease, particularly when the extended cDNA is associated with the
disease using techniques described in Example 53.
[0527] 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.
[0528] In some embodiments, the natural (beta) anomers of the
oligonucleotide units can be replaced with alpha anomers to render
the oligonucleotide more resistant to nucleases. Further, an
intercalating agent such as ethidium bromide, or the like, can be
attached to the 3' end of the alpha oligonucleotide to stabilize
the triple helix. For information on the generation of
oligonucleotides suitable for triple helix formation see Griffin et
al. (Science 245:967-971 (1989), which is hereby incorporated by
this reference).
EXAMPLE 61
Use of Extended cDNAs to Express an Encoded Protein in a Host
Organism
[0529] 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.
[0530] 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.
[0531] 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.
[0532] 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
[0533] 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)).
[0534] 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.
[0535] This method may be applied to study diverse intracellular
functions and cellular processes. For instance, it has been used to
probe functionally relevant domains of intracellular proteins and
to examine protein-protein interactions involved in signal
transduction pathways (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)).
[0536] 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.
[0537] 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
[0538] 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.
[0539] 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.
[0540] 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.
[0541] 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).
[0542] 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
[0543] 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 Oct., 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.
[0544] 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.
[0545] 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.
[0546] 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.
[0547] 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.
[0548] 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.
A) Proteins which are Closely Related to Known Proteins
Protein of SEQ ID NO:120 (internal designation 26-44-1-B5-CL31)
[0549] 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)).
[0550] 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)).
[0551] 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.
Proteins of SEQ ID NOs: 121 (Internal Designation
47-4-4-C6-CL2.sub.--3)
[0552] 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 murine 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)).
[0553] 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)).
[0554] 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.
Proteins of SEQ ID NO:128 (Internal Designation
58-34-2-H8-CL1.sub.--3)
[0555] 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)).
[0556] 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).
[0557] 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.
B) Proteins which are Remotely Related to Proteins with known
Functions
Protein of SEQ ID NO: 97 (Internal Designation
108-004-5-O-G6-FL)
[0558] 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.
[0559] 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.
[0560] 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.
Protein of SEQ ID NO:111 (Internal Designation
108-008-5-O-G12-FL)
[0561] 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.
[0562] 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.
Protein of SEQ ID NO: 94 (Internal Designation
108-004-5-0-D10-FL)
[0563] 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.
[0564] 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)).
[0565] 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.
Protein of SEQ ID NO:104 (Internal Designation
108-006-5-0-G2-FL)
[0566] 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.
[0567] 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.
C) Proteins Homologous to a Domain of a Protein with known
Function
Protein of SEQ ID NO:113 (Internal Designation
108-009-5-0-A2-FL)
[0568] 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).
[0569] 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.
Proteins of SEQ ID NO:129 (Internal Designation
76-13-3-A9-CL11)
[0570] 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)).
[0571] 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.
Proteins of SEQ ID NO: 95 (Internal Designation
108-004-5-0-E8-FL)
[0572] The protein of SEQ ID NO: 95 encoded by the extended cDNA
SEQ ID NO: 50 exhibit the typical PROSITE signature for amino acid
permeases (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)).
[0573] 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.
[0574] 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 patterns;
to raise anti-protein antibodies using DNA immunization techniques;
and as an antigen to raise anti-DNA antibodies or elicit another
immune response. Where the polynucleotide encodes a protein 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.
[0575] 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.
[0576] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0577] 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.
[0578] 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.
[0579] 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.
TABLE-US-00003 TABLE 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
[0580] TABLE-US-00004 TABLE II Parameters used for each step of EST
analysis Selection Characteristics Iden- Search Characteristics
tity 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.dagger. Vertebrate fasta* both S = 108 90 30 ESTs Blatsn both S
= 108 X = 16 90 30 Proteins blastx top E = 0.001 -- -- *use "Quick
Fast" Database Scanner .dagger.alignment further constrained to
begin closer than 10 bp to EST\5' end using BLOSUM62 substitution
matrix
[0581] TABLE-US-00005 TABLE 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, 90 8 in the last 20 E = 1000
nucleotides Polyadenylation -- top AATAAA allowing 1 mismatch in
the 50 signal nucleotides preceding the 5' end of the polA
Vertibrate* BLASTN both -- 90 then 30 first BLASTN and then 70 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
[0582] TABLE-US-00006 TABLE IV Mature Stop FCS SigPep Polypeptide
Codon PolyA Signal PolyA Site Id Location Location Location
Location Location Location 40 35 through 35 through 100 101 through
569 667 through 685 through 568 568 672 699 41 68 through 68
through 124 125 through 338 462 through 482 through 337 337 467 497
42 39 through 39 through 83 84 through 414 566 through 583 through
413 413 571 598 43 235 through 235 through 337 through 643 1540
through 1564 through 642 336 642 1545 1579 44 42 through 42 through
200 201 through 756 860 through 878 through 755 755 865 893 45 23
through 23 through 235 236 through 341 611 through 629 through 340
340 616 644 46 12 through 12 through 263 264 through 381 -- 523
through 380 380 538 47 8 through 232 8 through 154 155 through 233
-- 737 through 232 752 48 183 through 183 through 303 through 423
505 through 523 through 422 302 422 510 537 49 24 through 24
through 170 171 through 1005 -- 1586 through 1004 1004 1602 50 80
through 80 through 139 140 through 785 910 through 933 through 784
784 915 948 51 67 through 67 through 159 160 through 223 -- 673
through 222 222 687 52 46 through 46 through 186 187 through 733
781 through 806 through 732 732 786 821 53 81 through 81 through
152 153 through 357 406 through 429 through 356 356 411 445 54 72
through 72 through 140 141 through 1347 1482 through 1502 through
1346 1346 1487 1517 55 194 through 194 through 380 through 455 --
1545 through 454 379 454 1560 56 48 through 48 through 347 348
through 495 1031 through 1051 through 494 494 1036 1066 57 111
through 111 through 216 through 672 990 through 1045 through 671
215 671 995 1061 58 5 through 373 5 through 82 83 through 374 1986
through 2010 through 373 1991 2025 59 14 through 14 through 319 320
through 473 555 through 576 through 472 472 560 591 60 2 through
217 -- 2 through 217 218 489 through 529 through 494 544 61 51
through 51 through 110 111 through 576 1653 through 1674 through
575 575 1658 1689 62 69 through 69 through 128 129 through 978 1076
through 1096 through 977 977 1081 1111 63 44 through 44 through 160
161 through 239 443 through 540 through 238 238 448 554 64 114
through 114 through 165 through 525 1739 through 1758 through 524
164 524 1744 1773 65 26 through 26 through 64 65 through 488 883
through 901 through 487 487 888 917 66 80 through 80 through 187
188 through 389 609 through 627 through 388 388 614 641 67 186
through 186 through 408 through 444 827 through 839 through 443 407
443 832 854 68 75 through 75 through 1005 through 1260 1536 through
1553 through 1259 1004 1259 1541 1568 69 98 through 98 through 151
152 through 377 471 through 491 through 376 376 476 506 70 72
through 72 through 134 135 through 255 506 through 528 through 254
254 511 542 71 148 through 148 through 241 through 1141 1590
through 1614 through 1140 240 1140 1595 1629 72 109 through 109
through 406 through 739 1633 through 1650 through 738 405 738 1638
1665 73 55 through 55 through 255 256 through 292 390 through 410
through 291 291 395 425 74 25 through -- 25 through 277 508 through
533 through 276 276 513 546 75 32 through 32 through 91 92 through
308 452 through 472 through 307 307 457 485 76 46 through 46
through 87 88 through 676 1363 through 1382 through 675 675 1368
1394 77 329 through 329 through 746 through 944 -- 1322 through 943
745 943 1333 78 27 through 27 through 77 78 through 282 -- -- 281
281 79 61 through 61 through 213 214 through 406 675 through 692
through 405 405 680 703 80 137 through 137 through 230 through 380
728 through 755 through 379 229 379 733 768 81 37 through 37
through 153 154 through 742 969 through 994 through 741 741 974
1007 82 80 through 80 through 142 143 through 266 491 through 517
through 265 265 496 527 83 612 through -- 612 through 645 829
through 850 through 644 644 834 861 84 61 through 61 through 162
163 through 229 208 through -- 228 228 213 130 15 through 15
through 110 111 through 312 507 through 531 through 311 311 512 542
131 50 through 50 through 130 131 through 530 877 through 899
through 529 529 882 909 132 240 through 240 through 306 through 417
1117 through 1139 through 416 305 416 1122 1149 133 111 through 111
through 255 through 447 890 through 909 through 446 254 446 895 921
134 123 through 123 through 291 through 456 886 through 904 through
455 290 455 891 916 135 2 through 433 2 through 232 233 through 434
488 through 510 through 433 493 520 136 34 through 34 through 87 88
through 364 536 through 558 through 363 363 541 568 137 50 through
50 through 157 158 through 287 385 through 405 through 286 286 390
416 138 50 through 50 through 151 152 through 638 -- 1277 through
637 637 1289 139 72 through 72 through 125 126 through 603 -- 704
through 602 602 715 140 120 through 120 through 186 through 435 899
through 918 through 434 185 434 904 931 141 4 through 447 4 through
147 148 through 448 858 through 880 through 447 863 891 142 28
through 28 through 96 97 through 805 -- 806 through 804 804 817 143
27 through 27 through 212 213 through 360 988 through 1009 through
359 359 993 1020 144 25 through 25 through 93 94 through 958 1368
through 1388 through 957 957 1373 1399 145 47 through 47 through
226 227 through 320 -- 656 through 319 319 666 146 80 through 80
through 130 131 through 941 1101 through 1119 through 940 940 1106
1130 147 146 through 146 through 293 through 458 442 through 465
through 457 292 457 447 475 148 100 through 100 through 208 through
352 -- 940 through 351 207 351 949 149 177 through 177 through 237
through 570 -- 931 through 569 236 569 939 150 67 through 67
through 135 136 through 460 856 through 875 through 459 459 861 887
151 65 through 65 through 112 113 through 1070 1978 through 1999
through 1069 1069 1983 2010 152 70 through 70 through 234 235
through 322 364 through 375 through 321 321 369 387 153 38 through
38 through 91 92 through 878 947 through 974 through 877 877 952
983 154 51 through 51 through 203 204 through 471 1585 through 1604
through 470 470 1590 1614
[0583] TABLE-US-00007 TABLE 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
[0584] TABLE-US-00008 TABLE 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 SignalTag 28011 999 72
ECACC# XXXX SignalTag 28011 999 73 ECACC# XXXX SignalTag 28011 999
74 ECACC# XXXX SignalTag 28011 999 75 ECACC# XXXX SignalTag 28011
999 76 ECACC# XXXX SignalTag 28011 999 77 ECACC# XXXX SignalTag
28011 999 78 ECACC# XXXX SignalTag 28011 999 79 ECACC# XXXX
SignalTag 28011 999 80 ECACC# XXXX SignalTag 28011 999 81 ECACC#
XXXX SignalTag 28011 999 82 ECACC# XXXX SignalTag 28011 999 83
ECACC# XXXX SignalTag 28011 999 84 ECACC# XXXX SignalTag 28011
999
[0585] TABLE-US-00009 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
[0586] TABLE-US-00010 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
[0587]
Sequence CWU 1
1
182 1 47 RNA Artificial Sequence in vitro transcription product
modified_base 1 m7g 1 ggcauccuac ucccauccaa uuccacccua acuccuccca
ucuccac 47 2 46 RNA Artificial Sequence in vitro transcription
product 2 gcauccuacu cccauccaau uccacccuaa cuccucccau cuccac 46 3
25 DNA Artificial Sequence oligonucleotide 3 atcaagaatt cgcacgagac
catta 25 4 25 DNA Artificial Sequence oligonucleotide 4 taatggtctc
gtgcgaattc ttgat 25 5 25 DNA Artificial Sequence oligonucleotide 5
ccgacaagac caacgtcaag gccgc 25 6 25 DNA Artificial Sequence
oligonucleotide 6 tcaccagcag gcagtggctt aggag 25 7 25 DNA
Artificial Sequence oligonucleotide 7 agtgattcct gctactttgg atggc
25 8 25 DNA Artificial Sequence oligonucleotide 8 gcttggtctt
gttctggagt ttaga 25 9 25 DNA Artificial Sequence oligonucleotide 9
tccagaatgg gagacaagcc aattt 25 10 25 DNA Artificial Sequence
oligonucleotide 10 agggaggagg aaacagcgtg agtcc 25 11 25 DNA
Artificial Sequence oligonucleotide 11 atgggaaagg aaaagactca tatca
25 12 25 DNA Artificial Sequence oligonucleotide 12 agcagcaaca
atcaggacag cacag 25 13 25 DNA Artificial Sequence oligonucleotide
13 atcaagaatt cgcacgagac catta 25 14 67 DNA Artificial Sequence
oligonucleotide misc_feature 67 n=a, g, c or t 14 atcgttgaga
ctcgtaccag cagagtcacg agagagacta cacggtactg gttttttttt 60 tttttvn
67 15 29 DNA Artificial Sequence oligonucleotide 15 ccagcagagt
cacgagagag actacacgg 29 16 25 DNA Artificial Sequence
oligonucleotide 16 cacgagagag actacacggt actgg 25 17 526 DNA Homo
Sapiens misc_feature complement(261..376) blastn misc_feature
complement(380..486) blastn misc_feature complement(110..145)
blastn misc_feature complement(196..229) blastn sig_peptide 90..140
Von Heijne matrix misc_feature 290 n=a, g, c or t 17 aatatrarac
agctacaata ttccagggcc artcacttgc catttctcat aacagcgtca 60
gagagaaaga actgactgar acgtttgag atg aag aaa gtt ctc ctc ctg atc 113
Met Lys Lys Val Leu Leu Leu Ile -15 -10 aca gcc atc ttg gca gtg gct
gtw ggt ttc cca gtc tct caa gac cag 161 Thr Ala Ile Leu Ala Val Ala
Val Gly Phe Pro Val Ser Gln Asp Gln -5 1 5 gaa cga gaa aaa aga agt
atc agt gac agc gat gaa tta gct tca ggr 209 Glu Arg Glu Lys Arg Ser
Ile Ser Asp Ser Asp Glu Leu Ala Ser Gly 10 15 20 wtt ttt gtg ttc
cct tac cca tat cca ttt cgc cca ctt cca cca att 257 Xaa Phe Val Phe
Pro Tyr Pro Tyr Pro Phe Arg Pro Leu Pro Pro Ile 25 30 35 cca ttt
cca aga ttt cca tgg ttt aga cgt aan ttt cct att cca ata 305 Pro Phe
Pro Arg Phe Pro Trp Phe Arg Arg Xaa Phe Pro Ile Pro Ile 40 45 50 55
cct gaa tct gcc cct aca act ccc ctt cct agc gaa aag taaacaaraa 354
Pro Glu Ser Ala Pro Thr Thr Pro Leu Pro Ser Glu Lys 60 65
ggaaaagtca crataaacct ggtcacctga aattgaaatt gagccacttc cttgaaraat
414 caaaattcct gttaataaaa raaaaacaaa tgtaattgaa atagcacaca
gcattctcta 474 gtcaatatct ttagtgatct tctttaataa acatgaaagc
aaaaaaaaaa aa 526 18 17 PRT Homo Sapiens SIGNAL 1..17 Von Heijne
matrix score 8.2 seq LLLITAILAVAVG/FP 18 Met Lys Lys Val Leu Leu
Leu Ile Thr Ala Ile Leu Ala Val Ala Val 1 5 10 15 Gly 19 822 DNA
Homo Sapiens misc_feature 260..464 blastn misc_feature 118..184
blastn misc_feature 56..113 blastn misc_feature 454..485 blastn
misc_feature 118..545 blastn misc_feature 65..369 blastn
misc_feature 61..399 blastn misc_feature 408..458 blastn
misc_feature 60..399 blastn misc_feature 393..432 blastn
sig_peptide 346..408 Von Heijne matrix misc_feature 115 n=a, g, c
or t 19 actcctttta gcataggggc ttcggcgcca gcggccagcg ctagtcggtc
tggtaagtgc 60 ctgatgccga gttccgtctc tcgcgtcttt tcctggtccc
aggcaaagcg gasgnagatc 120 ctcaaacggc ctagtgcttc gcgcttccgg
agaaaatcag cggtctaatt aattcctctg 180 gtttgttgaa gcagttacca
agaatcttca accctttccc acaaaagcta attgagtaca 240 cgttcctgtt
gagtacacgt tcctgttgat ttacaaaagg tgcaggtatg agcaggtctg 300
aagactaaca ttttgtgaag ttgtaaaaca gaaaacctgt tagaa atg tgg tgg ttt
357 Met Trp Trp Phe -20 cag caa ggc ctc agt ttc ctt cct tca gcc ctt
gta att tgg aca tct 405 Gln Gln Gly Leu Ser Phe Leu Pro Ser Ala Leu
Val Ile Trp Thr Ser -15 -10 -5 gct gct ttc ata ttt tca tac att act
gca gta aca ctc cac cat ata 453 Ala Ala Phe Ile Phe Ser Tyr Ile Thr
Ala Val Thr Leu His His Ile 1 5 10 15 gac ccg gct tta cct tat atc
agt gac act ggt aca gta gct cca raa 501 Asp Pro Ala Leu Pro Tyr Ile
Ser Asp Thr Gly Thr Val Ala Pro Xaa 20 25 30 aaa tgc tta ttt ggg
gca atg cta aat att gcg gca gtt tta tgt caa 549 Lys Cys Leu Phe Gly
Ala Met Leu Asn Ile Ala Ala Val Leu Cys Gln 35 40 45 aaa tagaaatcag
gaarataatt caacttaaag aakttcattt catgaccaaa 602 Lys ctcttcaraa
acatgtcttt acaagcatat ctcttgtatt gctttctaca ctgttgaatt 662
gtctggcaat atttctgcag tggaaaattt gatttarmta gttcttgact gataaatatg
722 gtaaggtggg cttttccccc tgtgtaattg gctactatgt cttactgagc
caagttgtaw 782 tttgaaataa aatgatatga gagtgacaca aaaaaaaaaa 822 20
21 PRT Homo Sapiens SIGNAL 1..21 Von Heijne matrix score 5.5 seq
SFLPSALVIWTSA/AF 20 Met Trp Trp Phe Gln Gln Gly Leu Ser Phe Leu Pro
Ser Ala Leu Val 1 5 10 15 Ile Trp Thr Ser Ala 20 21 405 DNA Homo
Sapiens misc_feature complement(103..398) blastn sig_peptide
185..295 Von Heijne matrix 21 atcaccttct tctccatcct tstctgggcc
agtccccarc ccagtccctc tcctgacctg 60 cccagcccaa gtcagccttc
agcacgcgct tttctgcaca cagatattcc aggcctacct 120 ggcattccag
gacctccgma atgatgctcc agtcccttac aagcgcttcc tggatgaggg 180 tggc atg
gtg ctg acc acc ctc ccc ttg ccc tct gcc aac agc cct gtg 229 Met Val
Leu Thr Thr Leu Pro Leu Pro Ser Ala Asn Ser Pro Val -35 -30 -25 aac
atg ccc acc act ggc ccc aac agc ctg agt tat gct agc tct gcc 277 Asn
Met Pro Thr Thr Gly Pro Asn Ser Leu Ser Tyr Ala Ser Ser Ala -20 -15
-10 ctg tcc ccc tgt ctg acc gct cca aak tcc ccc cgg ctt gct atg atg
325 Leu Ser Pro Cys Leu Thr Ala Pro Xaa Ser Pro Arg Leu Ala Met Met
-5 1 5 10 cct gac aac taaatatcct tatccaaatc aataaarwra raatcctccc
374 Pro Asp Asn tccaraaggg tttctaaaaa caaaaaaaaa a 405 22 37 PRT
Homo Sapiens SIGNAL 1..37 Von Heijne matrix score 5.9 seq
LSYASSALSPCLT/AP 22 Met Val Leu Thr Thr Leu Pro Leu Pro Ser Ala Asn
Ser Pro Val Asn 1 5 10 15 Met Pro Thr Thr Gly Pro Asn Ser Leu Ser
Tyr Ala Ser Ser Ala Leu 20 25 30 Ser Pro Cys Leu Thr 35 23 496 DNA
Homo Sapiens misc_feature 149..331 blastn misc_feature 328..485
blastn misc_feature complement(182..496) blastn sig_peptide
196..240 Von Heijne matrix misc_feature 101 n=a, g, c or t 23
aaaaaattgg tcccagtttt caccctgccg cagggctggc tggggagggc agcggtttag
60 attagccgtg gcctaggccg tttaacgggg tgacacgagc ntgcagggcc
gagtccaagg 120 cccggagata ggaccaaccg tcaggaatgc gaggaatgtt
tttcttcgga ctctatcgag 180 gcacacagac agacc atg ggg att ctg tct aca
gtg aca gcc tta aca ttt 231 Met Gly Ile Leu Ser Thr Val Thr Ala Leu
Thr Phe -15 -10 -5 gcc ara gcc ctg gac ggc tgc aga aat ggc att gcc
cac cct gca agt 279 Ala Xaa Ala Leu Asp Gly Cys Arg Asn Gly Ile Ala
His Pro Ala Ser 1 5 10 gag aag cac aga ctc gag aaa tgt agg gaa ctc
gag asc asc cac tcg 327 Glu Lys His Arg Leu Glu Lys Cys Arg Glu Leu
Glu Xaa Xaa His Ser 15 20 25 gcc cca gga tca acc cas cac cga aga
aaa aca acc aga aga aat tat 375 Ala Pro Gly Ser Thr Xaa His Arg Arg
Lys Thr Thr Arg Arg Asn Tyr 30 35 40 45 tct tca gcc tgaaatgaak
ccgggatcaa atggttgctg atcaragccc 424 Ser Ser Ala atatttaaat
tggaaaagtc aaattgasca ttattaaata aagcttgttt aatatgtctc 484
aaacaaaaaa aa 496 24 15 PRT Homo Sapiens SIGNAL 1..15 Von Heijne
matrix score 5.5 seq ILSTVTALTFAXA/LD UNSURE 14 Xaa = any one of
the twenty amino acids 24 Met Gly Ile Leu Ser Thr Val Thr Ala Leu
Thr Phe Ala Xaa Ala 1 5 10 15 25 623 DNA Homo Sapiens sig_peptide
49..96 Von Heijne matrix 25 aaagatccct gcagcccggc aggagagaag
gctgagcctt ctggcgtc atg gag agg 57 Met Glu Arg -15 ctc gtc cta acc
ctg tgc acc ctc ccg ctg gct gtg gcg tct gct ggc 105 Leu Val Leu Thr
Leu Cys Thr Leu Pro Leu Ala Val Ala Ser Ala Gly -10 -5 1 tgc gcc
acg acg cca gct cgc aac ctg agc tgc tac cag tgc ttc aag 153 Cys Ala
Thr Thr Pro Ala Arg Asn Leu Ser Cys Tyr Gln Cys Phe Lys 5 10 15 gtc
agc agc tgg acg gag tgc ccg ccc acc tgg tgc agc ccg ctg gac 201 Val
Ser Ser Trp Thr Glu Cys Pro Pro Thr Trp Cys Ser Pro Leu Asp 20 25
30 35 caa gtc tgc atc tcc aac gag gtg gtc gtc tct ttt aaa tgg agt
gta 249 Gln Val Cys Ile Ser Asn Glu Val Val Val Ser Phe Lys Trp Ser
Val 40 45 50 cgc gtc ctg ctc agc aaa cgc tgt gct ccc aga tgt ccc
aac gac aac 297 Arg Val Leu Leu Ser Lys Arg Cys Ala Pro Arg Cys Pro
Asn Asp Asn 55 60 65 atg aak ttc gaa tgg tcg ccg gcc ccc atg gtg
caa ggc gtg atc acc 345 Met Xaa Phe Glu Trp Ser Pro Ala Pro Met Val
Gln Gly Val Ile Thr 70 75 80 agg cgc tgc tgt tcc tgg gct ctc tgc
aac agg gca ctg acc cca cag 393 Arg Arg Cys Cys Ser Trp Ala Leu Cys
Asn Arg Ala Leu Thr Pro Gln 85 90 95 gag ggg cgc tgg gcc ctg cra
ggg ggg ctc ctg ctc cag gac cct tcg 441 Glu Gly Arg Trp Ala Leu Xaa
Gly Gly Leu Leu Leu Gln Asp Pro Ser 100 105 110 115 agg ggc ara aaa
acc tgg gtg cgg cca cag ctg ggg ctc cca ctc tgc 489 Arg Gly Xaa Lys
Thr Trp Val Arg Pro Gln Leu Gly Leu Pro Leu Cys 120 125 130 ctt ccc
awt tcc aac ccc ctc tgc cca rgg gaa acc cag gaa gga 534 Leu Pro Xaa
Ser Asn Pro Leu Cys Pro Xaa Glu Thr Gln Glu Gly 135 140 145
taacactgtg ggtgccccca cctgtgcatt gggaccacra cttcaccctc ttggaracaa
594 taaactctca tgcccccaaa aaaaaaaaa 623 26 16 PRT Homo Sapiens
SIGNAL 1..16 Von Heijne matrix score 10.1 seq LVLTLCTLPLAVA/SA 26
Met Glu Arg Leu Val Leu Thr Leu Cys Thr Leu Pro Leu Ala Val Ala 1 5
10 15 27 848 DNA Homo Sapiens sig_peptide 32..73 Von Heijne matrix
27 aactttgcct tgtgttttcc accctgaaag a atg ttg tgg ctg ctc ttt ttt
52 Met Leu Trp Leu Leu Phe Phe -10 ctg gtg act gcc att cat gct gaa
ctc tgt caa cca ggt gca gaa aat 100 Leu Val Thr Ala Ile His Ala Glu
Leu Cys Gln Pro Gly Ala Glu Asn -5 1 5 gct ttt aaa gtg aga ctt agt
atc aga aca gct ctg gga gat aaa gca 148 Ala Phe Lys Val Arg Leu Ser
Ile Arg Thr Ala Leu Gly Asp Lys Ala 10 15 20 25 tat gcc tgg gat acc
aat gaa gaa tac ctc ttc aaa gcg atg gta gct 196 Tyr Ala Trp Asp Thr
Asn Glu Glu Tyr Leu Phe Lys Ala Met Val Ala 30 35 40 ttc tcc atg
aga aaa gtt ccc aac aga gaa gca aca gaa att tcc cat 244 Phe Ser Met
Arg Lys Val Pro Asn Arg Glu Ala Thr Glu Ile Ser His 45 50 55 gtc
cta ctt tgc aat gta acc cag agg gta tca ttc tgg ttt gtg gtt 292 Val
Leu Leu Cys Asn Val Thr Gln Arg Val Ser Phe Trp Phe Val Val 60 65
70 aca gac cct tca aaa aat cac acc ctt cct gct gtt gag gtg caa tca
340 Thr Asp Pro Ser Lys Asn His Thr Leu Pro Ala Val Glu Val Gln Ser
75 80 85 gcc ata aga atg aac aag aac cgg atc aac aat gcc ttc ttt
cta aat 388 Ala Ile Arg Met Asn Lys Asn Arg Ile Asn Asn Ala Phe Phe
Leu Asn 90 95 100 105 gac caa act ctg gaa ttt tta aaa atc cct tcc
aca ctt gca cca ccc 436 Asp Gln Thr Leu Glu Phe Leu Lys Ile Pro Ser
Thr Leu Ala Pro Pro 110 115 120 atg gac cca tct gtg ccc atc tgg att
att ata ttt ggt gtg ata ttt 484 Met Asp Pro Ser Val Pro Ile Trp Ile
Ile Ile Phe Gly Val Ile Phe 125 130 135 tgc atc atc ata gtt gca att
gca cta ctg att tta tca ggg atc tgg 532 Cys Ile Ile Ile Val Ala Ile
Ala Leu Leu Ile Leu Ser Gly Ile Trp 140 145 150 caa cgt ada ara aag
aac aaa gaa cca tct gaa gtg gat gac gct gaa 580 Gln Arg Xaa Xaa Lys
Asn Lys Glu Pro Ser Glu Val Asp Asp Ala Glu 155 160 165 rat aak tgt
gaa aac atg atc aca att gaa aat ggc atc ccc tct gat 628 Xaa Xaa Cys
Glu Asn Met Ile Thr Ile Glu Asn Gly Ile Pro Ser Asp 170 175 180 185
ccc ctg gac atg aag gga ggg cat att aat gat gcc ttc atg aca gag 676
Pro Leu Asp Met Lys Gly Gly His Ile Asn Asp Ala Phe Met Thr Glu 190
195 200 gat gag agg ctc acc cct ctc tgaagggctg ttgttctgct
tcctcaaraa 727 Asp Glu Arg Leu Thr Pro Leu 205 attaaacatt
tgtttctgtg tgactgctga gcatcctgaa ataccaagag cagatcatat 787
wttttgtttc accattcttc ttttgtaata aattttgaat gtgcttgaaa aaaaaaaaaa
847 c 848 28 14 PRT Homo Sapiens SIGNAL 1..14 Von Heijne matrix
score 10.7 seq LWLLFFLVTAIHA/EL 28 Met Leu Trp Leu Leu Phe Phe Leu
Val Thr Ala Ile His Ala 1 5 10 29 25 DNA Artificial Sequence
oligonucleotide 29 gggaagatgg agatagtatt gcctg 25 30 26 DNA
Artificial Sequence oligonucleotide 30 ctgccatgta catgatagag agattc
26 31 546 DNA Homo Sapiens promoter 1..517
codon_start="518"protein_bind 17..25 matinspector prediction name
CMYB_01 score 0.983 sequence tgtcagttg protein_bind
complement(18..27) matinspector prediction name MYOD_Q6 score 0.961
sequence cccaactgac protein_bind complement(75..85) matinspector
prediction name S8_01 score 0.960 sequence aatagaattag protein_bind
94..104 matinspector prediction name S8_01 score 0.966 sequence
aactaaattag protein_bind complement(129..139) matinspector
prediction name DELTAEF1_01 score 0.960 sequence gcacacctcag
protein_bind complement(155..165) matinspector prediction name
GATA_C score 0.964 sequence agataaatcca protein_bind 170..178
matinspector prediction name CMYB_01 score 0.958 sequence cttcagttg
protein_bind 176..189 matinspector prediction name GATA1_02 score
0.959 sequence ttgtagataggaca protein_bind 180..190 matinspector
prediction name GATA_C score 0.953 sequence agataggacat
protein_bind 284..299 matinspector prediction name TAL1ALPHAE47_01
score 0.973 sequence cataacagatggtaag protein_bind 284..299
matinspector prediction name TAL1BETAE47_01 score 0.983 sequence
cataacagatggtaag protein_bind 284..299 matinspector prediction name
TAL1BETAITF2_01 score 0.978 sequence cataacagatggtaag protein_bind
complement(287..296) matinspector prediction name MYOD_Q6 score
0.954 sequence accatctgtt protein_bind complement(302..314)
matinspector prediction name GATA1_04 score 0.953 sequence
tcaagataaagta protein_bind 393..405 matinspector prediction name
IK1_01 score 0.963 sequence agttgggaattcc protein_bind 393..404
matinspector prediction name IK2_01 score 0.985 sequence
agttgggaattc protein_bind 396..405 matinspector prediction name
CREL_01 score 0.962 sequence tgggaattcc protein_bind 423..436
matinspector prediction name GATA1_02 score 0.950 sequence
tcagtgatatggca protein_bind complement(478..489) matinspector
prediction name SRY_02 score 0.951 sequence taaaacaaaaca
protein_bind 486..493 matinspector prediction name E2F_02 score
0.957 sequence tttagcgc protein_bind complement(514..521)
matinspector prediction name MZF1_01 score 0.975 sequence
tgagggga
31 tgagtgcagt gttacatgtc agttgggtta agtttgttaa tgtcattcaa
atcttctatg 60 tcttgatttg cctgctaatt ctattatttc tggaactaaa
ttagtttgat ggttctatta 120 gttattgact gaggtgtgct aatctcccat
tatgtggatt tatctatttc ttcagttgta 180 gataggacat tgatagatac
ataagtacca ggacaaaagc agggagatct tttttccaaa 240 atcaggagaa
aaaaatgaca tctggaaaac ctatagggaa aggcataaca gatggtaagg 300
atactttatc ttgagtagga gagccttcct gtggcaacgt ggagaaggga agaggtcgta
360 gaattgagga gtcagctcag ttagaagcag ggagttggga attccgttca
tgtgatttag 420 catcagtgat atggcaaatg tgggactaag ggtagtgatc
agagggttaa aattgtgtgt 480 tttgttttag cgctgctggg gcatcgcctt
gggtcccctc aaacagattc ccatgaatct 540 cttcat 546 32 23 DNA
Artificial Sequence oligonucleotide 32 gtaccaggga ctgtgaccat tgc 23
33 24 DNA Artificial Sequence oligonucleotide 33 ctgtgaccat
tgctcccaag agag 24 34 861 DNA Homo Sapiens promoter 1..806
codon_start="807"protein_bind complement(60..70) matinspector
prediction name NFY_Q6 score 0.956 sequence ggaccaatcat
protein_bind 70..77 matinspector prediction name MZF1_01 score
0.962 sequence cctgggga protein_bind 124..132 matinspector
prediction name CMYB_01 score 0.994 sequence tgaccgttg protein_bind
complement(126..134) matinspector prediction name VMYB_02 score
0.985 sequence tccaacggt protein_bind 135..143 matinspector
prediction name STAT_01 score 0.968 sequence ttcctggaa protein_bind
complement(135..143) matinspector prediction name STAT_01 score
0.951 sequence ttccaggaa protein_bind complement(252..259)
matinspector prediction name MZF1_01 score 0.956 sequence ttggggga
protein_bind 357..368 matinspector prediction name IK2_01 score
0.965 sequence gaatgggatttc protein_bind 384..391 matinspector
prediction name MZF1_01 score 0.986 sequence agagggga protein_bind
complement(410..421) matinspector prediction name SRY_02 score
0.955 sequence gaaaacaaaaca protein_bind 592..599 matinspector
prediction name MZF1_01 score 0.960 sequence gaagggga protein_bind
618..627 matinspector prediction name MYOD_Q6 score 0.981 sequence
agcatctgcc protein_bind 632..642 matinspector prediction name
DELTAEF1_01 score 0.958 sequence tcccaccttcc protein_bind
complement(813..823) matinspector prediction name S8_01 score 0.992
sequence gaggcaattat protein_bind complement(824..831) matinspector
prediction name MZF1_01 score 0.986 sequence agagggga misc_feature
335,376 n=a, g, c or t 34 tactataggg cacgcgtggt cgacggccgg
gctgttctgg agcagagggc atgtcagtaa 60 tgattggtcc ctggggaagg
tctggctggc tccagcacag tgaggcattt aggtatctct 120 cggtgaccgt
tggattcctg gaagcagtag ctgttctgtt tggatctggt agggacaggg 180
ctcagagggc taggcacgag ggaaggtcag aggagaaggs aggsarggcc cagtgagarg
240 ggagcatgcc ttcccccaac cctggcttsc ycttggymam agggcgktty
tgggmacttr 300 aaytcagggc ccaascagaa scacaggccc aktcntggct
smaagcacaa tagcctgaat 360 gggatttcag gttagncagg gtgagagggg
aggctctctg gcttagtttt gttttgtttt 420 ccaaatcaag gtaacttgct
cccttctgct acgggccttg gtcttggctt gtcctcaccc 480 agtcggaact
ccctaccact ttcaggagag tggttttagg cccgtggggc tgttctgttc 540
caagcagtgt gagaacatgg ctggtagagg ctctagctgt gtgcggggcc tgaaggggag
600 tgggttctcg cccaaagagc atctgcccat ttcccacctt cccttctccc
accagaagct 660 tgcctgagct gtttggacaa aaatccaaac cccacttggc
tactctggcc tggcttcagc 720 ttggaaccca atacctaggc ttacaggcca
tcctgagcca ggggcctctg gaaattctct 780 tcctgatggt cctttaggtt
tgggcacaaa atataattgc ctctcccctc tcccattttc 840 tctcttggga
gcaatggtca c 861 35 20 DNA Artificial Sequence oligonucleotide 35
ctgggatgga aggcacggta 20 36 20 DNA Artificial Sequence
oligonucleotide 36 gagaccacac agctagacaa 20 37 555 DNA Homo Sapiens
promoter 1..500 codon_start="501"protein_bind 191..206 matinspector
prediction name ARNT_01 score 0.964 sequence ggactcacgtgctgct
protein_bind 193..204 matinspector prediction name NMYC_01 score
0.965 sequence actcacgtgctg protein_bind 193..204 matinspector
prediction name USF_01 score 0.985 sequence actcacgtgctg
protein_bind complement(193..204) matinspector prediction name
USF_01 score 0.985 sequence cagcacgtgagt protein_bind
complement(193..204) matinspector prediction name NMYC_01 score
0.956 sequence cagcacgtgagt protein_bind complement(193..204)
matinspector prediction name MYCMAX_02 score 0.972 sequence
cagcacgtgagt protein_bind 195..202 matinspector prediction name
USF_C score 0.997 sequence tcacgtgc protein_bind
complement(195..202) matinspector prediction name USF_C score 0.991
sequence gcacgtga protein_bind complement(210..217) matinspector
prediction name MZF1_01 score 0.968 sequence catgggga protein_bind
397..410 matinspector prediction name ELK1_02 score 0.963 sequence
ctctccggaagcct protein_bind 400..409 matinspector prediction name
CETS1P54_01 score 0.974 sequence tccggaagcc protein_bind
complement(460..470) matinspector prediction name AP1_Q4 score
0.963 sequence agtgactgaac protein_bind complement(460..470)
matinspector prediction name AP1FJ_Q2 score 0.961 sequence
agtgactgaac protein_bind 547..555 matinspector prediction name
PADS_C score 1.000 sequence tgtggtctc 37 ctatagggca cgcktggtcg
acggcccggg ctggtctggt ctgtkgtgga gtcgggttga 60 aggacagcat
ttgtkacatc tggtctactg caccttccct ctgccgtgca cttggccttt 120
kawaagctca gcaccggtgc ccatcacagg gccggcagca cacacatccc attactcaga
180 aggaactgac ggactcacgt gctgctccgt ccccatgagc tcagtggacc
tgtctatgta 240 gagcagtcag acagtgcctg ggatagagtg agagttcagc
cagtaaatcc aagtgattgt 300 cattcctgtc tgcattagta actcccaacc
tagatgtgaa aacttagttc tttctcatag 360 gttgctctgc ccatggtccc
actgcagacc caggcactct ccggaagcct ggaaatcacc 420 cgtgtcttct
gcctgctccc gctcacatcc cacacttgtg ttcagtcact gagttacaga 480
ttttgcctcc tcaatttctc ttgtcttagt cccatcctct gttcccctgg ccagtttgtc
540 tagctgtgtg gtctc 555 38 19 DNA Artificial Sequence
oligonucleotide 38 ggccatacac ttgagtgac 19 39 19 DNA Artificial
Sequence oligonucleotide 39 atatagacaa acgcacacc 19 40 699 DNA Homo
sapiens CDS 35..568 sig_peptide 35..100 Von Heijne matrix score
10.7 seq LLTLALLGGPTWA/GK polyA_signal 667..672 polyA_site 685..699
40 aaccagacgc ccagtcacag gcgagagccc tggg atg cac cgg cca gag gcc
atg 55 Met His Arg Pro Glu Ala Met -20 ctg ctg ctg ctc acg ctt gcc
ctc ctg ggg ggc ccc acc tgg gca ggg 103 Leu Leu Leu Leu Thr Leu Ala
Leu Leu Gly Gly Pro Thr Trp Ala Gly -15 -10 -5 1 aag atg tat ggc
cct gga gga ggc aag tat ttc agc acc act gaa gac 151 Lys Met Tyr Gly
Pro Gly Gly Gly Lys Tyr Phe Ser Thr Thr Glu Asp 5 10 15 tac gac cat
gaa atc aca ggg ctg cgg gtg tct gta ggt ctt ctc ctg 199 Tyr Asp His
Glu Ile Thr Gly Leu Arg Val Ser Val Gly Leu Leu Leu 20 25 30 gtg
aaa agt gtc cag gtg aaa ctt gga gac tcc tgg gac gtg aaa ctg 247 Val
Lys Ser Val Gln Val Lys Leu Gly Asp Ser Trp Asp Val Lys Leu 35 40
45 gga gcc tta ggt ggg aat acc cag gaa gtc acc ctg cag cca ggc gaa
295 Gly Ala Leu Gly Gly Asn Thr Gln Glu Val Thr Leu Gln Pro Gly Glu
50 55 60 65 tac atc aca aaa gtc ttt gtc gcc ttc caa act ttc ctc cgg
ggt atg 343 Tyr Ile Thr Lys Val Phe Val Ala Phe Gln Thr Phe Leu Arg
Gly Met 70 75 80 gtc atg tac acc agc aag gac cgc tat ttc tat ttt
ggg aag ctt gat 391 Val Met Tyr Thr Ser Lys Asp Arg Tyr Phe Tyr Phe
Gly Lys Leu Asp 85 90 95 ggc cag atc tcc tct gcc tac ccc agc caa
gag ggg cag gtg ctg gtg 439 Gly Gln Ile Ser Ser Ala Tyr Pro Ser Gln
Glu Gly Gln Val Leu Val 100 105 110 ggc atc tat ggc cag tat caa ctc
ctt ggc atc aag agc att ggc ttt 487 Gly Ile Tyr Gly Gln Tyr Gln Leu
Leu Gly Ile Lys Ser Ile Gly Phe 115 120 125 gaa tgg aat tat cca cta
gag gag ccg acc act gag cca cca gtt aat 535 Glu Trp Asn Tyr Pro Leu
Glu Glu Pro Thr Thr Glu Pro Pro Val Asn 130 135 140 145 ctc aca tac
tca gca aac tca ccc gtg ggt cgc tagggtgggg tatggggcca 588 Leu Thr
Tyr Ser Ala Asn Ser Pro Val Gly Arg 150 155 tccgagctga ggccatctgg
gtggtggtgg ctgatggtac tggagtaact gagtcgggac 648 gctgaatctg
aatccaccaa taaataaagg ttctgcaaaa aaaaaaaaaa a 699 41 497 DNA Homo
sapiens CDS 68..337 sig_peptide 68..124 Von Heijne matrix score 10
seq LVLLGVSIFLVSA/QN polyA_signal 462..467 polyA_site 482..497 41
agcgccttgc cttctcttag gctttgaagc atttttgtct gtgctccctg atcttcaggt
60 caccacc atg aag ttc tta gca gtc ctg gta ctc ttg gga gtt tcc atc
109 Met Lys Phe Leu Ala Val Leu Val Leu Leu Gly Val Ser Ile -15 -10
ttt ctg gtc tct gcc cag aat ccg aca aca gct gct cca gct gac acg 157
Phe Leu Val Ser Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr -5
1 5 10 tat cca gct act ggt cct gct gat gat gaa gcc cct gat gct gaa
acc 205 Tyr Pro Ala Thr Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu
Thr 15 20 25 act gct gct gca acc act gcg acc act gct gct cct acc
act gca acc 253 Thr Ala Ala Ala Thr Thr Ala Thr Thr Ala Ala Pro Thr
Thr Ala Thr 30 35 40 acc gct gct tct acc act gct cgt aaa gac att
cca gtt tta ccc aaa 301 Thr Ala Ala Ser Thr Thr Ala Arg Lys Asp Ile
Pro Val Leu Pro Lys 45 50 55 tgg gtt ggg gat ctc ccg aat ggt aga
gtg tgt ccc tgagatggaa 347 Trp Val Gly Asp Leu Pro Asn Gly Arg Val
Cys Pro 60 65 70 tcagcttgag tcttctgcaa ttggtcacaa ctattcatgc
ttcctgtgat ttcatccaac 407 tacttacctt gcctacgata tcccctttat
ctctaatcag tttattttct ttcaaataaa 467 aaataactat gagcaaaaaa
aaaaaaaaaa 497 42 598 DNA Homo sapiens CDS 39..413 sig_peptide
39..83 Von Heijne matrix score 4.6 seq LLTHNLLSSHVRG/VG
polyA_signal 566..571 polyA_site 583..598 42 ttttccggtt ccggcctggc
gagagtttgt gcggcgac atg aaa ctg ctt acc cac 56 Met Lys Leu Leu Thr
His -15 -10 aat ctg ctg agc tcg cat gtg cgg ggg gtg ggg tcc cgt ggc
ttc ccc 104 Asn Leu Leu Ser Ser His Val Arg Gly Val Gly Ser Arg Gly
Phe Pro -5 1 5 ctg cgc ctc cag gcc acc gag gtc cgt atc tgc cct gtg
gaa ttc aac 152 Leu Arg Leu Gln Ala Thr Glu Val Arg Ile Cys Pro Val
Glu Phe Asn 10 15 20 ccc aac ttc gtg gcg cgt atg ata cct aaa gtg
gag tgg tcg gcg ttc 200 Pro Asn Phe Val Ala Arg Met Ile Pro Lys Val
Glu Trp Ser Ala Phe 25 30 35 ctg gag gcg gcc gat aac ttg cgt ctg
atc cag gtg ccg aaa ggg ccg 248 Leu Glu Ala Ala Asp Asn Leu Arg Leu
Ile Gln Val Pro Lys Gly Pro 40 45 50 55 gtt gag gga tat gag gag aat
gag gag ttt ctg agg acc atg cac cac 296 Val Glu Gly Tyr Glu Glu Asn
Glu Glu Phe Leu Arg Thr Met His His 60 65 70 ctg ctg ctg gag gtg
gaa gtg ata gag ggc acc ctg cag tgc ccg gaa 344 Leu Leu Leu Glu Val
Glu Val Ile Glu Gly Thr Leu Gln Cys Pro Glu 75 80 85 tct gga cgt
atg ttc ccc atc agc cgc ggg atc ccc aac atg ctg ctg 392 Ser Gly Arg
Met Phe Pro Ile Ser Arg Gly Ile Pro Asn Met Leu Leu 90 95 100 agt
gaa gag gaa act gag agt tgattgtgcc aggcgccagt ttttcttgtt 443 Ser
Glu Glu Glu Thr Glu Ser 105 110 atgactgtgt atttttgttg atctataccc
tgtttccgaa ttctgccgtg tgtatcccca 503 acccttgacc caatgacacc
aaacacagtg tttttgagct cggtattata tatttttttc 563 tcattaaagg
tttaaaacca aaaaaaaaaa aaaaa 598 43 1579 DNA Homo sapiens CDS
235..642 sig_peptide 235..336 Von Heijne matrix score 8.7 seq
HLLALLVFSVLLA/LR polyA_signal 1540..1545 polyA_site 1564..1579 43
gtgggggcat ggcgtccgat cgaggcgggc gttcacgggc ggccagggtt gagtcccggg
60 tcggggccgg gggattgccg gcgcatcagg gccgagggct ggggctggcg
gggccgctcg 120 ctgcctctcg ctcgcagcag cggcggcagg cgcgggcgag
ggccacgggg agaggagacg 180 cagccccgcg ggtggcacgc tcggccgggc
cccggcccgc gctcaacggg cgcg atg 237 Met ctc ttc tcg ctc cgg gag ctg
gtg cag tgg cta ggc ttc gcc acc ttc 285 Leu Phe Ser Leu Arg Glu Leu
Val Gln Trp Leu Gly Phe Ala Thr Phe -30 -25 -20 gag atc ttc gtg cac
ctg ctg gcc ctg ttg gtg ttc tct gtg ctg ctg 333 Glu Ile Phe Val His
Leu Leu Ala Leu Leu Val Phe Ser Val Leu Leu -15 -10 -5 gca ctg cgt
gtg gat ggc ctg gtc ccg ggc ctc tcc tgg tgg aac gtg 381 Ala Leu Arg
Val Asp Gly Leu Val Pro Gly Leu Ser Trp Trp Asn Val 1 5 10 15 ttc
gtg cct ttc ttc gcc gct gac ggg ctc agc acc tac ttc acc acc 429 Phe
Val Pro Phe Phe Ala Ala Asp Gly Leu Ser Thr Tyr Phe Thr Thr 20 25
30 atc gtg tcc gtg cgc ctc ttc cag gat gga gag aag cgg ctg gcg gtg
477 Ile Val Ser Val Arg Leu Phe Gln Asp Gly Glu Lys Arg Leu Ala Val
35 40 45 ctc cgc ctt ttc tgg gta ctt acg gtc ctg agt ctc aag ttc
gtc ttc 525 Leu Arg Leu Phe Trp Val Leu Thr Val Leu Ser Leu Lys Phe
Val Phe 50 55 60 gag atg ctg ttg tgc cag aag ctg gcg gag cag act
cgg gag ctc tgg 573 Glu Met Leu Leu Cys Gln Lys Leu Ala Glu Gln Thr
Arg Glu Leu Trp 65 70 75 ttc ggc ctc att acg tcc ccg ctc ttc att
ctc ctg cag ctg ctc atg 621 Phe Gly Leu Ile Thr Ser Pro Leu Phe Ile
Leu Leu Gln Leu Leu Met 80 85 90 95 atc cgc gcc tgt cgg gtc aac
tagcctcacc gaggtgccgg agagggagcg 672 Ile Arg Ala Cys Arg Val Asn
100 ctggacaact agaatgttga cctcgagccg aggccctact tgcagcgcac
cggaggagag 732 gctctctagt ctgaaggcac cgccggcttg cgccgagctg
agtgccgggt ttccctattc 792 caatcctgtt tgaaatggtt tcttcagcag
ggcttaaaag agcagccttc atcctgaaaa 852 tgtatttcct tttgtttaat
gctttgagta gataatcctg aattgaggtc atgaggaggc 912 cccccaggcc
agacagtcct gaacccctct gacacttgga aactgaatat aagtaaaatg 972
tccaggtgga ctctgagtat ttcctgtgga tcctgggaaa gtactgttgc acaaaggctg
1032 caaagctgga ctcaggaatg tcctccaacc agcagcgcta acctaagagc
tccctgtgcc 1092 gtctatccag accagacttc ggtagatgcc tttgttagat
ctatcacatg taaacgagct 1152 tgtatctcct tccctgtgcc acgagagaga
ttggcttttt attccagtct aggcagagac 1212 agaagaatgt tgaataagag
cacgattaga gtcctgtctg gttatctgtt gcccaagaaa 1272 agaactctgc
tgtccaggca ctgcttggct tactatccca gcaaagactg cagttttgtg 1332
gacttttgac caccttgggc tggcactctt agcacacctg agacagattt aagcctccct
1392 aagagactga agagaggaac aggtgtcaga tactcatagg cactgagatc
tacaaatggg 1452 aagcttgtga gtggcccatc tttgttggcc tacgaacttt
ggtttgatgc cagtcaggtg 1512 ccacatgaga acctttgctg agatgcaaat
aaagtaagag aatgttttcc caaaaaaaaa 1572 aaaaaaa 1579 44 893 DNA Homo
sapiens CDS 42..755 sig_peptide 42..200 Von Heijne matrix score 5.8
seq ILSLQVLLTTVTS/TV polyA_signal 860..865 polyA_site 878..893 44
gcggttagtg gaccgggacc ggtaggggtg ctgttgccat c atg gct gac ccc gac
56 Met Ala Asp Pro Asp -50 ccc cgg tac cct cgc tcc tcg atc gag gac
gac ttc aac tat ggc agc 104 Pro Arg Tyr Pro Arg Ser Ser Ile Glu Asp
Asp Phe Asn Tyr Gly Ser -45 -40 -35 agc gtg gcc tcc gcc acc gtg cac
atc cga atg gcc ttt ctg aga aaa 152 Ser Val Ala Ser Ala Thr Val His
Ile Arg Met Ala Phe Leu Arg Lys -30 -25 -20 gtc tac agc att ctt tct
ctg cag gtt ctc tta act aca gtg act tca 200 Val Tyr Ser Ile Leu Ser
Leu Gln Val Leu Leu Thr Thr Val Thr Ser -15 -10 -5 aca gtt ttt tta
tac ttt gag tct gta cgg aca ttt gta cat gag agt 248 Thr Val Phe Leu
Tyr Phe Glu Ser Val Arg Thr Phe Val His Glu Ser 1 5 10 15 cct gcc
tta att ttg ctg ttt gcc ctc gga tct ctg ggt ttg att ttt 296 Pro Ala
Leu Ile Leu Leu Phe Ala Leu Gly Ser Leu Gly Leu Ile Phe 20 25 30
gcg ttg att tta aac aga cat aag tat ccc ctt aac ctg tac cta ctt 344
Ala Leu Ile Leu Asn Arg His Lys Tyr Pro Leu Asn Leu Tyr Leu Leu 35
40 45 ttt gga ttt acg ctg ttg gaa gct ctg act gtg gca gtt gtt gtt
act 392 Phe Gly Phe Thr Leu Leu Glu Ala Leu Thr Val Ala Val Val Val
Thr 50 55 60 ttc tat gat gta tat att att ctg caa gct ttc ata ctg
act act aca
440 Phe Tyr Asp Val Tyr Ile Ile Leu Gln Ala Phe Ile Leu Thr Thr Thr
65 70 75 80 gta ttt ttt ggt ttg act gtg tat act cta caa tct aag aag
gat ttc 488 Val Phe Phe Gly Leu Thr Val Tyr Thr Leu Gln Ser Lys Lys
Asp Phe 85 90 95 agc aaa ttt gga gca ggg ctg ttt gct ctt ttg tgg
ata ttg tgc ctg 536 Ser Lys Phe Gly Ala Gly Leu Phe Ala Leu Leu Trp
Ile Leu Cys Leu 100 105 110 tca gga ttc ttg aag ttt ttt tta tat agt
gag ata atg gag ttg gtc 584 Ser Gly Phe Leu Lys Phe Phe Leu Tyr Ser
Glu Ile Met Glu Leu Val 115 120 125 tta gcc gct gca gga gcc ctt ctt
ttc tgt gga ttc atc atc tat gac 632 Leu Ala Ala Ala Gly Ala Leu Leu
Phe Cys Gly Phe Ile Ile Tyr Asp 130 135 140 aca cac tca ctg atg cat
aaa ctg tca cct gaa gag tac gta tta gct 680 Thr His Ser Leu Met His
Lys Leu Ser Pro Glu Glu Tyr Val Leu Ala 145 150 155 160 gcc atc agc
ctc tac ttg gat atc atc aat cta ttc ctg cac ctg tta 728 Ala Ile Ser
Leu Tyr Leu Asp Ile Ile Asn Leu Phe Leu His Leu Leu 165 170 175 cgg
ttt ctg gaa gca gtt aat aaa aag taattaaaag tatctcagct 775 Arg Phe
Leu Glu Ala Val Asn Lys Lys 180 185 caactgaaga acaacaaaaa
aaatttaacg agaaaaaagg attaaagtaa ttggaagcag 835 tatatagaaa
ctgtttcatt aagtaataaa gtttgaacca ataaaaaaaa aaaaaaaa 893 45 644 DNA
Homo sapiens CDS 23..340 sig_peptide 23..235 Von Heijne matrix
score 3.9 seq VAVYCSFISFANS/RS polyA_signal 611..616 polyA_site
629..644 45 gtgatctggc cttcgactcg ct atg tcc act aac aat atg tcg
gac cca cgg 52 Met Ser Thr Asn Asn Met Ser Asp Pro Arg -70 -65 agg
ccg aac aaa gtg ctg agg tac aag ccc ccg ccg agc gaa tgt aac 100 Arg
Pro Asn Lys Val Leu Arg Tyr Lys Pro Pro Pro Ser Glu Cys Asn -60 -55
-50 ccg gcc ttg gac gac ccg acg ccg gac tac atg aac ctg ctg ggc atg
148 Pro Ala Leu Asp Asp Pro Thr Pro Asp Tyr Met Asn Leu Leu Gly Met
-45 -40 -35 -30 atc ttc agc atg tgc ggc ctc atg ctt aag ctg aag tgg
tgt gct tgg 196 Ile Phe Ser Met Cys Gly Leu Met Leu Lys Leu Lys Trp
Cys Ala Trp -25 -20 -15 gtc gct gtc tac tgc tcc ttc atc agc ttt gcc
aac tct cgg agc tcg 244 Val Ala Val Tyr Cys Ser Phe Ile Ser Phe Ala
Asn Ser Arg Ser Ser -10 -5 1 gag gac acg aag caa atg atg agt agc
ttc atg ctg tcc atc tct gcc 292 Glu Asp Thr Lys Gln Met Met Ser Ser
Phe Met Leu Ser Ile Ser Ala 5 10 15 gtg gtg atg tcc tat ctg cag aat
cct cag ccc atg acg ccc cca tgg 340 Val Val Met Ser Tyr Leu Gln Asn
Pro Gln Pro Met Thr Pro Pro Trp 20 25 30 35 tgataccagc ctagaagggt
cacattttgg accctgtcta tccactaggc ctgggctttg 400 gctgctaaac
ctgctgcctt cagctgccat cctggacttc cctgaatgag gccgtctcgg 460
tgcccccagc tggatagagg gaacctggcc ctttcctagg gaacacccta ggcttacccc
520 tcctgcctcc cttcccctgc ctgctgctgg gggagatgct gtccatgttt
ctaggggtat 580 tcatttgctt tctcgttgaa acctgttgtt aataaagttt
ttcactctaa aaaaaaaaaa 640 aaaa 644 46 538 DNA Homo sapiens CDS
12..380 sig_peptide 12..263 Von Heijne matrix score 6.2 seq
GLFRAAWLPGSRP/SP polyA_site 523..538 46 ctgaattcct t atg tcc ggt
ggg cca gaa gcc cgt cct cct atg ctg gtg 50 Met Ser Gly Gly Pro Glu
Ala Arg Pro Pro Met Leu Val -80 -75 gaa ggc gga gga ccg gag tcc ctg
cag aag gcc ccg tgc act cgg ggg 98 Glu Gly Gly Gly Pro Glu Ser Leu
Gln Lys Ala Pro Cys Thr Arg Gly -70 -65 -60 cct ccc tca cat ccc gtg
ccc cct gcg ctg gcc ttc aca gta ggt aat 146 Pro Pro Ser His Pro Val
Pro Pro Ala Leu Ala Phe Thr Val Gly Asn -55 -50 -45 -40 ggc tcc ggc
ccg ggt gtt cgc tgt cca cgg aac atg gca gag ggg cac 194 Gly Ser Gly
Pro Gly Val Arg Cys Pro Arg Asn Met Ala Glu Gly His -35 -30 -25 ccc
ggc ccg gaa aga cgc cag agc cag cag ggg ctg ttt cgg gcc gcg 242 Pro
Gly Pro Glu Arg Arg Gln Ser Gln Gln Gly Leu Phe Arg Ala Ala -20 -15
-10 tgg ctc ccc ggg tct cgg ccg tct ccc ctc ttc tgc gtc tgt tcc gtg
290 Trp Leu Pro Gly Ser Arg Pro Ser Pro Leu Phe Cys Val Cys Ser Val
-5 1 5 act tcg cct ggg tgg gat gta ccg cag gtg cat cgc gtc gag gtg
ggg 338 Thr Ser Pro Gly Trp Asp Val Pro Gln Val His Arg Val Glu Val
Gly 10 15 20 25 cac ggc cgc cgg caa gaa acc cac cct gtc cgg agg cgg
gcg 380 His Gly Arg Arg Gln Glu Thr His Pro Val Arg Arg Arg Ala 30
35 tgagacaagc ccagcccgca cgcgctcatc tttcttcgtt ttttgatcag
tttattcaga 440 attgctctat aatttaccaa ttgtatgtat ttaacctatt
cttgtggaaa aaaaaggtct 500 ttcattatat ctttatttct gcaaaaaaaa aaaaaaaa
538 47 752 DNA Homo sapiens CDS 8..232 sig_peptide 8..154 Von
Heijne matrix score 4.7 seq DTFLLSFLSTTWL/KT polyA_site 737..752 47
gggggtg atg ccg cgc ggt cgc agg ctt ggg atg gtg ttc gcg cct ccg 49
Met Pro Arg Gly Arg Arg Leu Gly Met Val Phe Ala Pro Pro -45 -40 aga
ccc gga cag agg caa gca ggg gcg ccg tgg gtg cca gag agg cgg 97 Arg
Pro Gly Gln Arg Gln Ala Gly Ala Pro Trp Val Pro Glu Arg Arg -35 -30
-25 -20 aag agg agg cct gat ggg gat acc ttc ctg ctg tcc ttc ctg agc
aca 145 Lys Arg Arg Pro Asp Gly Asp Thr Phe Leu Leu Ser Phe Leu Ser
Thr -15 -10 -5 acc tgg ctg aaa acc tgg agg tca caa cag tac aaa gaa
tca aag tca 193 Thr Trp Leu Lys Thr Trp Arg Ser Gln Gln Tyr Lys Glu
Ser Lys Ser 1 5 10 aga tct tgt gcc aga gag caa atg aac tct tcc tct
tgc tgagaaaacc 242 Arg Ser Cys Ala Arg Glu Gln Met Asn Ser Ser Ser
Cys 15 20 25 caccctgctc acctaaaccc tggccttgcc tggtaattcc atccatgcgc
ctggaaggcc 302 ccagacatca aggctctgag gggccaggca cggggagaac
ccagcagtgc cctgccctgc 362 agtctgagct accagattcc ttgtgaagat
aatttgagga ccatgactca cccaaccaca 422 tttcctgggg cctcaaattg
aaaattcagg atgggctttt ctatatgact ggctgatatc 482 caactatgcc
atggtcttta catgccatga acattctttc ctgccagagt tctaagaatc 542
tgtgttctct gccttagacc ttctgcagat gagcccacag gaagctccac gtgtagctga
602 gctacatgca ccaggcctca gtttgcccca agtcccctgt gtactctctc
atggcctgtg 662 gccaagaaat gtattctctc actttggact taggagtcca
aagagaagcc cagaaacaaa 722 attgcttgaa cttgaaaaaa aaaaaaaaaa 752 48
537 DNA Homo sapiens CDS 183..422 sig_peptide 183..302 Von Heijne
matrix score 5.8 seq VLFALFVAFLLRG/KL polyA_signal 505..510
polyA_site 523..537 48 agtatctcac catttctttc tctttctgaa ccacattggg
tgccaacaga acttgctctc 60 tgttctcttt caaaattacc aacatggacc
ccacccaatt cttcccttgg aactaaggaa 120 cgcctgactg atcatctgat
acagcagttc ctgagcagaa caaaacaaca aaaacaggac 180 ag atg gat gga ata
ccc atg tca atg aag aat gaa atg ccc atc tcc 227 Met Asp Gly Ile Pro
Met Ser Met Lys Asn Glu Met Pro Ile Ser -40 -35 -30 caa cta ctg atg
atc atc gcc ccc tcc ttg gga ttt gtg ctc ttc gca 275 Gln Leu Leu Met
Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala -25 -20 -15 -10 ttg
ttt gtg gcg ttt ctc ctg aga ggg aaa ctc atg gaa acc tat tgt 323 Leu
Phe Val Ala Phe Leu Leu Arg Gly Lys Leu Met Glu Thr Tyr Cys -5 1 5
tcg cag aaa cac aca agg cta gac tac att gga gat agt aaa aat gtc 371
Ser Gln Lys His Thr Arg Leu Asp Tyr Ile Gly Asp Ser Lys Asn Val 10
15 20 ctc aat gac gtg cag cat gga agg gaa gac gaa gac ggc ctt ttt
acc 419 Leu Asn Asp Val Gln His Gly Arg Glu Asp Glu Asp Gly Leu Phe
Thr 25 30 35 ctc taacaacgca gtagcatgtt agattgagga tgggggcatg
acactccagt 472 Leu 40 gtcaaaataa gtcttagtag atttccttgt ttcataaaaa
agactcactc aaaaaaaaaa 532 aaaaa 537 49 1602 DNA Homo sapiens CDS
24..1004 sig_peptide 24..170 Von Heijne matrix score 5.6 seq
ACLSLGFFSLLWL/QL polyA_site 1586..1602 49 atgcgccgcc gcctctccgc acg
atg ttc ccc tcg cgg agg aaa gcg gcg cag 53 Met Phe Pro Ser Arg Arg
Lys Ala Ala Gln -45 -40 ctg ccc tgg gag gac ggc agg tcc ggg ttg ctc
tcc ggc ggc ctc cct 101 Leu Pro Trp Glu Asp Gly Arg Ser Gly Leu Leu
Ser Gly Gly Leu Pro -35 -30 -25 cgg aag tgt tcc gtc ttc cac ctg ttc
gtg gcc tgc ctc tcg ctg ggc 149 Arg Lys Cys Ser Val Phe His Leu Phe
Val Ala Cys Leu Ser Leu Gly -20 -15 -10 ttc ttc tcc cta ctc tgg ctg
cag ctc agc tgc tct ggg gac gtg gcc 197 Phe Phe Ser Leu Leu Trp Leu
Gln Leu Ser Cys Ser Gly Asp Val Ala -5 1 5 cgg gca gtc agg gga caa
ggg cag gag acc tcg ggc cct ccc cgt gcc 245 Arg Ala Val Arg Gly Gln
Gly Gln Glu Thr Ser Gly Pro Pro Arg Ala 10 15 20 25 tgc ccc cca gag
ccg ccc cct gag cac tgg gaa gaa gac gca tcc tgg 293 Cys Pro Pro Glu
Pro Pro Pro Glu His Trp Glu Glu Asp Ala Ser Trp 30 35 40 ggc ccc
cac cgc ctg gca gtg ctg gtg ccc ttc cgc gaa cgc ttc gag 341 Gly Pro
His Arg Leu Ala Val Leu Val Pro Phe Arg Glu Arg Phe Glu 45 50 55
gag ctc ctg gtc ttc gtg ccc cac atg cgc cgc ttc ctg agc agg aag 389
Glu Leu Leu Val Phe Val Pro His Met Arg Arg Phe Leu Ser Arg Lys 60
65 70 aag atc cgg cac cac atc tac gtg ctc aac cag gtg gac cac ttc
agg 437 Lys Ile Arg His His Ile Tyr Val Leu Asn Gln Val Asp His Phe
Arg 75 80 85 ttc aac cgg gca gcg ctc atc aac gtg ggc ttc ctg gag
agc agc aac 485 Phe Asn Arg Ala Ala Leu Ile Asn Val Gly Phe Leu Glu
Ser Ser Asn 90 95 100 105 agc acg gac tac att gcc atg cac gac gtt
gac ctg ctc cct ctc aac 533 Ser Thr Asp Tyr Ile Ala Met His Asp Val
Asp Leu Leu Pro Leu Asn 110 115 120 gag gag ctg gac tat ggc ttt cct
gag gct ggg ccc ttc cac gtg gcc 581 Glu Glu Leu Asp Tyr Gly Phe Pro
Glu Ala Gly Pro Phe His Val Ala 125 130 135 tcc ccg gag ctc cac cct
ctc tac cac tac aag acc tat gtc ggc ggc 629 Ser Pro Glu Leu His Pro
Leu Tyr His Tyr Lys Thr Tyr Val Gly Gly 140 145 150 atc ctg ctg ctc
tcc aag cag cac tac cgg ctg tgc aat ggg atg tcc 677 Ile Leu Leu Leu
Ser Lys Gln His Tyr Arg Leu Cys Asn Gly Met Ser 155 160 165 aac cgc
ttc tgg ggc tgg ggc cgc gag gac gac gag ttc tac cgg cgc 725 Asn Arg
Phe Trp Gly Trp Gly Arg Glu Asp Asp Glu Phe Tyr Arg Arg 170 175 180
185 att aag gga gct ggg ctc cag ctt ttc cgc ccc tcg gga atc aca act
773 Ile Lys Gly Ala Gly Leu Gln Leu Phe Arg Pro Ser Gly Ile Thr Thr
190 195 200 ggg tac aag aca ttt cgc cac ctg cat gac cca gcc tgg cgg
aag agg 821 Gly Tyr Lys Thr Phe Arg His Leu His Asp Pro Ala Trp Arg
Lys Arg 205 210 215 gac cag aag cgc atc gca gct caa aaa cag gag cag
ttc aag gtg gac 869 Asp Gln Lys Arg Ile Ala Ala Gln Lys Gln Glu Gln
Phe Lys Val Asp 220 225 230 agg gag gga ggc ctg aac act gtg aag tac
cat gtg gct tcc cgc act 917 Arg Glu Gly Gly Leu Asn Thr Val Lys Tyr
His Val Ala Ser Arg Thr 235 240 245 gcc ctg tct gtg ggc ggg gcc ccc
tgc act gtc ctc aac atc atg ttg 965 Ala Leu Ser Val Gly Gly Ala Pro
Cys Thr Val Leu Asn Ile Met Leu 250 255 260 265 gac tgt gac aag acc
gcc aca ccc tgg tgc aca ttc agc tgagctggat 1014 Asp Cys Asp Lys Thr
Ala Thr Pro Trp Cys Thr Phe Ser 270 275 ggacagtgag gaagcctgta
cctacaggcc atattgctca ggctcaggac aaggcctcag 1074 gtcgtgggcc
cagctctgac aggatgtgga gtggccagga ccaagacagc aagctacgca 1134
attgcagcca cccggccgcc aaggcaggct tgggctgggc caggacacgt ggggtgcctg
1194 ggacgctgct tgccatgcac agtgatcaga gagaggctgg ggtgtgtcct
gtccgggacc 1254 ccccctgcct tcctgctcac cctactctga cctccttcac
gtgcccaggc ctgtgggtag 1314 tggggagggc tgaacaggac aacctctcat
cacccccact tttgttcctt cctgctgggc 1374 tgcctcgtgc agagacacag
tgtaggggcc atgcagctgg cgtaggtggc agttgggcct 1434 ggtgagggtt
aggacttcag aaaccagagc acaagcccca cagaggggga acagccagca 1494
ccgctctagc tggttgttgc catgccggaa tgtgggccta gtgttgccag atcttctgat
1554 ttttcgaaag aaactagaat gctggattct caaaaaaaaa aaaaaaaa 1602 50
948 DNA Homo sapiens CDS 80..784 sig_peptide 80..139 Von Heijne
matrix score 4 seq LLKVVFVVFASLC/AW polyA_signal 910..915
polyA_site 933..948 50 cttcctgacc caggggctcc gctggctgcg gtcgcctggg
agctgccgcc agggccagga 60 ggggagcggc acctggaag atg cgc cca ttg gct
ggt ggc ctg ctc aag gtg 112 Met Arg Pro Leu Ala Gly Gly Leu Leu Lys
Val -20 -15 -10 gtg ttc gtg gtc ttc gcc tcc ttg tgt gcc tgg tat tcg
ggg tac ctg 160 Val Phe Val Val Phe Ala Ser Leu Cys Ala Trp Tyr Ser
Gly Tyr Leu -5 1 5 ctc gca gag ctc att cca gat gca ccc ctg tcc agt
gct gcc tat agc 208 Leu Ala Glu Leu Ile Pro Asp Ala Pro Leu Ser Ser
Ala Ala Tyr Ser 10 15 20 atc cgc agc atc ggg gag agg cct gtc ctc
aaa gct cca gtc ccc aaa 256 Ile Arg Ser Ile Gly Glu Arg Pro Val Leu
Lys Ala Pro Val Pro Lys 25 30 35 agg caa aaa tgt gac cac tgg act
ccc tgc cca tct gac acc tat gcc 304 Arg Gln Lys Cys Asp His Trp Thr
Pro Cys Pro Ser Asp Thr Tyr Ala 40 45 50 55 tac agg tta ctc agc gga
ggt ggc aga agc aag tac gcc aaa atc tgc 352 Tyr Arg Leu Leu Ser Gly
Gly Gly Arg Ser Lys Tyr Ala Lys Ile Cys 60 65 70 ttt gag gat aac
cta ctt atg gga gaa cag ctg gga aat gtt gcc aga 400 Phe Glu Asp Asn
Leu Leu Met Gly Glu Gln Leu Gly Asn Val Ala Arg 75 80 85 gga ata
aac att gcc att gtc aac tat gta act ggg aat gtg aca gca 448 Gly Ile
Asn Ile Ala Ile Val Asn Tyr Val Thr Gly Asn Val Thr Ala 90 95 100
aca cga tgt ttt gat atg tat gaa ggc gat aac tct gga ccg atg aca 496
Thr Arg Cys Phe Asp Met Tyr Glu Gly Asp Asn Ser Gly Pro Met Thr 105
110 115 aag ttt att cag agt gct gct cca aaa tcc ctg ctc ttc atg gtg
acc 544 Lys Phe Ile Gln Ser Ala Ala Pro Lys Ser Leu Leu Phe Met Val
Thr 120 125 130 135 tat gac gac gga agc aca aga ctg aat aac gat gcc
aag aat gcc ata 592 Tyr Asp Asp Gly Ser Thr Arg Leu Asn Asn Asp Ala
Lys Asn Ala Ile 140 145 150 gaa gca ctt gga agt aaa gaa atc agg aac
atg aaa ttc agg tct agc 640 Glu Ala Leu Gly Ser Lys Glu Ile Arg Asn
Met Lys Phe Arg Ser Ser 155 160 165 tgg gta ttt att gca gca aaa ggc
ttg gaa ctc cct tcc gaa att cag 688 Trp Val Phe Ile Ala Ala Lys Gly
Leu Glu Leu Pro Ser Glu Ile Gln 170 175 180 aga gaa aag atc aac cac
tct gat gct aag aac aac aga tat tct ggc 736 Arg Glu Lys Ile Asn His
Ser Asp Ala Lys Asn Asn Arg Tyr Ser Gly 185 190 195 tgg cct gca gag
atc cag ata gaa ggc tgc ata ccc aaa gaa cga agc 784 Trp Pro Ala Glu
Ile Gln Ile Glu Gly Cys Ile Pro Lys Glu Arg Ser 200 205 210 215
tgacactgca gggtcctgag taaatgtgtt ctgtataaac aaatgcagct ggaatcgctc
844 aagaatctta tttttctaaa tccaacagcc catatttgat gagtattttg
ggtttgttgt 904 aaaccaatga acatttgcta gttgtaccaa aaaaaaaaaa aaaa 948
51 687 DNA Homo sapiens CDS 67..222 sig_peptide 67..159 Von Heijne
matrix score 5.8 seq VLFSASSFPSISG/NI polyA_site 673..687 51
tacaattgga aaatctttat acattgaaaa aagcaacttt tcctccccct ctcaataggt
60 acaaga atg cgg gtt tat aaa agg aca cag ttg agg caa gag acc gga
108 Met Arg Val Tyr Lys Arg Thr Gln Leu Arg Gln Glu Thr Gly -30 -25
-20 ccc aaa agt tat gtg ctc ttt agt gcc tca agt ttt cca agc atc tct
156 Pro Lys Ser Tyr Val Leu Phe Ser Ala Ser Ser Phe Pro Ser Ile Ser
-15 -10 -5 ggt aac ata agg agt aga aat tat ttt caa aaa caa aat aat
cac tgg 204 Gly Asn Ile Arg Ser Arg Asn Tyr Phe Gln Lys Gln Asn Asn
His Trp 1 5 10 15 ttc cag acc agt gat tat taaccctttt tgaattatga
acccctttaa 252 Phe Gln Thr Ser Asp Tyr 20 aacctaatga aatttaagga
ccctctcccc caaaatatac atataaaaaa acaaggcagt 312 ctatggacct
actgagtaac tctcaagata gtaagtaagg agagaaagat ctatgtttcc 372
ctctttgata agtatgaaat atttggagga gatgctaatt tttgcacgtt tatgatattt
432 gcaatctttc atttttgtag cagattatac tcaaaaattt gatccagaac
ttggccccta 492 ttcttttatc agcactttaa cttgtaaact gaaaagttta
ccatcatctg tatgacatcc 552 taatgaggtt aaaaagataa aatgcagtta
tgattatgat aggtataact gtatccaggt 612 ttccacagca aaaacaaaac
aaaacataca ccatgttctg gggttattga cagcctcctc 672 aaaaaaaaaa aaaaa
687 52 821 DNA Homo sapiens CDS 46..732 sig_peptide 46..186 Von
Heijne matrix score 9.4 seq LILLILCVGMVVG/LV polyA_signal 781..786
polyA_site 806..821 52 gcaaagtcat tgaactctga gctcagttgc agtactcggg
aagcc atg cag gat gaa 57 Met Gln Asp Glu -45 gat gga tac atc acc
tta aat att aaa act cgg aaa cca gct ctc gtc 105 Asp Gly Tyr Ile Thr
Leu Asn Ile Lys Thr Arg Lys Pro Ala Leu Val -40 -35 -30 tcc gtt ggc
cct gca tcc tcc ttc tgg tgg cgt gtg atg gct ttg att 153 Ser Val Gly
Pro Ala Ser Ser Phe Trp Trp Arg Val Met Ala Leu Ile -25 -20 -15 ctg
ctg atc ctg tgc gtg ggg atg gtt gtc ggg ctg gtg gct ctg ggg 201 Leu
Leu Ile Leu Cys Val Gly Met Val Val Gly Leu Val Ala Leu Gly -10 -5
1 5 att tgg tct gtc atg cag cgc aat tac cta caa gat gag aat gaa aat
249 Ile Trp Ser Val Met Gln Arg Asn Tyr Leu Gln Asp Glu Asn Glu Asn
10 15 20 cgc aca gga act ctg caa caa tta gca aag cgc ttc tgt caa
tat gtg 297 Arg Thr Gly Thr Leu Gln Gln Leu Ala Lys Arg Phe Cys Gln
Tyr Val 25 30 35 gta aaa caa tca gaa cta aag ggc act ttc aaa ggt
cat aaa tgc agc 345 Val Lys Gln Ser Glu Leu Lys Gly Thr Phe Lys Gly
His Lys Cys Ser 40 45 50 ccc tgt gac aca aac tgg aga tat tat gga
gat agc tgc tat ggg ttc 393 Pro Cys Asp Thr Asn Trp Arg Tyr Tyr Gly
Asp Ser Cys Tyr Gly Phe 55 60 65 ttc agg cac aac tta aca tgg gaa
gag agt aag cag tac tgc act gac 441 Phe Arg His Asn Leu Thr Trp Glu
Glu Ser Lys Gln Tyr Cys Thr Asp 70 75 80 85 atg aat gct act ctc ctg
aag att gac aac cgg aac att gtg gag tac 489 Met Asn Ala Thr Leu Leu
Lys Ile Asp Asn Arg Asn Ile Val Glu Tyr 90 95 100 atc aaa gcc agg
act cat tta att cgt tgg gtc gga tta tct cgc cag 537 Ile Lys Ala Arg
Thr His Leu Ile Arg Trp Val Gly Leu Ser Arg Gln 105 110 115 aag tcg
aat gag gtc tgg aag tgg gag gat ggc tcg gtt atc tca gaa 585 Lys Ser
Asn Glu Val Trp Lys Trp Glu Asp Gly Ser Val Ile Ser Glu 120 125 130
aat atg ttt gag ttt ttg gaa gat gga aaa gga aat atg aat tgt gct 633
Asn Met Phe Glu Phe Leu Glu Asp Gly Lys Gly Asn Met Asn Cys Ala 135
140 145 tat ttt cat aat ggg aaa atg cac cct acc ttc tgt gag aac aaa
cat 681 Tyr Phe His Asn Gly Lys Met His Pro Thr Phe Cys Glu Asn Lys
His 150 155 160 165 tat tta atg tgt gag agg aag gct ggc atg acc aag
gtg gac caa cta 729 Tyr Leu Met Cys Glu Arg Lys Ala Gly Met Thr Lys
Val Asp Gln Leu 170 175 180 cct taatgcaaag aggtggacag gataacacag
ataagggctt tattgtacaa 782 Pro taaaagatat gtatgaatgc aacaaaaaaa
aaaaaaaaa 821 53 445 DNA Homo sapiens CDS 81..356 sig_peptide
81..152 Von Heijne matrix score 6.2 seq AILGSTWVALTTG/AL
polyA_signal 406..411 polyA_site 429..445 misc_feature 1 n=a, g, c
or t 53 ngaaaaaaaa catccgggcc gcgcggggaa ggggagacgt ggggtagagg
ggagcattgc 60 ttccttctct cgcagtgacc atg acg aaa tta gcg cag tgg ctt
tgg gga cta 113 Met Thr Lys Leu Ala Gln Trp Leu Trp Gly Leu -20 -15
gcg atc ctg ggc tcc acc tgg gtg gcc ctg acc acg gga gcc ttg ggc 161
Ala Ile Leu Gly Ser Thr Trp Val Ala Leu Thr Thr Gly Ala Leu Gly -10
-5 1 ctg gag ctg ccc ttg tcc tgc cag gaa gtc ctg tgg cca ctg ccc
gcc 209 Leu Glu Leu Pro Leu Ser Cys Gln Glu Val Leu Trp Pro Leu Pro
Ala 5 10 15 tac ttg ctg gtg tcc gcc ggc tgc tat gcc ctg ggc act gtg
ggc tat 257 Tyr Leu Leu Val Ser Ala Gly Cys Tyr Ala Leu Gly Thr Val
Gly Tyr 20 25 30 35 cgt gtg gcc act ttt cat gac tgc gag gac gcc gca
cgc gag ctg cag 305 Arg Val Ala Thr Phe His Asp Cys Glu Asp Ala Ala
Arg Glu Leu Gln 40 45 50 agc cag ata cag gag gcc cga gcc gac tta
gcc cgc agg ggg ctg cgc 353 Ser Gln Ile Gln Glu Ala Arg Ala Asp Leu
Ala Arg Arg Gly Leu Arg 55 60 65 ttc tgacagccta accccattcc
tgtgcggaca gcccttcctc ccatttccca 406 Phe ttaaagagcc agtttatttt
ctaaaaaaaa aaaaaaaaa 445 54 1517 DNA Homo sapiens CDS 72..1346
sig_peptide 72..140 Von Heijne matrix score 5.9 seq
SCDCFVSVPPASA/IP polyA_signal 1482..1487 polyA_site 1502..1517 54
atggggcggc cctggccaga agcggaggag gtggcacccg ggaccgagct ggggtcttgg
60 aggaagagag g atg gcg tcg tcg agc cct gac tcc cca tgt tcc tgc gac
110 Met Ala Ser Ser Ser Pro Asp Ser Pro Cys Ser Cys Asp -20 -15 tgc
ttt gtc tcc gtg ccc ccg gcc tca gcc atc ccg gct gtg atc ttt 158 Cys
Phe Val Ser Val Pro Pro Ala Ser Ala Ile Pro Ala Val Ile Phe -10 -5
1 5 gcc aag aac tcg gac cga ccc cgg gac gag gtg cag gag gtg gtg ttt
206 Ala Lys Asn Ser Asp Arg Pro Arg Asp Glu Val Gln Glu Val Val Phe
10 15 20 gtc ccc gca ggc act cac act cct ggg agc cgg ctc cag tgc
acc tac 254 Val Pro Ala Gly Thr His Thr Pro Gly Ser Arg Leu Gln Cys
Thr Tyr 25 30 35 att gaa gtg gaa cag gtg tcg aag acg cac gct gtg
att ctg agc cgt 302 Ile Glu Val Glu Gln Val Ser Lys Thr His Ala Val
Ile Leu Ser Arg 40 45 50 cct tct tgg cta tgg ggg gct gag atg ggc
gcc aac gag cat ggt gtc 350 Pro Ser Trp Leu Trp Gly Ala Glu Met Gly
Ala Asn Glu His Gly Val 55 60 65 70 tgc att ggc aac gag gct gtg tgg
acg aag gag cca gtt ggg gag ggg 398 Cys Ile Gly Asn Glu Ala Val Trp
Thr Lys Glu Pro Val Gly Glu Gly 75 80 85 gaa gcc ctg ctg ggc atg
gac cta ctc agg ctg gct ttg gaa cgg agc 446 Glu Ala Leu Leu Gly Met
Asp Leu Leu Arg Leu Ala Leu Glu Arg Ser 90 95 100 agc tct gcc cag
gag gcc ttg cat gtg atc aca ggg tta ctg gag cac 494 Ser Ser Ala Gln
Glu Ala Leu His Val Ile Thr Gly Leu Leu Glu His 105 110 115 tat ggg
cag ggg ggc aac tgc ctg gag gat gct gcg cca ttc tcc tac 542 Tyr Gly
Gln Gly Gly Asn Cys Leu Glu Asp Ala Ala Pro Phe Ser Tyr 120 125 130
cat agc acc ttc ctg ctg gct gac cgc act gag gcg tgg gtg ctg gag 590
His Ser Thr Phe Leu Leu Ala Asp Arg Thr Glu Ala Trp Val Leu Glu 135
140 145 150 aca gct ggg agg ctc tgg gct gca cag agg atc cag gag ggg
gcc cgc 638 Thr Ala Gly Arg Leu Trp Ala Ala Gln Arg Ile Gln Glu Gly
Ala Arg 155 160 165 aac atc tcc aac cag ctg agc att ggc acg gac atc
tcg gcc caa cac 686 Asn Ile Ser Asn Gln Leu Ser Ile Gly Thr Asp Ile
Ser Ala Gln His 170 175 180 ccg gag ctg cgg act cat gcc cag gcc aag
ggc tgg tgg gat ggg cag 734 Pro Glu Leu Arg Thr His Ala Gln Ala Lys
Gly Trp Trp Asp Gly Gln 185 190 195 ggt gcc ttt gac ttt gct cag atc
ttc tcc ctg acc cag cag cct gtg 782 Gly Ala Phe Asp Phe Ala Gln Ile
Phe Ser Leu Thr Gln Gln Pro Val 200 205 210 cgc atg gag gct gcc aag
gcc cgc ttc cag gca ggg cgg gag ctg ctg 830 Arg Met Glu Ala Ala Lys
Ala Arg Phe Gln Ala Gly Arg Glu Leu Leu 215 220 225 230 cgg caa cgg
caa ggg ggc atc acg gca gag gtg atg atg ggc atc ctc 878 Arg Gln Arg
Gln Gly Gly Ile Thr Ala Glu Val Met Met Gly Ile Leu 235 240 245 aga
gac aag gag agt ggt atc tgt atg gac tcg gga ggc ttt cgc acc 926 Arg
Asp Lys Glu Ser Gly Ile Cys Met Asp Ser Gly Gly Phe Arg Thr 250 255
260 acg gcc agc atg gtg tct gtc ctg ccc cag gat ccc acg cag ccc tgc
974 Thr Ala Ser Met Val Ser Val Leu Pro Gln Asp Pro Thr Gln Pro Cys
265 270 275 gtg cac ttt ctt acc gcc acg cca gac cca tcc agg tct gtg
ttc aaa 1022 Val His Phe Leu Thr Ala Thr Pro Asp Pro Ser Arg Ser
Val Phe Lys 280 285 290 cct ttc atc ttc ggg gtg ggg gtg gcc cag gcc
ccc cag gtg ctg tcc 1070 Pro Phe Ile Phe Gly Val Gly Val Ala Gln
Ala Pro Gln Val Leu Ser 295 300 305 310 ccc act ttt gga gca caa gac
cct gtt cgg acc ctg ccc cga ttc cag 1118 Pro Thr Phe Gly Ala Gln
Asp Pro Val Arg Thr Leu Pro Arg Phe Gln 315 320 325 act cag gta gat
cgt cgg cat acc ctc tac cgt gga cac cag gca gcc 1166 Thr Gln Val
Asp Arg Arg His Thr Leu Tyr Arg Gly His Gln Ala Ala 330 335 340 ctg
ggg ctg atg gag aga gat cag gat cgg ggg cag cag ctc cag cag 1214
Leu Gly Leu Met Glu Arg Asp Gln Asp Arg Gly Gln Gln Leu Gln Gln 345
350 355 aaa cag cag gat ctg gag cag gaa ggc ctc gag gcc aca cag ggg
ctg 1262 Lys Gln Gln Asp Leu Glu Gln Glu Gly Leu Glu Ala Thr Gln
Gly Leu 360 365 370 ctg gcc ggc gag tgg gcc cca ccc ctc tgg gag ctg
ggc agc ctc ttc 1310 Leu Ala Gly Glu Trp Ala Pro Pro Leu Trp Glu
Leu Gly Ser Leu Phe 375 380 385 390 cag gcc ttc gtg aag agg gag agc
cag gct tat gcg taagcttcat 1356 Gln Ala Phe Val Lys Arg Glu Ser Gln
Ala Tyr Ala 395 400 agcttctgct ggcctggggt ggacccagga cccctggggc
ctgggtgccc tgagtggtgg 1416 taaagtggag caatcccttc acgctccttg
gccatgttct gagcggccag cttggccttt 1476 gccttaataa atgtgcttta
ttttcaaaaa aaaaaaaaaa a 1517 55 1560 DNA Homo sapiens CDS 194..454
sig_peptide 194..379 Von Heijne matrix score 4.6 seq
HILTVPLLEPARC/SG polyA_site 1545..1560 55 cattcataaa tattctctta
ccattttact tgacaattat tttaggctta cagaaaagtg 60 gccagagtag
tgcagggctc ctatagttgg cttcccctgt tgccatcatc tcgtctgatc 120
gtagggcagg ttagcattgc tacaggcctc ttacccggcc tacagctctt aggcacatct
180 gtccatttga cta atg gcc att ttc tgg ata gtc cat gct cac ttc tgg
229 Met Ala Ile Phe Trp Ile Val His Ala His Phe Trp -60 -55 agc ccc
ctc cca ccc agg ctc cca cat ggc cgg tgc tgt tgc ctg aag 277 Ser Pro
Leu Pro Pro Arg Leu Pro His Gly Arg Cys Cys Cys Leu Lys -50 -45 -40
-35 gcc cct ctt cct cct gac gtg gga ccc ctt cag gta gcc ccg cat ctt
325 Ala Pro Leu Pro Pro Asp Val Gly Pro Leu Gln Val Ala Pro His Leu
-30 -25 -20 ttc agc gtg ccc ctt cac att ctg act gtt cct ctt ctg gaa
cct gca 373 Phe Ser Val Pro Leu His Ile Leu Thr Val Pro Leu Leu Glu
Pro Ala -15 -10 -5 aga tgc tct ggg atc ctt gta ttt ttc ctg cac cag
ccc gtt tca tcc 421 Arg Cys Ser Gly Ile Leu Val Phe Phe Leu His Gln
Pro Val Ser Ser 1 5 10 ctg agc ttc tgt tat ttt att gga gga tgg tgc
tagaaacaca ggtctggatg 474 Leu Ser Phe Cys Tyr Phe Ile Gly Gly Trp
Cys 15 20 25 caggcaggag acacacgcgt ccacactagc atgcgtgtgt acacacatct
acatgtgctt 534 atcccccgcg ttcatgttaa aaaccatggg atcataccgg
tgtttcagat tcacatccac 594 cccagcaggg tttctcgccc ccattgctta
taaccttagc aggtgttgag aaccctggcg 654 ctcactgtcc acagtgagtt
tgcttattcg ttgaaaccta gcgtgcctgt agagtgtgga 714 gagttgccgg
cccgcacccc tgcgagacac agactttctg accgcagccc tcatgtgtgt 774
ggctcttctt gtccttggcc ttacagtgca gtcggatcgc tgctttccag agttgcctgg
834 gggtaggtcc ctcctcttct gtgctctgcg gcgcagtgag cggcctttgc
ctcaggcctc 894 ccgcggcttc cttaagcctc tggcctgccc ggtccctggc
gccaggtctg ttttccctgc 954 tcccttctct ctgatcctgc tttggtctga
gccgtgcctc tgggccccag cattgctggg 1014 ccgcattgtc gttttatttc
tcttgtgtcg ttgcgtctag tgtaagacat tcagtggatc 1074 attgtggatg
gtcattagtg gtccagagtg gaaagtgagg tcgttgttgg tggtgtacct 1134
acagtgcctg ttagggagct gttcctggtg ttgcccgtga atattagact tgctcccgag
1194 cctgcgccac agcccatccc tagcgactta gcgacagtgg ctgccaggtg
cgggtggctg 1254 tgtcttgtat acactgtgtg ggcagcccag ggccaggggc
ctcctccttc catggcagcc 1314 tctgtctgca tcacagagat aaggccgcgg
ctgccaccag gataaggagc cagcagctgc 1374 tctcggagga gccgccctga
cccctcccca tcatgccgcc gtggggtttc catgcagaat 1434 tttccttggg
cagagttgct ttttgattct agtttttaaa aaaactgttc tttccatcat 1494
gataaaaaga aagacatgct catttcaaat agtttaggag atgtggaagc aaaaaaaaaa
1554 aaaaaa 1560 56 1066 DNA Homo sapiens CDS 48..494 sig_peptide
48..347 Von Heijne matrix score 3.7 seq LASSFLFTMGGLG/FI
polyA_signal 1031..1036 polyA_site 1051..1066 56 gaggcgcgtg
gggcttgagg ccgagaacgg cccttgctgc caccaac atg gag act 56 Met Glu Thr
-100 ttg tac cgt gtc ccg ttc tta gtg ctc gaa tgt ccc aac ctg aag
ctg 104 Leu Tyr Arg Val Pro Phe Leu Val Leu Glu Cys Pro Asn Leu Lys
Leu -95 -90 -85 aag aag ccg ccc tgg ttg cac atg ccg tcg gcc atg act
gtg tat gct 152 Lys Lys Pro Pro Trp Leu His Met Pro Ser Ala Met Thr
Val Tyr Ala -80 -75 -70 ctg gtg gtg gtg tct tac ttc ctc atc acc gga
gga ata att tat gat 200 Leu Val Val Val Ser Tyr Phe Leu Ile Thr Gly
Gly Ile Ile Tyr Asp -65 -60 -55 -50 gtt att gtt gaa cct cca agt gtc
ggt tct atg act gat gaa cat ggg 248 Val Ile Val Glu Pro Pro Ser Val
Gly Ser Met Thr Asp Glu His Gly -45 -40 -35 cat cag agg cca gta gct
ttc ttg gcc tac aga gta aat gga caa tat 296 His Gln Arg Pro Val Ala
Phe Leu Ala Tyr Arg Val Asn Gly Gln Tyr -30 -25 -20 att atg gaa gga
ctt gca tcc agc ttc cta ttt aca atg gga ggt tta 344 Ile Met Glu Gly
Leu Ala Ser Ser Phe Leu Phe Thr Met Gly Gly Leu -15 -10 -5 ggt ttc
ata atc ctg gac cga tcg aat gca cca aat atc cca aaa ctc 392 Gly Phe
Ile Ile Leu Asp Arg Ser Asn Ala Pro Asn Ile Pro Lys Leu 1 5 10 15
aat aga ttc ctt ctt ctg ttc att gga ttc gtc tgt gtc cta ttg agt 440
Asn Arg Phe Leu Leu Leu Phe Ile Gly Phe Val Cys Val Leu Leu Ser 20
25 30 ttt ttc atg gct aga gta ttc atg aga atg aaa ctg ccg ggc tat
ctg 488 Phe Phe Met Ala Arg Val Phe Met Arg Met Lys Leu Pro Gly Tyr
Leu 35 40 45 atg ggt tagagtgcct ttgagaagaa atcagtggat actggatttg
ctcctgtcaa 544 Met Gly tgaagtttta aaggctgtac caatcctcta atatgaaatg
tggaaaagaa tgaagagcag 604 cagtaaaaga aatatctagt gaaaaaacag
gaagcgtatt gaagcttgga ctagaatttc 664 ttcttggtat taaagagaca
agtttatcac agaatttttt ttcctgctgg cctattgcta 724 taccaatgat
gttgagtggc attttctttt tagtttttca ttaaaatata ttccatatct 784
acaactataa tatcaaataa agtgattatt ttttacaacc ctcttaacat tttttggaga
844 tgacatttct gattttcaga aattaacata aaatccagaa gcaagattcc
gtaagctgag 904 aactctggac agttgatcag ctttacctat ggtgctttgc
ctttaactag agtgtgtgat 964 ggtagattat ttcagatatg tatgtaaaac
tgtttcctga acaataagat gtatgaacgg 1024 agcagaaata aatacttttt
ctaattaaaa aaaaaaaaaa aa 1066 57 1061 DNA Homo sapiens CDS 111..671
sig_peptide 111..215 Von Heijne matrix score 4.5 seq
SFTVSMAIGLVLG/GF polyA_signal 990..995 polyA_site 1045..1061 57
attatttttc tcttgctgta ctacaaagag atagaatcaa actgcttttt ttcgacatac
60 tggtttttct ttctgttttt cttctctttc ttctatttct tgtggatatt atg gct
116 Met Ala -35 aat aac aca aca agt tta ggg agt cca tgg cca gaa aac
ttt tgg gag 164 Asn Asn Thr Thr Ser Leu Gly Ser Pro Trp Pro Glu Asn
Phe Trp Glu -30 -25 -20 gac ctt atc atg tcc ttc act gta tcc atg gca
atc ggg ctg gta ctt 212 Asp Leu Ile Met Ser Phe Thr Val Ser Met Ala
Ile Gly Leu Val Leu -15 -10 -5 gga gga ttt att tgg gct gtg ttc att
tgt ctg tct cga aga aga aga 260 Gly Gly Phe Ile Trp Ala Val Phe Ile
Cys Leu Ser Arg Arg Arg Arg 1 5 10 15 gcc agt gct ccc atc tca cag
tgg agt tca agc agg aga tct agg tct 308 Ala Ser Ala Pro Ile Ser Gln
Trp Ser Ser Ser Arg Arg Ser Arg Ser 20 25 30 tct tac acc cac ggc
ctc aac aga act gga ttt tac cgc cac agt ggc 356 Ser Tyr Thr His Gly
Leu Asn Arg Thr Gly Phe Tyr Arg His Ser Gly 35 40 45 tgt gaa cgt
cga agc aac ctc agc ctg gcc agt ctc acc ttc cag cga 404 Cys Glu Arg
Arg Ser Asn Leu Ser Leu Ala Ser Leu Thr Phe Gln Arg 50 55 60 caa
gct tcc ctg gaa caa gca aat tcc ttt cca aga aaa tca agt ttc 452 Gln
Ala Ser Leu Glu Gln Ala Asn Ser Phe Pro Arg Lys Ser Ser Phe 65 70
75 aga gct tct act ttc cat ccc ttt ctg caa tgt cca cca ctt cct gtg
500 Arg Ala Ser Thr Phe His Pro Phe Leu Gln Cys Pro Pro Leu Pro Val
80 85
90 95 gaa act gag agt cag ctg gtg act ctc cct tct tcc aat atc tct
ccc 548 Glu Thr Glu Ser Gln Leu Val Thr Leu Pro Ser Ser Asn Ile Ser
Pro 100 105 110 acc atc agc act tcc cac agt ctg agc cgt cct gac tac
tgg tcc agt 596 Thr Ile Ser Thr Ser His Ser Leu Ser Arg Pro Asp Tyr
Trp Ser Ser 115 120 125 aac agt ctt cga gtg ggc ctt tca aca ccg ccc
cca cct gcc tat gag 644 Asn Ser Leu Arg Val Gly Leu Ser Thr Pro Pro
Pro Pro Ala Tyr Glu 130 135 140 tcc atc atc aag gca ttc cca gat tcc
tgagtagggt ggcttttggt 691 Ser Ile Ile Lys Ala Phe Pro Asp Ser 145
150 ttttgtttct ttcttgtctt gtcttttatt gaaaggaaat caaaaatagg
ctaaacagaa 751 ttttgagggc atggcccaaa taactcatga gttccaagtt
gaaacatggt tgtgcaagtt 811 ggacattaca atgtaaaaca cattttcttc
aaacacgttt tcccttttgt ttcaaaaaat 871 gtaatatttt cccccaagcg
ttttatattt atgtattttg tattcaatgt gaggcttatt 931 aaaaatagtg
attctaatgt aagaatcagc taagatgcat tatatatatt ttaattaaaa 991
ttaaaacttc agatatttgt ggattacaat cctcatttac ttccaatgtg actaaaaaaa
1051 aaaaaaaaaa 1061 58 2025 DNA Homo sapiens CDS 5..373
sig_peptide 5..82 Von Heijne matrix score 4 seq SLFWFTVITLSFG/YY
polyA_signal 1986..1991 polyA_site 2010..2025 58 agcc atg gct acg
gca gcc ggc gcg acc tac ttt cag cga ggc agt ctg 49 Met Ala Thr Ala
Ala Gly Ala Thr Tyr Phe Gln Arg Gly Ser Leu -25 -20 -15 ttc tgg ttc
aca gtc atc acc ctc agc ttt ggc tac tac aca tgg gtt 97 Phe Trp Phe
Thr Val Ile Thr Leu Ser Phe Gly Tyr Tyr Thr Trp Val -10 -5 1 5 gtc
ttc tgg cct cag agt atc cct tat cag aac ctt ggg ccc ctg ggc 145 Val
Phe Trp Pro Gln Ser Ile Pro Tyr Gln Asn Leu Gly Pro Leu Gly 10 15
20 ccc ttc act cag tac ttg gtg gac cac cat cac acc ctc ctg tgc aat
193 Pro Phe Thr Gln Tyr Leu Val Asp His His His Thr Leu Leu Cys Asn
25 30 35 ggg tat tgg ctt gcc tgg ctg att cat gtg gga gag tcc ttg
tat gcc 241 Gly Tyr Trp Leu Ala Trp Leu Ile His Val Gly Glu Ser Leu
Tyr Ala 40 45 50 ata gta ttg tgc aag cat aaa ggc atc aca agt ggt
cgg gct cag cta 289 Ile Val Leu Cys Lys His Lys Gly Ile Thr Ser Gly
Arg Ala Gln Leu 55 60 65 ctc tgg ttc cta cag act ttc ttc ttt ggg
ata gcg tct ctc acc atc 337 Leu Trp Phe Leu Gln Thr Phe Phe Phe Gly
Ile Ala Ser Leu Thr Ile 70 75 80 85 ttg att gct tac aaa cgg aag cgc
caa aaa caa act tgaagttgtc 383 Leu Ile Ala Tyr Lys Arg Lys Arg Gln
Lys Gln Thr 90 95 tgaaagcttg ctctacactt ttacattcat cctcaccctt
ttttttgtgg ggtagaggag 443 gtgcagtaat ttactcagtg atctttctac
tttctagaaa ctgtccttca aagctcttta 503 agaccccctc gttagtcagt
tttttctctt atatgctctg gttgagcttg aatagaccag 563 ttgttactta
agaaagaaac agagaaagat tttagctttt caatcctatt tggcagagga 623
cttcagctac cttcttacag tctttggctg tgttggtacc ctcgtgtgct ctgagctaag
683 ccacatacta aactgacttt ttggtttgta tacccttgct cccgccttct
gatgaaaaca 743 ccttaccctc acaaccacca tctttcctct cctttccaaa
gctctttcca ccttgctgca 803 ctaagataaa gtgacacttc cactatatgt
caattccaca cacatttatt aggtacctgt 863 gaggtaggat cctatcctct
caaacttcca tttctcatgc tacagagaaa gataaggaag 923 atgagcaagt
gcctggaatg gggcaggctg agcagtcaca caggcataga ggcacgctga 983
gaacctggag gggagactgc agagtgcctt ccctgatgct gcagccggaa gtgatccttc
1043 cctccacctg gcccctggga cactgtgctc tgcagtgtgc agggcctgat
ggcactgcta 1103 gattgctcct tcagctcagg gccacagctt aaacagcttt
acctttcccc tcagcacctg 1163 tcccactatc ttgcacacag gtgctctaac
catgtttatt gaacaaagga gggaaactga 1223 tttcactttc acttgttcat
tatcattcca atttttatgt gaaaatggca caacccattt 1283 ggggtaccct
caccccaaaa taaaagccca agtctacctt tgactggtac cacctttttt 1343
gtggtttcgt tggtgagaaa cctttatctt tttcatacct ttctattctc aatcacttct
1403 ccaaaagtgt gtctttccag ctctgattta ttcaaaacac aagcatttct
gtttagagat 1463 tctagcccat gggttatctg gctagttatt acctctcctg
ttcacttagt tatactttat 1523 tattgctcac aggctgggga ggcagaatga
ctctgtcacc actaggagcc attagggctt 1583 cttccctgga ggactgcctg
cttgctttct ggggacacta gccctcattt cccttctgtg 1643 gtacagtggg
gcaaattatt tgtattaagc aaacatttat gggaaacaac ccgctcccga 1703
aaacggagcc cccaagtaaa gcacaaccct gaaagattat gaactatgaa ttgtctctgg
1763 tagagataaa tttctgcaaa catatctcag tcttccctct gtttctctgg
tgattaagaa 1823 gttccttttt ggtaaggaaa aggattttta accatagagt
taggcatcat ggaaattcaa 1883 accagatttc ttaatacctg gtcttcctca
aagagaaata ataacagtaa tagtggtgct 1943 gggaacaata tggcagatta
ttgaatgaaa ttgattaact tgaataaaat gctgtgaatt 2003 ttctctaaaa
aaaaaaaaaa aa 2025 59 591 DNA Homo sapiens CDS 14..472 sig_peptide
14..319 Von Heijne matrix score 4.9 seq VFFFGVSIILVLG/ST
polyA_signal 555..560 polyA_site 576..591 59 agcaccatct gtc atg gcg
gct ggg ctg ttt ggt ttg agc gct cgc cgt 49 Met Ala Ala Gly Leu Phe
Gly Leu Ser Ala Arg Arg -100 -95 ctt ttg gcg gca gcg gcg acg cga
ggg ctc ccg gcc gcc cgc gtc cgc 97 Leu Leu Ala Ala Ala Ala Thr Arg
Gly Leu Pro Ala Ala Arg Val Arg -90 -85 -80 -75 tgg gaa tct agc ttc
tcc agg act gtg gtc gcc ccg tcc gct gtg gcg 145 Trp Glu Ser Ser Phe
Ser Arg Thr Val Val Ala Pro Ser Ala Val Ala -70 -65 -60 gga aag cgg
ccc cca gaa ccg acc aca ccg tgg caa gag gac cca gaa 193 Gly Lys Arg
Pro Pro Glu Pro Thr Thr Pro Trp Gln Glu Asp Pro Glu -55 -50 -45 ccc
gag gac gaa aac ttg tat gag aag aac cca gac tcc cat ggt tat 241 Pro
Glu Asp Glu Asn Leu Tyr Glu Lys Asn Pro Asp Ser His Gly Tyr -40 -35
-30 gac aag gac ccc gtt ttg gac gtc tgg aac atg cga ctt gtc ttc ttc
289 Asp Lys Asp Pro Val Leu Asp Val Trp Asn Met Arg Leu Val Phe Phe
-25 -20 -15 ttt ggc gtc tcc atc atc ctg gtc ctt ggc agc acc ttt gtg
gcc tat 337 Phe Gly Val Ser Ile Ile Leu Val Leu Gly Ser Thr Phe Val
Ala Tyr -10 -5 1 5 ctg cct gac tac agg atg aaa gag tgg tcc cgc cgc
gaa gct gag agg 385 Leu Pro Asp Tyr Arg Met Lys Glu Trp Ser Arg Arg
Glu Ala Glu Arg 10 15 20 ctt gtg aaa tac cga gag gcc aat ggc ctt
ccc atc atg gaa tcc aac 433 Leu Val Lys Tyr Arg Glu Ala Asn Gly Leu
Pro Ile Met Glu Ser Asn 25 30 35 tgc ttc gac ccc agc aag atc cag
ctg cca gag gat gag tgaccagttg 482 Cys Phe Asp Pro Ser Lys Ile Gln
Leu Pro Glu Asp Glu 40 45 50 ctaagtgggg ctcaagaagc accgccttcc
ccaccacctg cctgccattc tgacctcttc 542 tcagagcacc taattaaagg
ggctgaaagt ctgaaaaaaa aaaaaaaaa 591 60 544 DNA Homo sapiens CDS
2..217 polyA_signal 489..494 polyA_site 529..544 60 t cta cct gtg
agt act agg atc atc aat cat atc tac agc ttc ccc tca 49 Leu Pro Val
Ser Thr Arg Ile Ile Asn His Ile Tyr Ser Phe Pro Ser 1 5 10 15 gtt
gat tta tgg ata gtt tgt att ttc act gta tct gtc tca cac ctt 97 Val
Asp Leu Trp Ile Val Cys Ile Phe Thr Val Ser Val Ser His Leu 20 25
30 ttt gaa aag gga aca ttg tat ggc tac ttt tat gtg att aac tcc tcc
145 Phe Glu Lys Gly Thr Leu Tyr Gly Tyr Phe Tyr Val Ile Asn Ser Ser
35 40 45 atc aat tta tgt gtc aat gat tgc ctt cct gta atg gat tca
att tct 193 Ile Asn Leu Cys Val Asn Asp Cys Leu Pro Val Met Asp Ser
Ile Ser 50 55 60 ctg tct cca ttg ttt ctt tct cac tagagaagtt
ctttaaaatt ctatgaaaat 247 Leu Ser Pro Leu Phe Leu Ser His 65 70
gaaactgtgc taaattaaaa atctactcat gataacagga gacactcaaa attatgggtt
307 tcagtttcag gcttctcacc atgtcctcag attgtactcc ctttctagcc
cttctgcagc 367 aaataaacct ttgccatcag ttcaccaaaa gcactcatga
gaggaaaaat ggcatatcac 427 taaatataga gttctttgtc acttcttgat
ttcaaattta caactaatac tcaacacttt 487 aattaaatct ttcttttctc
ttcttcctaa aacatacatg caaaaaaaaa aaaaaaa 544 61 1689 DNA Homo
sapiens CDS 51..575 sig_peptide 51..110 Von Heijne matrix score
11.2 seq AFLLLVALSYTLA/RD polyA_signal 1653..1658 polyA_site
1674..1689 61 agaagcttgg accgcatcct agccgccgac tcacacaagg
cagagttgcc atg gag 56 Met Glu -20 aaa att cca gtg tca gca ttc ttg
ctc ctt gtg gcc ctc tcc tac act 104 Lys Ile Pro Val Ser Ala Phe Leu
Leu Leu Val Ala Leu Ser Tyr Thr -15 -10 -5 ctg gcc aga gat acc aca
gtc aaa cct gga gcc aaa aag gac aca aag 152 Leu Ala Arg Asp Thr Thr
Val Lys Pro Gly Ala Lys Lys Asp Thr Lys 1 5 10 gac tct cga ccc aaa
ctg ccc cag acc ctc tcc aga ggt tgg ggt gac 200 Asp Ser Arg Pro Lys
Leu Pro Gln Thr Leu Ser Arg Gly Trp Gly Asp 15 20 25 30 caa ctc atc
tgg act cag aca tat gaa gaa gct cta tat aaa tcc aag 248 Gln Leu Ile
Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys Ser Lys 35 40 45 aca
agc aac aaa ccc ttg atg att att cat cac ttg gat gag tgc cca 296 Thr
Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu Cys Pro 50 55
60 cac agt caa gct tta aag aaa gtg ttt gct gaa aat aaa gaa atc cag
344 His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn Lys Glu Ile Gln
65 70 75 aaa ttg gca gag cag ttt gtc ctc ctc aat ctg gtt tat gaa
aca act 392 Lys Leu Ala Glu Gln Phe Val Leu Leu Asn Leu Val Tyr Glu
Thr Thr 80 85 90 gac aaa cac ctt tct cct gat ggc cag tat gtc ccc
agg att atg ttt 440 Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Val Pro
Arg Ile Met Phe 95 100 105 110 gtt gac cca tct ctg aca gtt aga gcc
gat atc act gga aga tat tca 488 Val Asp Pro Ser Leu Thr Val Arg Ala
Asp Ile Thr Gly Arg Tyr Ser 115 120 125 aat cgt ctc tat gct tac gaa
cct gca gat aca gct ctg ttg ctt gac 536 Asn Arg Leu Tyr Ala Tyr Glu
Pro Ala Asp Thr Ala Leu Leu Leu Asp 130 135 140 aac atg aag aaa gct
ctc aag ttg ctg aag act gaa ttg taaagaaaaa 585 Asn Met Lys Lys Ala
Leu Lys Leu Leu Lys Thr Glu Leu 145 150 155 aaatctccaa gcccttctgt
ctgtcaggcc ttgagacttg aaaccagaag aagtgtgaga 645 agactggcta
gtgtggaagc atagtgaaca cactgattag gttatggttt aatgttacaa 705
caactatttt ttaagaaaaa caagttttag aaatttggtt tcaagtgtac atgtgtgaaa
765 acaatattgt atactaccat agtgagccat gattttctaa aaaaaaaata
aatgttttgg 825 gggtgttctg ttttctccaa cttggtcttt cacagtggtt
cgtttaccaa ataggattaa 885 acacacacaa aatgctcaag gaagggacaa
gacaaaacca aaactagttc aaatgatgaa 945 gaccaaagac caagttatca
tctcaccaca ccacaggttc tcactagatg actgtaagta 1005 gacacgagct
taatcaacag aagtatcaag ccatgtgctt tagcataaaa gaatatttag 1065
aaaaacatcc caagaaaatc acatcactac ctagagtcaa ctctggccag gaactctaag
1125 gtacacactt tcatttagta attaaatttt agtcagattt tgcccaacct
aatgctctca 1185 gggaaagcct ctggcaagta gctttctcct tcagaggtct
aatttagtag aaaggtcatc 1245 caaagaacat ctgcactcct gaacacaccc
tgaagaaatc ctgggaattg accttgtaat 1305 cgatttgtct gtcaaggtcc
taaagtactg gagtgaaata aattcagcca acatgtgact 1365 aattggaaga
agagcaaagg gtggtgacgt gttgatgagg cagatggaga tcagaggtta 1425
ctagggttta ggaaacgtga aaggctgtgg catcagggta ggggagcatt ctgcctaaca
1485 gaaattagaa ttgtgtgtta atgtcttcac tctatactta atctcacatt
cattaatata 1545 tggaattcct ctactgccca gcccctactg atttctttgg
cccctggact atggtgctgt 1605 atataatgct ttgcagtatc tgttgcttgt
cttgattaac ttttttggat aaaacctttt 1665 ttgaacagaa aaaaaaaaaa aaaa
1689 62 1111 DNA Homo sapiens CDS 69..977 sig_peptide 69..128 Von
Heijne matrix score 5.3 seq VLLGSGLTILSQP/LM polyA_signal
1076..1081 polyA_site 1096..1111 62 acctaggacc ggctcaccgg
gtcgcttggt ggctccgtct gtctgtccgt ccgcccgcgg 60 gtgccatc atg gcg gac
gcg gcc agt cag gtg ctc ctg ggc tcc ggt ctc 110 Met Ala Asp Ala Ala
Ser Gln Val Leu Leu Gly Ser Gly Leu -20 -15 -10 acc atc ctg tcc cag
ccg ctc atg tac gtg aaa gtg ctc atc cag gtg 158 Thr Ile Leu Ser Gln
Pro Leu Met Tyr Val Lys Val Leu Ile Gln Val -5 1 5 10 gga tat gag
cct ctt cct cca aca ata gga cga aat att ttt ggg cgg 206 Gly Tyr Glu
Pro Leu Pro Pro Thr Ile Gly Arg Asn Ile Phe Gly Arg 15 20 25 caa
gtg tgt cag ctt cct ggt ctc ttt agt tat gct cag cac att gcc 254 Gln
Val Cys Gln Leu Pro Gly Leu Phe Ser Tyr Ala Gln His Ile Ala 30 35
40 agt atc gat ggg agg cgc ggg ttg ttc aca ggc tta act cca aga ctg
302 Ser Ile Asp Gly Arg Arg Gly Leu Phe Thr Gly Leu Thr Pro Arg Leu
45 50 55 tgt tcg gga gtc ctt gga act gtg gtc cat ggt aaa gtt tta
cag cat 350 Cys Ser Gly Val Leu Gly Thr Val Val His Gly Lys Val Leu
Gln His 60 65 70 tac cag gag agt gac aag ggt gag gag tta gga cct
gga aat gta cag 398 Tyr Gln Glu Ser Asp Lys Gly Glu Glu Leu Gly Pro
Gly Asn Val Gln 75 80 85 90 aaa gaa gtc tca tct tcc ttt gac cac gtt
atc aag gag aca act cga 446 Lys Glu Val Ser Ser Ser Phe Asp His Val
Ile Lys Glu Thr Thr Arg 95 100 105 gag atg atc gct cgt tct gct gct
acc ctc atc aca cat ccc ttc cat 494 Glu Met Ile Ala Arg Ser Ala Ala
Thr Leu Ile Thr His Pro Phe His 110 115 120 gtg atc act ctg aga tct
atg gta cag ttc att ggc aga gaa tcc aag 542 Val Ile Thr Leu Arg Ser
Met Val Gln Phe Ile Gly Arg Glu Ser Lys 125 130 135 tac tgt gga ctt
tgt gat tcc ata ata acc atc tat cgg gaa gag ggc 590 Tyr Cys Gly Leu
Cys Asp Ser Ile Ile Thr Ile Tyr Arg Glu Glu Gly 140 145 150 att cta
gga ttt ttc gcg ggt ctt gtt cct cgc ctt cta ggt gac atc 638 Ile Leu
Gly Phe Phe Ala Gly Leu Val Pro Arg Leu Leu Gly Asp Ile 155 160 165
170 ctt tct ttg tgg ctg tgt aac tca ctg gcc tac ctc gtc aat acc tat
686 Leu Ser Leu Trp Leu Cys Asn Ser Leu Ala Tyr Leu Val Asn Thr Tyr
175 180 185 gca ctg gac agt ggg gtt tct acc atg aat gaa atg aag agt
tat tct 734 Ala Leu Asp Ser Gly Val Ser Thr Met Asn Glu Met Lys Ser
Tyr Ser 190 195 200 caa gct gtc aca gga ttt ttt gcg agt atg ttg acc
tat ccc ttt gtg 782 Gln Ala Val Thr Gly Phe Phe Ala Ser Met Leu Thr
Tyr Pro Phe Val 205 210 215 ctt gtc tcc aat ctt atg gct gtc aac aac
tgt ggt ctt gct ggt gga 830 Leu Val Ser Asn Leu Met Ala Val Asn Asn
Cys Gly Leu Ala Gly Gly 220 225 230 tgc cct cct tac tcc cca ata tat
acg tct tgg ata gac tgt tgg tgc 878 Cys Pro Pro Tyr Ser Pro Ile Tyr
Thr Ser Trp Ile Asp Cys Trp Cys 235 240 245 250 atg cta caa aaa gag
ggg aat atg agc cga gga aat agc tta ttt ttc 926 Met Leu Gln Lys Glu
Gly Asn Met Ser Arg Gly Asn Ser Leu Phe Phe 255 260 265 cgg aag gtc
ccc ttt ggg aag act tat tgt tgt gac ctg aaa atg tta 974 Arg Lys Val
Pro Phe Gly Lys Thr Tyr Cys Cys Asp Leu Lys Met Leu 270 275 280 att
tgaagatgtg gggcagggac agtgacattt ctgtagtccc agatgcacag 1027 Ile
aattatggga gagaatgttg atttctatac agtgtggcgc gcttttttaa taatcattta
1087 atcttggcaa aaaaaaaaaa aaaa 1111 63 554 DNA Homo sapiens CDS
44..238 sig_peptide 44..160 Von Heijne matrix score 3.9 seq
FKTIAFLLLYVSA/GP polyA_signal 443..448 polyA_site 540..554 63
atcctcaaca gaataattgc tgacaaactc tcttgcccag aaa atg tct act gga 55
Met Ser Thr Gly att atg gag tac aaa aaa act aca aaa gca atg aaa aaa
aag aag gat 103 Ile Met Glu Tyr Lys Lys Thr Thr Lys Ala Met Lys Lys
Lys Lys Asp -35 -30 -25 -20 gtt tta ttt aca tcc tat ttc aaa acc att
gct ttc ttg cta ttg tat 151 Val Leu Phe Thr Ser Tyr Phe Lys Thr Ile
Ala Phe Leu Leu Leu Tyr -15 -10 -5 gtc tct gca ggc cca ata tcg cga
atc ttc ata aga agt tta gaa ttg 199 Val Ser Ala Gly Pro Ile Ser Arg
Ile Phe Ile Arg Ser Leu Glu Leu 1 5 10 ttc ctt atg ttt cct tct aac
aaa cac tgg tat att tca tgaaagtgta 248 Phe Leu Met Phe Pro Ser Asn
Lys His Trp Tyr Ile Ser 15 20 25 tattttattc acttccaaaa cagttagctc
ataattcaga acattgaggt ttgcaaaatg 308 actgaaggaa actttaccta
aacaatagtt gccagttctg ctgagaatta tcacgggccc 368 acaacggctg
tgtgtttttc catacagata ttctaatttt tttattatgc agctaatttt 428
tttttagact cgcgaataaa atagcaagtc agtctgtgca taagcatatg tttaaatcta
488 ccaggagaaa tgtctggaat ctttttggtt attaaaatta aaattcagga
taaaaaaaaa 548 aaaaaa 554 64 1773 DNA Homo sapiens CDS 114..524
sig_peptide 114..164 Von Heijne matrix score 5.2 seq
ATLAVGLTIFVLS/VV polyA_signal 1739..1744 polyA_site 1758..1773 64
gatttgcttt ctttttctcc aaaaggggag gaaattgaaa
ctgagtggcc cacgatggga 60 agaggggaaa gcccaggggt acaggaggcc
tctgggtgaa ggcagaggct aac atg 116 Met ggg ttc gga gcg acc ttg gcc
gtt ggc ctg acc atc ttt gtg ctg tct 164 Gly Phe Gly Ala Thr Leu Ala
Val Gly Leu Thr Ile Phe Val Leu Ser -15 -10 -5 gtc gtc act atc atc
atc tgc ttc acc tgc tcc tgc tgc tgc ctt tac 212 Val Val Thr Ile Ile
Ile Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr 1 5 10 15 aag acg tgc
cgc cga cca cgt ccg gtt gtc acc acc acc aca tcc acc 260 Lys Thr Cys
Arg Arg Pro Arg Pro Val Val Thr Thr Thr Thr Ser Thr 20 25 30 act
gtg gtg cat gcc cct tat cct cag cct cca agt gtg ccg ccc agc 308 Thr
Val Val His Ala Pro Tyr Pro Gln Pro Pro Ser Val Pro Pro Ser 35 40
45 tac cct gga cca agc tac cag ggc tac cac acc atg ccg cct cag cca
356 Tyr Pro Gly Pro Ser Tyr Gln Gly Tyr His Thr Met Pro Pro Gln Pro
50 55 60 ggg atg cca gca gca ccc tac cca atg cag tac cca cca cct
tac cca 404 Gly Met Pro Ala Ala Pro Tyr Pro Met Gln Tyr Pro Pro Pro
Tyr Pro 65 70 75 80 gcc cag ccc atg ggc cca ccg gcc tac cac gag acc
ctg gct gga gga 452 Ala Gln Pro Met Gly Pro Pro Ala Tyr His Glu Thr
Leu Ala Gly Gly 85 90 95 gca gcc gcg ccc tac ccc gcc agc cag cct
cct tac aac ccg gcc tac 500 Ala Ala Ala Pro Tyr Pro Ala Ser Gln Pro
Pro Tyr Asn Pro Ala Tyr 100 105 110 atg gat gcc ccg aag gcg gcc ctc
tgagcattcc ctggcctctc tggctgccac 554 Met Asp Ala Pro Lys Ala Ala
Leu 115 120 ttggttatgt tgtgtgtgtg cgtgagtggt gtgcaggcgc ggttccttac
gccccatgtg 614 tgctgtgtgt gtccaggcac ggttccttac gccccatgtg
tgctgtgtgt gtcctgcctg 674 tatatgtggc ttcctctgat gctgacaagg
tggggaacaa tccttgccag agtgggctgg 734 gaccagactt tgttctcttc
ctcacctgaa attatgcttc ctaaaatctc aagccaaact 794 caaagaatgg
ggtggtgggg ggcaccctgt gaggtggccc ctgagaggtg ggggcctctc 854
cagggcacat ctggagttct tctccagctt accctagggt gaccaagtag ggcctgtcac
914 accagggtgg cgcagctttc tgtgtgatgc agatgtgtcc tggtttcggc
agcgtagcca 974 gctgctgctt gaggccatgg ctcgtccccg gagttggggg
tacccgttgc agagccaggg 1034 acatgatgca ggcgaagctt gggatctggc
caagttggac tttgatcctt tgggcagatg 1094 tcccattgct ccctggagcc
tgtcatgcct gttggggatc aggcagcctc ctgatgccag 1154 aacacctcag
gcagagccct actcagctgt acctgtctgc ctggactgtc ccctgtcccc 1214
gcatctcccc tgggaccagc tggagggcca catgcacaca cagcctagct gcccccaggg
1274 agctctgctg cccttgctgg ccctgccctt cccacaggtg agcagggctc
ctgtccacca 1334 gcacactcag ttctcttccc tgcagtgttt tcattttatt
ttagccaaac attttgcctg 1394 ttttctgttt caaacatgat agttgatatg
agactgaaac ccctgggttg tggagggaaa 1454 ttggctcaga gatggacaac
ctggcaactg tgagtccctg cttcccgaca ccagcctcat 1514 ggaatatgca
acaactcctg taccccagtc cacggtgttc tggcagcagg gacacctggg 1574
ccaatgggcc atctggacca aaggtggggt gtggggccct ggatggcagc tctggcccag
1634 acatgaatac ctcgtgttcc tcctccctct attactgttt caccagagct
gtcttagctc 1694 aaatctgttg tgtttctgag tctagggtct gtacacttgt
ttataataaa tgcaatcgtt 1754 tgcaaaaaaa aaaaaaaaa 1773 65 917 DNA
Homo sapiens CDS 26..487 sig_peptide 26..64 Von Heijne matrix score
6.4 seq MALLLSVLRVLLG/GF polyA_signal 883..888 polyA_site 901..917
65 aacccacggt ggggggagcg cggcc atg gcg ctc ctg ctt tcg gtg ctg cgt
52 Met Ala Leu Leu Leu Ser Val Leu Arg -10 -5 gta ctg ctg ggc ggc
ttc ttc gcg ctc gtg ggg ttg gcc aag ctc tcg 100 Val Leu Leu Gly Gly
Phe Phe Ala Leu Val Gly Leu Ala Lys Leu Ser 1 5 10 gag gag atc tcg
gct cca gtt tcg gag cgg atg aat gcc ctg ttc gtg 148 Glu Glu Ile Ser
Ala Pro Val Ser Glu Arg Met Asn Ala Leu Phe Val 15 20 25 cag ttt
gct gag gtg ttc ccg ctg aag gta ttt ggc tac cag cca gat 196 Gln Phe
Ala Glu Val Phe Pro Leu Lys Val Phe Gly Tyr Gln Pro Asp 30 35 40
ccc ctg aac tac caa ata gct gtg ggc ttt ctg gaa ctg ctg gct ggg 244
Pro Leu Asn Tyr Gln Ile Ala Val Gly Phe Leu Glu Leu Leu Ala Gly 45
50 55 60 ttg ctg ctg gtc atg ggc cca ccg atg ctg caa gag atc agt
aac ttg 292 Leu Leu Leu Val Met Gly Pro Pro Met Leu Gln Glu Ile Ser
Asn Leu 65 70 75 ttc ttg att ctg ctc atg atg ggg gct atc ttc acc
ttg gca gct ctg 340 Phe Leu Ile Leu Leu Met Met Gly Ala Ile Phe Thr
Leu Ala Ala Leu 80 85 90 aaa gag tca cta agc acc tgt atc cca gcc
att gtc tgc ctg ggg ttc 388 Lys Glu Ser Leu Ser Thr Cys Ile Pro Ala
Ile Val Cys Leu Gly Phe 95 100 105 ctg ctg ctg ctg aat gtc ggc cag
ctc tta gcc cag act aag aag gtg 436 Leu Leu Leu Leu Asn Val Gly Gln
Leu Leu Ala Gln Thr Lys Lys Val 110 115 120 gtc aga ccc act agg aag
aag act cta agt aca ttc aag gaa tcc tgg 484 Val Arg Pro Thr Arg Lys
Lys Thr Leu Ser Thr Phe Lys Glu Ser Trp 125 130 135 140 aag
tagagcatct ctgtctcttt atgccatgca gctgtcacag caggaacatg 537 Lys
gtagaacaca gagtctatca tcttgttacc agtataatat ccagggtcag ccagtgttga
597 aagagacatt ttgtctacct ggcactgctt tctcttttta gctttactac
tcttttgtga 657 ggagtacatg ttatgcatat taacattcct catatcatat
gaaaatacaa aataagcaga 717 aaagaaattt aaatcaacca aaattctgat
gccccaaata accactttta atgccttggt 777 gtaagtatac ctctgaactt
ttttctgtgc ctttaaacag atatatattt tttttaaatg 837 aaaataaaac
catatatcct attttatttc ctccttttaa aaccttataa actataacac 897
tgcaaaaaaa aaaaaaaaaa 917 66 641 DNA Homo sapiens CDS 80..388
sig_peptide 80..187 Von Heijne matrix score 3.6 seq
RALSTFLFGSIRG/AA polyA_signal 609..614 polyA_site 627..641 66
gccagtgcgc agacgcaggg gtcggcgccg ggtgagagcg tgcggccggg taagggcgtg
60 tggccggatt caccacaac atg gca aat ctt ttt ata agg aaa atg gtg aac
112 Met Ala Asn Leu Phe Ile Arg Lys Met Val Asn -35 -30 cct ctg ctc
tat ctc agt cgt cac acg gtg aag cct cga gcc ctc tcc 160 Pro Leu Leu
Tyr Leu Ser Arg His Thr Val Lys Pro Arg Ala Leu Ser -25 -20 -15 -10
aca ttt cta ttt gga tcc att cga ggt gca gcc ccc gtg gct gtg gaa 208
Thr Phe Leu Phe Gly Ser Ile Arg Gly Ala Ala Pro Val Ala Val Glu -5
1 5 ccc ggg gca gca gtg cgc tca ctt ctc tca ccc ggc ctc ctg ccc cat
256 Pro Gly Ala Ala Val Arg Ser Leu Leu Ser Pro Gly Leu Leu Pro His
10 15 20 ctg ctg cct gcg ctg ggg ttc aaa aac aag act gtc ctt aat
aag cgc 304 Leu Leu Pro Ala Leu Gly Phe Lys Asn Lys Thr Val Leu Asn
Lys Arg 25 30 35 tgc aag gac tgt tac ctg gtg aag agg cgg ggt cgg
tgg tac gtc tac 352 Cys Lys Asp Cys Tyr Leu Val Lys Arg Arg Gly Arg
Trp Tyr Val Tyr 40 45 50 55 tgt aaa acc cat ccg agg cac aag cag aga
cag atg tagacccttt 398 Cys Lys Thr His Pro Arg His Lys Gln Arg Gln
Met 60 65 ccctccagac tcacgcacat actcgtcatc gcatcacttg ggagaatggt
tgtatcttat 458 ggaaggaatt atcacatcaa ggagtcaggg gaaagtgact
ggaagcaaac gccctaaaag 518 ttacccatca cgtttcagtg taaatgagta
actatagaag acattgcgtt atcttatttc 578 caaaacgttc caactaaaaa
acattttcct attaaaatag accttccgaa aaaaaaaaaa 638 aaa 641 67 854 DNA
Homo sapiens CDS 186..443 sig_peptide 186..407 Von Heijne matrix
score 3.9 seq ISCTCLLLYLTHC/IL polyA_signal 827..832 polyA_site
839..854 67 aaatgttaat attagaaaga gtctcatagt gcttatgtga catcattctt
tgcctaaagc 60 ctttgtacct actgtaatga agctaaactc cttggcacag
gatataaggc tcacgatctg 120 gcctggactc attttcactc ccatcttcag
tcatccccta actcccccac agtcagtccc 180 caaag atg cca tat gct ttc act
tct cca tgc cct tgc tca ttt gtc tca 230 Met Pro Tyr Ala Phe Thr Ser
Pro Cys Pro Cys Ser Phe Val Ser -70 -65 -60 ttg cct gaa ata tcc ttt
tat ttc acc aaa ctg ctg ctc atc ctc aag 278 Leu Pro Glu Ile Ser Phe
Tyr Phe Thr Lys Leu Leu Leu Ile Leu Lys -55 -50 -45 gcc ctg cct gag
tca cct ttc ctt ctt gct tcc tcc ccc ttg cct cct 326 Ala Leu Pro Glu
Ser Pro Phe Leu Leu Ala Ser Ser Pro Leu Pro Pro -40 -35 -30 ctc ccc
act acc cta aga aaa ttc atc cct ccc cct tca tta ata tca 374 Leu Pro
Thr Thr Leu Arg Lys Phe Ile Pro Pro Pro Ser Leu Ile Ser -25 -20 -15
tgc aca tgc ttg tta tta tat tta aca cat tgt ata tta ggt att tgt 422
Cys Thr Cys Leu Leu Leu Tyr Leu Thr His Cys Ile Leu Gly Ile Cys -10
-5 1 5 ttt gct tat cct ttt atc cta tgaaattgtg aacaatttgt tgaataattg
473 Phe Ala Tyr Pro Phe Ile Leu 10 aataatcaca tatcaaaatg tagagaggtt
atttgtctct tccctgtagg actccatttt 533 caggcagtgt ctgctaagaa
tccccttgac ctgggattgg aagttgtttc tcccactgct 593 gagctccttt
atattagctc ttcacctctc actcctttgt ttcttctctt ggcactttac 653
gtctttctac ccatttaatt tgataaatgt ctcatgtcat ctttaaaact gaaggtgaca
713 catgtctggt ttatctttat aactcaaaaa tgttgagctt aatgcagaat
ggagaatagc 773 tacttagtaa atttttaaaa tacatgctac catttttaag
gggagaagaa gacaatatac 833 atgacaaaaa aaaaaaaaaa a 854 68 1568 DNA
Homo sapiens CDS 75..1259 sig_peptide 75..1004 Von Heijne matrix
score 4.4 seq VLILLFSLALIIL/PS polyA_signal 1536..1541 polyA_site
1553..1568 68 agaaaaggtg tagtgtttgg ggcggtcaac gggctatgct
ggcttgacag ggctgggctc 60 ttcagaacag aagc atg gat ctc gga atc cct
gac ctg ctg gac gcg tgg 110 Met Asp Leu Gly Ile Pro Asp Leu Leu Asp
Ala Trp -310 -305 -300 ctg gag ccc cca gag gat atc ttc tcg aca gga
tcc gtc ctg gag ctg 158 Leu Glu Pro Pro Glu Asp Ile Phe Ser Thr Gly
Ser Val Leu Glu Leu -295 -290 -285 gga ctc cac tgc ccc cct cca gag
gtt ccg gta act agg cta cag gaa 206 Gly Leu His Cys Pro Pro Pro Glu
Val Pro Val Thr Arg Leu Gln Glu -280 -275 -270 cag gga ctg caa ggc
tgg aag tcc ggt ggg gac cgt ggc tgt ggc ctt 254 Gln Gly Leu Gln Gly
Trp Lys Ser Gly Gly Asp Arg Gly Cys Gly Leu -265 -260 -255 caa gag
agt gag cct gaa gat ttc ttg aag ctt ttc att gat ccc aat 302 Gln Glu
Ser Glu Pro Glu Asp Phe Leu Lys Leu Phe Ile Asp Pro Asn -250 -245
-240 -235 gag gtg tac tgc tca gaa gca tct cct ggc agt gac agt ggc
atc tct 350 Glu Val Tyr Cys Ser Glu Ala Ser Pro Gly Ser Asp Ser Gly
Ile Ser -230 -225 -220 gag gac tcc tgc cat cca gac agt ccc cct gcc
ccc agg gca acc agt 398 Glu Asp Ser Cys His Pro Asp Ser Pro Pro Ala
Pro Arg Ala Thr Ser -215 -210 -205 tct cct atg ctc tat gag gtt gtc
tat gag gca ggg gcc ctg gag agg 446 Ser Pro Met Leu Tyr Glu Val Val
Tyr Glu Ala Gly Ala Leu Glu Arg -200 -195 -190 atg cag ggg gaa act
ggg cca aat gta ggc ctt atc tcc atc cag cta 494 Met Gln Gly Glu Thr
Gly Pro Asn Val Gly Leu Ile Ser Ile Gln Leu -185 -180 -175 gat cag
tgg agc cca gca ttt atg gtg cct gat tcc tgc atg gtc agt 542 Asp Gln
Trp Ser Pro Ala Phe Met Val Pro Asp Ser Cys Met Val Ser -170 -165
-160 -155 gag ctg ccc ttt gat gct cat gcc cac atc ctg ccc aga gca
ggc acc 590 Glu Leu Pro Phe Asp Ala His Ala His Ile Leu Pro Arg Ala
Gly Thr -150 -145 -140 gta gcc cca gtg ccc tgt aca acc ctg ctg ccc
tgt caa acc ctg ttc 638 Val Ala Pro Val Pro Cys Thr Thr Leu Leu Pro
Cys Gln Thr Leu Phe -135 -130 -125 ctg acc gat gag gag aag cgt ctg
ctg ggg cag gaa ggg gtt tcc ctg 686 Leu Thr Asp Glu Glu Lys Arg Leu
Leu Gly Gln Glu Gly Val Ser Leu -120 -115 -110 ccc tct cac ctg ccc
ctc acc aag gca gag gag agg gtc ctc aag aag 734 Pro Ser His Leu Pro
Leu Thr Lys Ala Glu Glu Arg Val Leu Lys Lys -105 -100 -95 gtc agg
agg aaa atc cgt aac aag cag tca gct cag gac agt cgg cgg 782 Val Arg
Arg Lys Ile Arg Asn Lys Gln Ser Ala Gln Asp Ser Arg Arg -90 -85 -80
-75 cgg aag aag gag tac att gat ggg ctg gag agc agg gtg gca gcc tgt
830 Arg Lys Lys Glu Tyr Ile Asp Gly Leu Glu Ser Arg Val Ala Ala Cys
-70 -65 -60 tct gca cag aac caa gaa tta cag aaa aaa gtc cag gag ctg
gag agg 878 Ser Ala Gln Asn Gln Glu Leu Gln Lys Lys Val Gln Glu Leu
Glu Arg -55 -50 -45 cac aac atc tcc ttg gta gct cag ctc cgc cag ctg
cag acg cta att 926 His Asn Ile Ser Leu Val Ala Gln Leu Arg Gln Leu
Gln Thr Leu Ile -40 -35 -30 gct caa act tcc aac aaa gct gcc cag acc
agc act tgt gtt ttg att 974 Ala Gln Thr Ser Asn Lys Ala Ala Gln Thr
Ser Thr Cys Val Leu Ile -25 -20 -15 ctt ctt ttt tcc ctg gct ctc atc
atc ctg ccc agc ttc agt cca ttc 1022 Leu Leu Phe Ser Leu Ala Leu
Ile Ile Leu Pro Ser Phe Ser Pro Phe -10 -5 1 5 cag agt cga cca gaa
gct ggg tct gag gat tac cag cct cac gga gtg 1070 Gln Ser Arg Pro
Glu Ala Gly Ser Glu Asp Tyr Gln Pro His Gly Val 10 15 20 act tcc
aga aat atc ctg acc cac aag gac gta aca gaa aat ctg gag 1118 Thr
Ser Arg Asn Ile Leu Thr His Lys Asp Val Thr Glu Asn Leu Glu 25 30
35 acc caa gtg gta gag tcc aga ctg agg gag cca cct gga gcc aag gat
1166 Thr Gln Val Val Glu Ser Arg Leu Arg Glu Pro Pro Gly Ala Lys
Asp 40 45 50 gca aat ggc tca aca agg aca ctg ctt gag aag atg gga
ggg aag cca 1214 Ala Asn Gly Ser Thr Arg Thr Leu Leu Glu Lys Met
Gly Gly Lys Pro 55 60 65 70 aga ccc agt ggg cgc atc cgg tcc gtg ctg
cat gca gat gag atg 1259 Arg Pro Ser Gly Arg Ile Arg Ser Val Leu
His Ala Asp Glu Met 75 80 85 tgagctggaa cagaccttcc tggcccactt
cctgatcaca aggaatcctg ggcttcctta 1319 tggctttctt cccactggga
ttcctactta ggtgtctgcc ctcaggggtc caaatcactt 1379 caggacaccc
caagagatgt cctttagtct ctgcctgagg cctagtctgc atttgtttgc 1439
atatatgaga gggtacctca aatacttctg ttatgtatct gtgattttat ttcttctttg
1499 ggtatagggt tgaggggaaa taagttttga gtgagaaata aacgttttag
ctgaaaaaaa 1559 aaaaaaaaa 1568 69 506 DNA Homo sapiens CDS 98..376
sig_peptide 98..151 Von Heijne matrix score 12.3 seq
HILFLLLLPVAAA/QT polyA_signal 471..476 polyA_site 491..506 69
gacatccgct attgctactt ctctgctccc ccacagttcc tctggacttc tctggaccac
60 agtcctctgc cagacccctg ccagacccca gtccacc atg atc cat ctg ggt cac
115 Met Ile His Leu Gly His -15 atc ctc ttc ctg ctt ttg ctc cca gtg
gct gca gct cag acg act cca 163 Ile Leu Phe Leu Leu Leu Leu Pro Val
Ala Ala Ala Gln Thr Thr Pro -10 -5 1 gga gag aga tca tca ctc cct
gcc ttt tac cct ggc act tca ggc tct 211 Gly Glu Arg Ser Ser Leu Pro
Ala Phe Tyr Pro Gly Thr Ser Gly Ser 5 10 15 20 tgt tcc gga tgt ggg
tcc ctc tct ctg ccg ctc ctg gca ggc ctc gtg 259 Cys Ser Gly Cys Gly
Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu Val 25 30 35 gct gct gat
gcg gtg gca tcg ctg ctc atc gtg ggg gcg gtg ttc ctg 307 Ala Ala Asp
Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe Leu 40 45 50 tgc
gca cgc cca cgc cgc agc ccc gcc caa gaa tat ggc aaa gtc tac 355 Cys
Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Tyr Gly Lys Val Tyr 55 60
65 atc aac atg cca ggc agg ggc tgaccctcct gcagcttgga cctttgactt 406
Ile Asn Met Pro Gly Arg Gly 70 75 ctgaccctct catcctggat ggtgtgtggt
ggcacaggaa cccccgcccc aacttttgga 466 ttgtaataaa acaattgaaa
caccaaaaaa aaaaaaaaaa 506 70 542 DNA Homo sapiens CDS 72..254
sig_peptide 72..134 Von Heijne matrix score 4.2 seq
LINLAASRTLSFC/IS polyA_signal 506..511 polyA_site 528..542 70
gaccttaaga agagctaaac gggctgccac ctgtagctga agagtgcctt aacgccgagg
60 cccacggctc c atg cga gag atg cct gtt cct tct ctg ata aat ttg gca
110 Met Arg Glu Met Pro Val Pro Ser Leu Ile Asn Leu Ala -20 -15 -10
gct tca cgt acc cta agt ttt tgc att tct gac aac cac gtg tcc tca 158
Ala Ser Arg Thr Leu Ser Phe Cys Ile Ser Asp Asn His Val Ser Ser -5
1 5 cct gga ccc gcc aac cca tcc tgt ggc ctc cac cct cac tgg ctt cgt
206 Pro Gly Pro Ala Asn Pro Ser Cys Gly Leu His Pro His Trp Leu Arg
10 15 20 cca ctt aaa ctt tta acg tac aca tgt aga gag ctg aaa ctc
cag ggg 254 Pro Leu Lys Leu Leu Thr Tyr Thr Cys Arg Glu Leu Lys Leu
Gln Gly 25 30 35 40 taacatggga caggtcctct tgatttaatg aaaacagaag
atcaactgga ccgggtagca 314 agaaataagg cttaagaagc actggtttct
ctgcagaaga
cagcaagatg ccccagggaa 374 tgtttgtgaa aaaggatgac tggatgggaa
gcaagctgaa gaaaaagaag gaaagaaaga 434 gagaaatcag taaatcacca
cacaagaggt ggagaagagg acttataaat attgtttcta 494 tgacatttga
aaataaatgt tttactccat gctaaaaaaa aaaaaaaa 542 71 1629 DNA Homo
sapiens CDS 148..1140 sig_peptide 148..240 Von Heijne matrix score
10 seq LVLLLVTRSPVNA/CL polyA_signal 1590..1595 polyA_site
1614..1629 71 gtctgctgcc gccattgtgc ggcgctggtc ccctcagagg
gttcctgctg ctgccggtgc 60 cttggaccct ccccctcgct tctcgttcta
ctgccccagg agcccggcgg gtccgggact 120 cccgtccgtg ccggtgcggg cgccggc
atg tgg ctg tgg gag gac cag ggc ggc 174 Met Trp Leu Trp Glu Asp Gln
Gly Gly -30 -25 ctc ctg ggc cct ttc tcc ttc ctg ctg cta gtg ctg ctg
ctg gtg acg 222 Leu Leu Gly Pro Phe Ser Phe Leu Leu Leu Val Leu Leu
Leu Val Thr -20 -15 -10 cgg agc ccg gtc aat gcc tgc ctc ctc acc ggc
agc ctc ttc gtt cta 270 Arg Ser Pro Val Asn Ala Cys Leu Leu Thr Gly
Ser Leu Phe Val Leu -5 1 5 10 ctg cgc gtc ttc agc ttt gag ccg gtg
ccc tct tgc agg gcc ctg cag 318 Leu Arg Val Phe Ser Phe Glu Pro Val
Pro Ser Cys Arg Ala Leu Gln 15 20 25 gtg ctc aag ccc cgg gac cgc
att tct gcc atc gcc cac cgt ggc ggc 366 Val Leu Lys Pro Arg Asp Arg
Ile Ser Ala Ile Ala His Arg Gly Gly 30 35 40 agc cac gac gcg ccc
gag aac acg ctg gcg gcc att cgg cag gca gct 414 Ser His Asp Ala Pro
Glu Asn Thr Leu Ala Ala Ile Arg Gln Ala Ala 45 50 55 aag aat gga
gca aca ggc gtg gag ttg gac att gag ttt act tct gac 462 Lys Asn Gly
Ala Thr Gly Val Glu Leu Asp Ile Glu Phe Thr Ser Asp 60 65 70 ggg
att cct gtc tta atg cac gat aac aca gta gat agg acg act gat 510 Gly
Ile Pro Val Leu Met His Asp Asn Thr Val Asp Arg Thr Thr Asp 75 80
85 90 ggg act ggg cga ttg tgt gat ttg aca ttt gaa caa att agg aag
ctg 558 Gly Thr Gly Arg Leu Cys Asp Leu Thr Phe Glu Gln Ile Arg Lys
Leu 95 100 105 aat cct gca gca aac cac aga ctc agg aat gat ttc cct
gat gaa aag 606 Asn Pro Ala Ala Asn His Arg Leu Arg Asn Asp Phe Pro
Asp Glu Lys 110 115 120 atc cct acc cta atg gaa gct gtt gca gag tgc
cta aac cat aac ctc 654 Ile Pro Thr Leu Met Glu Ala Val Ala Glu Cys
Leu Asn His Asn Leu 125 130 135 aca atc ttc ttt gat gtc aaa ggc cat
gca cac aag gct act gag gct 702 Thr Ile Phe Phe Asp Val Lys Gly His
Ala His Lys Ala Thr Glu Ala 140 145 150 cta aag aaa atg tat atg gaa
ttt cct caa ctg tat aat aat agt gtg 750 Leu Lys Lys Met Tyr Met Glu
Phe Pro Gln Leu Tyr Asn Asn Ser Val 155 160 165 170 gtc tgt tct ttc
ttg cca gaa gtt atc tac aag atg aga caa aca gat 798 Val Cys Ser Phe
Leu Pro Glu Val Ile Tyr Lys Met Arg Gln Thr Asp 175 180 185 cgg gat
gta ata aca gca tta act cac aga cct tgg agc cta agc cat 846 Arg Asp
Val Ile Thr Ala Leu Thr His Arg Pro Trp Ser Leu Ser His 190 195 200
aca gga gat ggg aaa cca cgc tat gat act ttc tgg aaa cat ttt ata 894
Thr Gly Asp Gly Lys Pro Arg Tyr Asp Thr Phe Trp Lys His Phe Ile 205
210 215 ttt gtt atg atg gac att ttg ctc gat tgg agc atg cat aat atc
ttg 942 Phe Val Met Met Asp Ile Leu Leu Asp Trp Ser Met His Asn Ile
Leu 220 225 230 tgg tac ctg tgt gga att tca gct ttc ctc atg caa aag
gat ttt gta 990 Trp Tyr Leu Cys Gly Ile Ser Ala Phe Leu Met Gln Lys
Asp Phe Val 235 240 245 250 tcc ccg gcc tac ttg aag aag tgg tca gct
aaa gga atc cag gtt gtt 1038 Ser Pro Ala Tyr Leu Lys Lys Trp Ser
Ala Lys Gly Ile Gln Val Val 255 260 265 ggt tgg act gtt aat acc ttt
gat gaa aag agt tac tac gaa tcc cat 1086 Gly Trp Thr Val Asn Thr
Phe Asp Glu Lys Ser Tyr Tyr Glu Ser His 270 275 280 ctt ggt tcc agc
tat atc act gac agc atg gta gaa gac tgc gaa cct 1134 Leu Gly Ser
Ser Tyr Ile Thr Asp Ser Met Val Glu Asp Cys Glu Pro 285 290 295 cac
ttc tagactttca cggtgggacg aaacgggttc agaaactgcc aggggcctca 1190 His
Phe 300 tacagggata tcaaaatacc ctttgtgcta gcccaggccc tggggaatca
ggtgactcac 1250 acaaatgcaa tagttggtca ctgcattttt acctgaacca
aagctaaacc cggtgttgcc 1310 accatgcacc atggcatgcc agagttcaac
actgttgctc ttgaaaatct gggtctgaaa 1370 aaacgcacaa gagcccctgc
cctgccctag ctgaggcaca cagggagacc cagtgaggat 1430 aagcacagat
tgaattgtac aatttgcaga tgcagatgta aatgcatggg acatgcatga 1490
taactcagag ttgacatttt aaaacttgcc acacttattt caaatatttg tactcagcta
1550 tgttaacatg tactgtagac atcaaacttg tggccatact aataaaatta
ttaaaaggag 1610 cacaaaaaaa aaaaaaaaa 1629 72 1665 DNA Homo sapiens
CDS 109..738 sig_peptide 109..405 Von Heijne matrix score 4.5 seq
LAPGSFLAAVVDA/LE polyA_signal 1633..1638 polyA_site 1650..1665 72
cccagcgttc ctcctccggc cccaggtcac cgccagcacg cgcctgcttc ccgtctgcgc
60 gagtccacgc agctccccag gcccttcacc agcacagcag cagcaggc atg gca gca
117 Met Ala Ala agc gtg gag cag cgc gag ggc acc atc cag gtg cag ggc
cag gcc ctc 165 Ser Val Glu Gln Arg Glu Gly Thr Ile Gln Val Gln Gly
Gln Ala Leu -95 -90 -85 ttc ttc cga gag gcc ctg ccc ggc agt ggg cag
gct cgc ttc tct gta 213 Phe Phe Arg Glu Ala Leu Pro Gly Ser Gly Gln
Ala Arg Phe Ser Val -80 -75 -70 -65 ctg ctg ctg cat ggt att cgc ttc
tcc tcc gag acc tgg cag aac ctg 261 Leu Leu Leu His Gly Ile Arg Phe
Ser Ser Glu Thr Trp Gln Asn Leu -60 -55 -50 ggt aca ctg cac agg ctg
gcc cag gct ggc tac cgg gct gtg gcc att 309 Gly Thr Leu His Arg Leu
Ala Gln Ala Gly Tyr Arg Ala Val Ala Ile -45 -40 -35 gac ctg cca ggt
ctg ggg cac tcc aag gaa gca gca gcc cct gcc cct 357 Asp Leu Pro Gly
Leu Gly His Ser Lys Glu Ala Ala Ala Pro Ala Pro -30 -25 -20 att ggg
gag ctg gcc cct ggc agc ttc ctg gcg gct gtg gtg gat gcc 405 Ile Gly
Glu Leu Ala Pro Gly Ser Phe Leu Ala Ala Val Val Asp Ala -15 -10 -5
ttg gag ctg ggc ccc ccg gtt gtg atc agt cca tca ctg agt ggc atg 453
Leu Glu Leu Gly Pro Pro Val Val Ile Ser Pro Ser Leu Ser Gly Met 1 5
10 15 tac tcc ctg ccc ttc ctc acg gcc cct ggc tcc cag ctc ccg ggc
ttt 501 Tyr Ser Leu Pro Phe Leu Thr Ala Pro Gly Ser Gln Leu Pro Gly
Phe 20 25 30 gtg cca gtg gcc ccc atc tgc act gac aaa atc aat gct
gcc aac tat 549 Val Pro Val Ala Pro Ile Cys Thr Asp Lys Ile Asn Ala
Ala Asn Tyr 35 40 45 gcc agt gtg aag act cca gct ctg att gta tat
gga gac cag gac ccc 597 Ala Ser Val Lys Thr Pro Ala Leu Ile Val Tyr
Gly Asp Gln Asp Pro 50 55 60 atg ggt cag acc agc ttt gag cac ctg
aag cag ctg ccc aac cac cgg 645 Met Gly Gln Thr Ser Phe Glu His Leu
Lys Gln Leu Pro Asn His Arg 65 70 75 80 gtg ctg atc atg aag ggg gcg
ggg cac ccc tgt tac ctg gac aaa cca 693 Val Leu Ile Met Lys Gly Ala
Gly His Pro Cys Tyr Leu Asp Lys Pro 85 90 95 gag gag tgg cat aca
ggg ctg ctg gac ttc ctg cag ggg ctc cag 738 Glu Glu Trp His Thr Gly
Leu Leu Asp Phe Leu Gln Gly Leu Gln 100 105 110 tgaagcccag
cactgctgca gggggtgggc tgcctgcctg ctctgagctc tctcttgcac 798
gctctctctt ctctcccagg ctctggctca tgcacatgca acaggtgcgt ctgtctatat
858 gtctgggttc ttgtcttttg tggtctgttt gtcttttcta cctctttctc
ttgcagtgat 918 agactgaggg ggtaaaatca agagaaaaaa ctctcaggaa
tcaaggaaca taatcctgtg 978 gagggtaatc cattacatga gcttctcctg
ttcttccact ttcctgcctg gctttcactc 1038 cttcccctgc tctgcccagc
ctttccctcc cacccactcc tacttctgca aatgccctga 1098 aggccagccc
ttaccccaac acccacttcc ccacctcctt aggccccaga tacatacatg 1158
cccacatgca cgcttacatg tttagagcca tccttgtttc caaatatgac ccttcgcttg
1218 agggcaactg cataggtaca tctaactctg gactggcatg cacattgtca
tgtgcagctt 1278 tgcatataca cacatgcata catgagcctc cacacaagca
cttgcacaca tgtggactcc 1338 taaccatgct aacctcactg gctgggaagg
tggggacccc atgggccagc ccttgcagga 1398 ggcccttttg caaggcttag
ggtgtggcca gccctgaaag ctacttggac acaggtttca 1458 gctggcccca
gcccagaagt gacccccaga aagggagggc caccgctttg ccccctgctt 1518
ttacccttcc ttctgggtgc tctacacctc aggttaccag gcctgaggca tctcagccaa
1578 gcttgtttcc tgctctgagg cttgtggggt gggagccaga gtggaggtcg
gtgaaataaa 1638 gtgatgcaat taaaaaaaaa aaaaaaa 1665 73 425 DNA Homo
sapiens CDS 55..291 sig_peptide 55..255 Von Heijne matrix score 4.4
seq LISLVASLFMGFG/VL polyA_signal 390..395 polyA_site 410..425 73
ctgccgacgt gttcttccgg tggcggagcg gcggattagc cttcgcgggg caaa atg 57
Met gag ctc gag gcc atg agc aga tat acc agc cca gtg aac cca gct gtc
105 Glu Leu Glu Ala Met Ser Arg Tyr Thr Ser Pro Val Asn Pro Ala Val
-65 -60 -55 ttc ccc cat ctg acc gtg gtg ctt ttg gcc att ggc atg ttc
ttc acc 153 Phe Pro His Leu Thr Val Val Leu Leu Ala Ile Gly Met Phe
Phe Thr -50 -45 -40 -35 gcc tgg ttc ttc gtt tac gag gtc acc tct acc
aag tac act cgt gat 201 Ala Trp Phe Phe Val Tyr Glu Val Thr Ser Thr
Lys Tyr Thr Arg Asp -30 -25 -20 atc tat aaa gag ctc ctc atc tcc tta
gtg gcc tca ctc ttc atg ggc 249 Ile Tyr Lys Glu Leu Leu Ile Ser Leu
Val Ala Ser Leu Phe Met Gly -15 -10 -5 ttt gga gtc ctc ttc ctg ctg
ctc tgg gtt ggc atc tac gtg 291 Phe Gly Val Leu Phe Leu Leu Leu Trp
Val Gly Ile Tyr Val 1 5 10 tgagcaccca agggtaacaa ccagatggct
tcactgaaac ctgcttttgt aaattacttt 351 tttttactgt tgctggaaat
gtcccacctg ctgctcataa taaatgcaga tgtataacaa 411 aaaaaaaaaa aaaa 425
74 546 DNA Homo sapiens CDS 25..276 polyA_signal 508..513
polyA_site 533..546 74 gttgcaccag gcgatgcaag acac atg gca gtc tgg
cct gaa gtt tcc caa 51 Met Ala Val Trp Pro Glu Val Ser Gln 1 5 aac
agg ctg act agg ggc cta ctg ctt ccc aac tac cag ctg agg ggg 99 Asn
Arg Leu Thr Arg Gly Leu Leu Leu Pro Asn Tyr Gln Leu Arg Gly 10 15
20 25 tcc gtc ccg aaa agg gag aag agg cct aag agg aaa cat caa cat
ctt 147 Ser Val Pro Lys Arg Glu Lys Arg Pro Lys Arg Lys His Gln His
Leu 30 35 40 ttt act cct agc gag cgg cat tct gtc tgc ctt gat tgt
ctt ctg gaa 195 Phe Thr Pro Ser Glu Arg His Ser Val Cys Leu Asp Cys
Leu Leu Glu 45 50 55 ata tcg ctt tca ggg aaa caa tgg cga aat gtc
atc agt ttc aac tgc 243 Ile Ser Leu Ser Gly Lys Gln Trp Arg Asn Val
Ile Ser Phe Asn Cys 60 65 70 ttt tgc act act aag acg ctt ttc tgg
gtt aat tagcagcaat acagacaacg 296 Phe Cys Thr Thr Lys Thr Leu Phe
Trp Val Asn 75 80 atcttttatt caacaacctc tctcgagata ttttaaataa
tttctcacac tcgaaaaaca 356 tgcagaagcg actattggca aacctgaaga
gggtggaata ccaaatggct gaactggaat 416 attttctagt tagcgagggt
ttgagaggtg cgtcaggtct ccagaaattc acctcaaaag 476 cgtacaggat
gtaatgccag tggtggaaat cattaaagac actttgagta gattcaaaaa 536
aaaaaaaaaa 546 75 485 DNA Homo sapiens CDS 32..307 sig_peptide
32..91 Von Heijne matrix score 7.4 seq LVFCVGLLTMAKA/ES
polyA_signal 452..457 polyA_site 472..485 75 ctttcagcag gggacagccc
gattggggac a atg gcg tct ctt ggc cac atc 52 Met Ala Ser Leu Gly His
Ile -20 -15 ttg gtt ttc tgt gtg ggt ctc ctc acc atg gcc aag gca gaa
agt cca 100 Leu Val Phe Cys Val Gly Leu Leu Thr Met Ala Lys Ala Glu
Ser Pro -10 -5 1 aag gaa cac gac ccg ttc act tac gac tac cag tcc
ctg cag atc gga 148 Lys Glu His Asp Pro Phe Thr Tyr Asp Tyr Gln Ser
Leu Gln Ile Gly 5 10 15 ggc ctc gtc atc gcc ggg atc ctc ttc atc ctg
ggc atc ctc atc gtg 196 Gly Leu Val Ile Ala Gly Ile Leu Phe Ile Leu
Gly Ile Leu Ile Val 20 25 30 35 ctg agc aga aga tgc cgg tgc aag ttc
aac cag cag cag agg act ggg 244 Leu Ser Arg Arg Cys Arg Cys Lys Phe
Asn Gln Gln Gln Arg Thr Gly 40 45 50 gaa ccc gat gaa gag gag gga
act ttc cgc agc tcc atc cgc cgt ctg 292 Glu Pro Asp Glu Glu Glu Gly
Thr Phe Arg Ser Ser Ile Arg Arg Leu 55 60 65 tcc acc cgc agg cgg
tagaaacacc tggagcgatg gaatccggcc aggactcccc 347 Ser Thr Arg Arg Arg
70 tggcacctga catctcccac gctccacctg cgcgcccacc gccccctccg
ccgccccttc 407 cccagccctg cccccgcaga ctccccctgc cgccaagact
tccaataaaa cgtgcgttcc 467 tctcaaaaaa aaaagaaa 485 76 1394 DNA Homo
sapiens CDS 46..675 sig_peptide 46..87 Von Heijne matrix score 5.9
seq LTLLGLSLILAGL/IV polyA_signal 1363..1368 polyA_site 1382..1394
76 ctccgagttg ccacccagga aaaagagggc tcctctggga gatgt atg ctt act
ctc 57 Met Leu Thr Leu tta ggc ctt tca ctc atc ttg gca gga ctt att
gtt ggt gga gcc tgc 105 Leu Gly Leu Ser Leu Ile Leu Ala Gly Leu Ile
Val Gly Gly Ala Cys -10 -5 1 5 att tac aag cac ttc atg ccc aag agc
acc att tac cgt gga gag atg 153 Ile Tyr Lys His Phe Met Pro Lys Ser
Thr Ile Tyr Arg Gly Glu Met 10 15 20 tgc ttt ttt gat tct gag gat
cct gca aat tcc ctt cgt gga gga gag 201 Cys Phe Phe Asp Ser Glu Asp
Pro Ala Asn Ser Leu Arg Gly Gly Glu 25 30 35 cct aac ttc ctg cct
gtg act gag gag gct gac att cgt gag gat gac 249 Pro Asn Phe Leu Pro
Val Thr Glu Glu Ala Asp Ile Arg Glu Asp Asp 40 45 50 aac att gca
atc att gat gtg cct gtc ccc agt ttc tct gat agt gac 297 Asn Ile Ala
Ile Ile Asp Val Pro Val Pro Ser Phe Ser Asp Ser Asp 55 60 65 70 cct
gca gca att att cat gac ttt gaa aag gga atg act gct tac ctg 345 Pro
Ala Ala Ile Ile His Asp Phe Glu Lys Gly Met Thr Ala Tyr Leu 75 80
85 gac ttg ttg ctg ggg aac tgc tat ctg atg ccc ctc aat act tct att
393 Asp Leu Leu Leu Gly Asn Cys Tyr Leu Met Pro Leu Asn Thr Ser Ile
90 95 100 gtt atg cct cca gaa aat ctg gta gag ctc ttt ggc aaa ctg
gcg agt 441 Val Met Pro Pro Glu Asn Leu Val Glu Leu Phe Gly Lys Leu
Ala Ser 105 110 115 ggc aga tat ctg cct caa act tat gtg gtt cga gaa
gac cta gtt gct 489 Gly Arg Tyr Leu Pro Gln Thr Tyr Val Val Arg Glu
Asp Leu Val Ala 120 125 130 gtg gag gaa att cgt gat gtt agt aac ctt
ggc atc ttt att tac caa 537 Val Glu Glu Ile Arg Asp Val Ser Asn Leu
Gly Ile Phe Ile Tyr Gln 135 140 145 150 ctt tgc aat aac aga aag tcc
ttc cgc ctt cgt cgc aga gac ctc ttg 585 Leu Cys Asn Asn Arg Lys Ser
Phe Arg Leu Arg Arg Arg Asp Leu Leu 155 160 165 ctg ggt ttc aac aaa
cgt gcc att gat aaa tgc tgg aag att aga cac 633 Leu Gly Phe Asn Lys
Arg Ala Ile Asp Lys Cys Trp Lys Ile Arg His 170 175 180 ttc ccc aac
gaa ttt att gtt gag acc aag atc tgt caa gag 675 Phe Pro Asn Glu Phe
Ile Val Glu Thr Lys Ile Cys Gln Glu 185 190 195 taagaggcaa
cagatagagt gtccttggta acaagaagtc agagatttac aatatgactt 735
taacattaag gtttatggga tactcaagat atttactcat gcatttactc tattgcttat
795 gctttaaaaa aaggaaaaaa aaaaactact aaccactgca agctcttgtc
aaattttagt 855 ttaattggca ttgcttgttt tttgaaactg aaattacctg
agtttcattt tttctttgaa 915 tttatagggt ttagatttct gaaagcagca
tgaatatatc acctaacatc ctgacaataa 975 attccatccg ttgttttttt
tgtttgtttg ttttttcttt tcctttaagt aagctcttta 1035 ttcatcttat
ggtgcagcaa ttttaaaatt tgaaatattt taaattgttt ttgaactttt 1095
tgtgtaaaat atatcagatc tcaacattgt tggtttcttt tgtttttcat tttgtacaac
1155 tttcttgaat ttagaaatta catctttgca gctctgttag gtgctctgta
attaacctga 1215 cttatatgtg aacaattttc atgagacagt catttttaaa
taatgcagtg attctttctc 1275 actactatct gtattgtgga atgcacaaaa
ttgtgtaggt gctgaatgct gtaaggagtt 1335 taggttgtat gaattctaca
accctataat aaattttact ctatacaaaa aaaaaaaaa 1394 77 1333 DNA Homo
sapiens CDS 329..943 sig_peptide 329..745 Von Heijne matrix score
4.2 seq SLSLALKTGPTSG/LC polyA_site 1322..1333 77 cgccagtgtc
agtggtgttg gcatcagctt gggcaggtgt gcgggctcag gatggggcgg 60
ccgtggtgag gaaccctgga ctctcagcat cacaagaggc aacaccagga gccaacatga
120 gctcgggact gaactgctgt ggcccggagc agcgctgctg gtgctgttgg
gggtggcagc 180 cagtctgtgt gtgcgctgct cacgcccagg tgcaaagagg
tcagagagaa tctaccagca 240 gagaagtctg cgtgaggacc aacagagctt
tacggggtcc cggacctact ccttggtcgg 300 gcaggcatgg ccaggacccc tggcggac
atg gca ccc aca agg aag gac aag
352 Met Ala Pro Thr Arg Lys Asp Lys -135 ctg ttg caa ttc tac ccc
agc ctg gag gat cca gca tct tcc agg tac 400 Leu Leu Gln Phe Tyr Pro
Ser Leu Glu Asp Pro Ala Ser Ser Arg Tyr -130 -125 -120 cag aac ttc
agc aaa gga agc aga cac ggg tcg gag gaa gcc tac ata 448 Gln Asn Phe
Ser Lys Gly Ser Arg His Gly Ser Glu Glu Ala Tyr Ile -115 -110 -105
-100 gac ccc att gcc atg gag tat tac aac tgg ggg cgg ttc tcg aag
ccc 496 Asp Pro Ile Ala Met Glu Tyr Tyr Asn Trp Gly Arg Phe Ser Lys
Pro -95 -90 -85 cca gaa ggt gag gcg aag gac aaa gcc gga ggt gga gga
agt ggt gtg 544 Pro Glu Gly Glu Ala Lys Asp Lys Ala Gly Gly Gly Gly
Ser Gly Val -80 -75 -70 gga gct cag ggc aga agc cat acc tcc agg cag
gag agg agg ctg ggc 592 Gly Ala Gln Gly Arg Ser His Thr Ser Arg Gln
Glu Arg Arg Leu Gly -65 -60 -55 ctg ggt tcg gat gat gat gcc aat tcc
tac gag aat gtg ctc att tgc 640 Leu Gly Ser Asp Asp Asp Ala Asn Ser
Tyr Glu Asn Val Leu Ile Cys -50 -45 -40 aag cag aaa acc aca gag aca
ggt gcc cag cag gag gac gta ggt ggc 688 Lys Gln Lys Thr Thr Glu Thr
Gly Ala Gln Gln Glu Asp Val Gly Gly -35 -30 -25 -20 ctc tgc aga ggg
gac ctc agc ctg tca ctg gcc ctg aag act ggc ccc 736 Leu Cys Arg Gly
Asp Leu Ser Leu Ser Leu Ala Leu Lys Thr Gly Pro -15 -10 -5 act tct
ggt ctc tgt ccc tct gcc tcc ccg gaa gaa gat ggg gaa tct 784 Thr Ser
Gly Leu Cys Pro Ser Ala Ser Pro Glu Glu Asp Gly Glu Ser 1 5 10 gag
gat tat cag aac tca gca tcc atc cat caa tgg cgc gag tcc agg 832 Glu
Asp Tyr Gln Asn Ser Ala Ser Ile His Gln Trp Arg Glu Ser Arg 15 20
25 aag gtc atg ggg caa ctc cag aga gaa gca tcc cct ggc ccg gtg gga
880 Lys Val Met Gly Gln Leu Gln Arg Glu Ala Ser Pro Gly Pro Val Gly
30 35 40 45 agc cca gac gag gag gac ggg gaa ccg gat tac gtg aat ggg
gag gtg 928 Ser Pro Asp Glu Glu Asp Gly Glu Pro Asp Tyr Val Asn Gly
Glu Val 50 55 60 gca gcc aca gaa gcc tagggcagac caagaagaaa
ggagccaagg caaagagggg 983 Ala Ala Thr Glu Ala 65 ccactgtgct
catggaccca tcgctgcctt ccaaggacca tttcccagag ctactcaact 1043
tttaagcccc tgccatggtt gctcctggaa ggagaaccag ccaccctgag gaccacctgg
1103 ccatgcgtgc acagcctggg aaaagacagt tactcacggg agctgcaggc
ccgtcaccaa 1163 gccctctccc gacccaggct ttgtggggca ggcacctggt
accatgggta acccggctcc 1223 tggtatggac ggatgcgcag gatttaggat
aagctgtcac ccagtcccca taacaaaacc 1283 actgtccaac actggtatct
gtgttctttt gtgctatgaa aaaaaaaaaa 1333 78 326 DNA Homo sapiens CDS
27..281 sig_peptide 27..77 Von Heijne matrix score 8.2 seq
LLLITAILAVAVG/FP 78 gaaaagaact gactgaaacg tttgag atg aag aaa gtt
ctc ctc ctg atc aca 53 Met Lys Lys Val Leu Leu Leu Ile Thr -15 -10
gcc atc ttg gca gtg gct gtt ggt ttc cca gtc tct caa gac cag gaa 101
Ala Ile Leu Ala Val Ala Val Gly Phe Pro Val Ser Gln Asp Gln Glu -5
1 5 cga gaa aaa aga agt atc agt gac agc gat gaa tta gct tca ggg ttt
149 Arg Glu Lys Arg Ser Ile Ser Asp Ser Asp Glu Leu Ala Ser Gly Phe
10 15 20 ttt gtg ttc cct tac cca tat cca ttt cgc cca ctt cca cca
att cca 197 Phe Val Phe Pro Tyr Pro Tyr Pro Phe Arg Pro Leu Pro Pro
Ile Pro 25 30 35 40 ttt cca aga ttt cca tgg ttt aga cgt aat ttt cct
att cca ata cct 245 Phe Pro Arg Phe Pro Trp Phe Arg Arg Asn Phe Pro
Ile Pro Ile Pro 45 50 55 gaa tct gcc cct aca act ccc ctt cct agc
gaa aag taaacaagaa 291 Glu Ser Ala Pro Thr Thr Pro Leu Pro Ser Glu
Lys 60 65 ggaaaagtca cgataaacct ggtcacctga aattg 326 79 703 DNA
Homo sapiens CDS 61..405 sig_peptide 61..213 Von Heijne matrix
score 8.1 seq VCLCGTFCFPCLG/CQ polyA_signal 675..680 polyA_site
692..703 79 catttcctgc tcggaacctt gtttactaat ttccactgct tttaaggccc
tgcactgaaa 60 atg caa gct cag gcg ccg gtg gtc gtt gtg acc caa cct
gga gtc ggt 108 Met Gln Ala Gln Ala Pro Val Val Val Val Thr Gln Pro
Gly Val Gly -50 -45 -40 ccc ggt ccg gcc ccc cag aac tcc aac tgg cag
aca ggc atg tgt gac 156 Pro Gly Pro Ala Pro Gln Asn Ser Asn Trp Gln
Thr Gly Met Cys Asp -35 -30 -25 -20 tgt ttc agc gac tgc gga gtc tgt
ctc tgt ggc aca ttt tgt ttc ccg 204 Cys Phe Ser Asp Cys Gly Val Cys
Leu Cys Gly Thr Phe Cys Phe Pro -15 -10 -5 tgc ctt ggg tgt caa gtt
gca gct gat atg aat gaa tgc tgt ctg tgt 252 Cys Leu Gly Cys Gln Val
Ala Ala Asp Met Asn Glu Cys Cys Leu Cys 1 5 10 gga aca agc gtc gca
atg agg act ctc tac agg acc cga tat ggc atc 300 Gly Thr Ser Val Ala
Met Arg Thr Leu Tyr Arg Thr Arg Tyr Gly Ile 15 20 25 cct gga cct
att tgt gat gac tat atg gca act ctt tgc tgt cct cat 348 Pro Gly Pro
Ile Cys Asp Asp Tyr Met Ala Thr Leu Cys Cys Pro His 30 35 40 45 tgt
act ctt tgc caa atc aag aga gat atc aac aga agg aga gcc atg 396 Cys
Thr Leu Cys Gln Ile Lys Arg Asp Ile Asn Arg Arg Arg Ala Met 50 55
60 cgt act ttc taaaaactga tggtgaaaag ctcttaccga agcaacaaaa 445 Arg
Thr Phe ttcagcagac acctctccag cttgagttct tcaccatctt ttgcaactga
aatatgatgg 505 atatgcttaa gtacaactga tggcatgaaa aaaatcaaat
ttttgattta ttataaatga 565 atgttgtccc tgaacttagc taaatggtgc
aacttagttt ctccttgctt tcatattatc 625 gaatttcctg gcttataaac
tttttaaatt acatttgaaa tataaaccaa atgaaatatt 685 ttactcaaaa aaaaaaaa
703 80 768 DNA Homo sapiens CDS 137..379 sig_peptide 137..229 Von
Heijne matrix score 4.4 seq TCCHLGLPHPVRA/PR polyA_signal 728..733
polyA_site 755..768 80 tcggagttgg aaagggacgc ctggtttccc cccaagcgaa
ccgggatggg aagtgacttc 60 aatgagattg aacttcagct ggattgaaag
agaggctaga agttccgctt gccagcagcc 120 cccttagtag agcgga atg agt aat
acc cac acg gtg ctt gtc tca ctt ccc 172 Met Ser Asn Thr His Thr Val
Leu Val Ser Leu Pro -30 -25 -20 cat ccg cac ccg gcc ctc acc tgc tgt
cac ctc ggc ctc cca cac ccg 220 His Pro His Pro Ala Leu Thr Cys Cys
His Leu Gly Leu Pro His Pro -15 -10 -5 gtc cgc gct ccc cgc cct ctt
cct cgc gta gaa ccg tgg gat cct agg 268 Val Arg Ala Pro Arg Pro Leu
Pro Arg Val Glu Pro Trp Asp Pro Arg 1 5 10 tgg cag gac tca gag cta
agg tat cca cag gcc atg aat tcc ttc cta 316 Trp Gln Asp Ser Glu Leu
Arg Tyr Pro Gln Ala Met Asn Ser Phe Leu 15 20 25 aat gag cgg tca
tcg ccg tgc agg acc tta agg caa gaa gca tcg gct 364 Asn Glu Arg Ser
Ser Pro Cys Arg Thr Leu Arg Gln Glu Ala Ser Ala 30 35 40 45 gac aga
tgt gat ctc tgaacctgat agattgctga ttttatctta ttttatcctt 419 Asp Arg
Cys Asp Leu 50 gacttggtac aagttttggg atttctgaaa agaccatgca
gataaccaca aatatcaaga 479 aagtcgtctt cagtattaag tagaatttag
atttaggttt ccttcctgct tcccacctcc 539 ttcgaataag gaaacgtctt
tgggaccaac tttatggaat aaataagctg agctgtattt 599 caagtaatat
agttataaat taacaatgta gcagttattg atagagaaat tgagaaaact 659
gaaacgtgac cggagtattg gaaataacgt agtacatcac ctagcacaat gacacatagt
719 aggtgctcaa taaatttatg cttataattt ttgtcaaaaa aaaaaataa 768 81
1007 DNA Homo sapiens CDS 37..741 sig_peptide 37..153 Von Heijne
matrix score 7.2 seq SALAKLLLTCCSA/LR polyA_signal 969..974
polyA_site 994..1007 81 cgcaggtccc gaggagcgca gactgtgtcc ctgaca atg
gga aca gcc gac agt 54 Met Gly Thr Ala Asp Ser -35 gat gag atg gcc
ccg gag gcc cca cag cac acc cac atc gat gtg cac 102 Asp Glu Met Ala
Pro Glu Ala Pro Gln His Thr His Ile Asp Val His -30 -25 -20 atc cac
cag gag tct gcc ctg gcc aag ctc ctg ctc acc tgc tgc tct 150 Ile His
Gln Glu Ser Ala Leu Ala Lys Leu Leu Leu Thr Cys Cys Ser -15 -10 -5
gcg ctg cgg ccc cgg gcc acc cag gcc agg ggc agc agc cgg ctg ctg 198
Ala Leu Arg Pro Arg Ala Thr Gln Ala Arg Gly Ser Ser Arg Leu Leu 1 5
10 15 gtg gcc tcg tgg gtg atg cag atc gtg ctg ggg atc ttg agt gca
gtc 246 Val Ala Ser Trp Val Met Gln Ile Val Leu Gly Ile Leu Ser Ala
Val 20 25 30 cta gga gga ttt ttc tac atc cgc gac tac acc ctc ctc
gtc acc tcg 294 Leu Gly Gly Phe Phe Tyr Ile Arg Asp Tyr Thr Leu Leu
Val Thr Ser 35 40 45 ggg gct gcc atc tgg aca ggg gct gtg gct gtg
ctg gct gga gct gct 342 Gly Ala Ala Ile Trp Thr Gly Ala Val Ala Val
Leu Ala Gly Ala Ala 50 55 60 gcc ttc att tac gag aaa cgg ggt ggt
aca tac tgg gcc ctg ctg agg 390 Ala Phe Ile Tyr Glu Lys Arg Gly Gly
Thr Tyr Trp Ala Leu Leu Arg 65 70 75 act ctg cta gcg ctg gca gct
ttc tcc aca gcc atc gct gcc ctc aaa 438 Thr Leu Leu Ala Leu Ala Ala
Phe Ser Thr Ala Ile Ala Ala Leu Lys 80 85 90 95 ctt tgg aat gaa gat
ttc cga tat ggc tac tct tat tac aac agt gcc 486 Leu Trp Asn Glu Asp
Phe Arg Tyr Gly Tyr Ser Tyr Tyr Asn Ser Ala 100 105 110 tgc cgc atc
tcc agc tcg agt gac tgg aac act cca gcc ccc act cag 534 Cys Arg Ile
Ser Ser Ser Ser Asp Trp Asn Thr Pro Ala Pro Thr Gln 115 120 125 agt
cca gaa gaa gtc aga agg cta cac cta tgt acc tcc ttc atg gac 582 Ser
Pro Glu Glu Val Arg Arg Leu His Leu Cys Thr Ser Phe Met Asp 130 135
140 atg ctg aag gcc ttg ttc aga acc ctt cag gcc atg ctc ttg ggt gtc
630 Met Leu Lys Ala Leu Phe Arg Thr Leu Gln Ala Met Leu Leu Gly Val
145 150 155 tgg att ctg ctg ctt ctg gca tct ctg gcc cct ctg tgg ctg
tac tgc 678 Trp Ile Leu Leu Leu Leu Ala Ser Leu Ala Pro Leu Trp Leu
Tyr Cys 160 165 170 175 tgg aga atg ttc cca acc aaa ggg aaa aga gac
cag aag gaa atg ttg 726 Trp Arg Met Phe Pro Thr Lys Gly Lys Arg Asp
Gln Lys Glu Met Leu 180 185 190 gaa gtg agt gga atc tagccatgcc
tctcctgatt attagtgcct ggtgcttctg 781 Glu Val Ser Gly Ile 195
caccgggcgt ccctgcatct gactgctgga agaagaacca gactgaggaa aagaggctct
841 tcaacagccc cagttatcct ggccccatga ccgtggccac agccctgctc
cagcagcact 901 tgcccattcc ttacacccct tccccatcct gctccgcttc
atgtcccctc ctgagtagtc 961 atgtgataat aaactctcat gttattgttc
ccaaaaaaaa aaaaaa 1007 82 527 DNA Homo sapiens CDS 80..265
sig_peptide 80..142 Von Heijne matrix score 5.4 seq
TFCLIFGLGAVWG/LG polyA_signal 491..496 polyA_site 517..527 82
cccgcttgat tccaagaacc tcttcgattt ttatttttat ttttaaagag ggagacgatg
60 gactgagctg atccgcacc atg gag tct cgg gtc tta ctg aga aca ttc tgt
112 Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys -20 -15 ttg atc ttc
ggt ctc gga gca gtt tgg ggg ctt ggt gtg gac cct tcc 160 Leu Ile Phe
Gly Leu Gly Ala Val Trp Gly Leu Gly Val Asp Pro Ser -10 -5 1 5 cta
cag att gac gtc tta aca gag tta gaa ctt ggg gag tcc acg acc 208 Leu
Gln Ile Asp Val Leu Thr Glu Leu Glu Leu Gly Glu Ser Thr Thr 10 15
20 gga gtg cgt cag gtc ccg ggg ctg cat aat ggg acg aaa gcc ttt ctc
256 Gly Val Arg Gln Val Pro Gly Leu His Asn Gly Thr Lys Ala Phe Leu
25 30 35 ttt caa gcg tgactgaagc agcagcctgc acatgtggat ggtcatcagt
305 Phe Gln Ala 40 gcctcgccca gagatacctg gccttcatcc aaagggaccc
tgctgccaca agtcctccag 365 gcagcacccg cactgtggct ccttcgcact
gagtatgttg gactctgcca tagactgacc 425 ctcttgtctg gctgctgcag
tttgtctgta atgccctgac atgttgcatt ctccccattt 485 ggataaataa
aaacaaacaa atgcttctgt caaaaaaaaa aa 527 83 861 DNA Homo sapiens CDS
612..644 polyA_signal 829..834 polyA_site 850..861 83 agctctggtg
gttctggctg ctctggactg tcctcatcct ctttagctgc tgttgcgcct 60
tccgccaccg acgagctaaa ctcaggctgc aacaacagca gcggcagcgt gaaatcaact
120 tgttggccta tcatggggca tgccatgggg ctggtccttt ccctaccggt
tcactgcttg 180 accttcgcct cctcagcacc ttcaagcccc cagcctacga
ggatgtggtt caccgcccag 240 gcacaccacc ccccccttat actgtggccc
caggccgccc cttgactgct tccagtgaac 300 aaacctgctg ttcctcctca
tccagctgcc ctgcccactt tgaaggaaca aatgtggaag 360 gtgtttcctc
ccaccagagt gccccccctc atcaggaggg tgagcccggg gcaggggtga 420
cccctgcctc cacacccccc tcctgccgct atcgccgttt aactggcgac tccggtattg
480 agctctgccc ttgtcctgcc tccggtgagg gtgagccagt caaggaggtg
agggttagtg 540 ccaccctgcc agatctggag gactactccc cgtgtgcact
acccccagag tctgtaccgc 600 agatctttcc c atg ggg ctg tct tcc agt gaa
ggg gac atc cca 644 Met Gly Leu Ser Ser Ser Glu Gly Asp Ile Pro 1 5
10 taagtagttt tgagagggtg gatgggttac ttgcccacca gaaacagccc
tagtcccaac 704 tccttgcgtt cctttggccc ctccctgcct acctagaatc
tgcctgaagg ggctggagag 764 ggacagtatt gggggactgt gctagcttta
cccccgcagg acatacacag gagcctttga 824 tctcattaaa gagatgtaaa
ccagcaaaaa aaaaaaa 861 84 239 DNA Homo sapiens CDS 61..228
sig_peptide 61..162 Von Heijne matrix score 4 seq IAVLYLHLYDVFG/DP
polyA_signal 208..213 84 aatctgactc ctgagttctc acaacgcttg
accaataaga ttcggaagct tcttcagcaa 60 atg gag aga ggc ctg aaa tca gca
gac cct cgg gat ggc acc ggt tac 108 Met Glu Arg Gly Leu Lys Ser Ala
Asp Pro Arg Asp Gly Thr Gly Tyr -30 -25 -20 act ggc tgg gca ggt att
gct gtg ctt tac tta cat ctt tat gat gta 156 Thr Gly Trp Ala Gly Ile
Ala Val Leu Tyr Leu His Leu Tyr Asp Val -15 -10 -5 ttt ggg gac cct
gcc tct atg ttc tgt aaa gta ttt gac tta cta gtt 204 Phe Gly Asp Pro
Ala Ser Met Phe Cys Lys Val Phe Asp Leu Leu Val 1 5 10 ctc aat aaa
att tta tta gga cta taaaaaaaaa a 239 Leu Asn Lys Ile Leu Leu Gly
Leu 15 20 85 178 PRT Homo sapiens SIGNAL -22..-1 85 Met His Arg Pro
Glu Ala Met Leu Leu Leu Leu Thr Leu Ala Leu Leu -20 -15 -10 Gly Gly
Pro Thr Trp Ala Gly Lys Met Tyr Gly Pro Gly Gly Gly Lys -5 1 5 10
Tyr Phe Ser Thr Thr Glu Asp Tyr Asp His Glu Ile Thr Gly Leu Arg 15
20 25 Val Ser Val Gly Leu Leu Leu Val Lys Ser Val Gln Val Lys Leu
Gly 30 35 40 Asp Ser Trp Asp Val Lys Leu Gly Ala Leu Gly Gly Asn
Thr Gln Glu 45 50 55 Val Thr Leu Gln Pro Gly Glu Tyr Ile Thr Lys
Val Phe Val Ala Phe 60 65 70 Gln Thr Phe Leu Arg Gly Met Val Met
Tyr Thr Ser Lys Asp Arg Tyr 75 80 85 90 Phe Tyr Phe Gly Lys Leu Asp
Gly Gln Ile Ser Ser Ala Tyr Pro Ser 95 100 105 Gln Glu Gly Gln Val
Leu Val Gly Ile Tyr Gly Gln Tyr Gln Leu Leu 110 115 120 Gly Ile Lys
Ser Ile Gly Phe Glu Trp Asn Tyr Pro Leu Glu Glu Pro 125 130 135 Thr
Thr Glu Pro Pro Val Asn Leu Thr Tyr Ser Ala Asn Ser Pro Val 140 145
150 Gly Arg 155 86 90 PRT Homo sapiens SIGNAL -19..-1 86 Met Lys
Phe Leu Ala Val Leu Val Leu Leu Gly Val Ser Ile Phe Leu -15 -10 -5
Val Ser Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Pro 1 5
10 Ala Thr Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Thr Thr Ala
15 20 25 Ala Ala Thr Thr Ala Thr Thr Ala Ala Pro Thr Thr Ala Thr
Thr Ala 30 35 40 45 Ala Ser Thr Thr Ala Arg Lys Asp Ile Pro Val Leu
Pro Lys Trp Val 50 55 60 Gly Asp Leu Pro Asn Gly Arg Val Cys Pro 65
70 87 125 PRT Homo sapiens SIGNAL -15..-1 87 Met Lys Leu Leu Thr
His Asn Leu Leu Ser Ser His Val Arg Gly Val -15 -10 -5 1 Gly Ser
Arg Gly Phe Pro Leu Arg Leu Gln Ala Thr Glu Val Arg Ile 5 10 15 Cys
Pro Val Glu Phe Asn Pro Asn Phe Val Ala Arg Met Ile Pro Lys 20 25
30 Val Glu Trp Ser Ala Phe Leu Glu Ala Ala Asp Asn Leu Arg Leu Ile
35 40 45 Gln Val Pro Lys Gly Pro Val Glu Gly Tyr Glu Glu Asn Glu
Glu Phe 50 55 60 65 Leu Arg Thr Met His His Leu Leu Leu Glu Val Glu
Val Ile Glu Gly 70 75 80 Thr Leu Gln Cys Pro Glu Ser Gly Arg Met
Phe Pro Ile Ser Arg Gly 85 90 95
Ile Pro Asn Met Leu Leu Ser Glu Glu Glu Thr Glu Ser 100 105 110 88
136 PRT Homo sapiens SIGNAL -34..-1 88 Met Leu Phe Ser Leu Arg Glu
Leu Val Gln Trp Leu Gly Phe Ala Thr -30 -25 -20 Phe Glu Ile Phe Val
His Leu Leu Ala Leu Leu Val Phe Ser Val Leu -15 -10 -5 Leu Ala Leu
Arg Val Asp Gly Leu Val Pro Gly Leu Ser Trp Trp Asn 1 5 10 Val Phe
Val Pro Phe Phe Ala Ala Asp Gly Leu Ser Thr Tyr Phe Thr 15 20 25 30
Thr Ile Val Ser Val Arg Leu Phe Gln Asp Gly Glu Lys Arg Leu Ala 35
40 45 Val Leu Arg Leu Phe Trp Val Leu Thr Val Leu Ser Leu Lys Phe
Val 50 55 60 Phe Glu Met Leu Leu Cys Gln Lys Leu Ala Glu Gln Thr
Arg Glu Leu 65 70 75 Trp Phe Gly Leu Ile Thr Ser Pro Leu Phe Ile
Leu Leu Gln Leu Leu 80 85 90 Met Ile Arg Ala Cys Arg Val Asn 95 100
89 238 PRT Homo sapiens SIGNAL -53..-1 89 Met Ala Asp Pro Asp Pro
Arg Tyr Pro Arg Ser Ser Ile Glu Asp Asp -50 -45 -40 Phe Asn Tyr Gly
Ser Ser Val Ala Ser Ala Thr Val His Ile Arg Met -35 -30 -25 Ala Phe
Leu Arg Lys Val Tyr Ser Ile Leu Ser Leu Gln Val Leu Leu -20 -15 -10
Thr Thr Val Thr Ser Thr Val Phe Leu Tyr Phe Glu Ser Val Arg Thr -5
1 5 10 Phe Val His Glu Ser Pro Ala Leu Ile Leu Leu Phe Ala Leu Gly
Ser 15 20 25 Leu Gly Leu Ile Phe Ala Leu Ile Leu Asn Arg His Lys
Tyr Pro Leu 30 35 40 Asn Leu Tyr Leu Leu Phe Gly Phe Thr Leu Leu
Glu Ala Leu Thr Val 45 50 55 Ala Val Val Val Thr Phe Tyr Asp Val
Tyr Ile Ile Leu Gln Ala Phe 60 65 70 75 Ile Leu Thr Thr Thr Val Phe
Phe Gly Leu Thr Val Tyr Thr Leu Gln 80 85 90 Ser Lys Lys Asp Phe
Ser Lys Phe Gly Ala Gly Leu Phe Ala Leu Leu 95 100 105 Trp Ile Leu
Cys Leu Ser Gly Phe Leu Lys Phe Phe Leu Tyr Ser Glu 110 115 120 Ile
Met Glu Leu Val Leu Ala Ala Ala Gly Ala Leu Leu Phe Cys Gly 125 130
135 Phe Ile Ile Tyr Asp Thr His Ser Leu Met His Lys Leu Ser Pro Glu
140 145 150 155 Glu Tyr Val Leu Ala Ala Ile Ser Leu Tyr Leu Asp Ile
Ile Asn Leu 160 165 170 Phe Leu His Leu Leu Arg Phe Leu Glu Ala Val
Asn Lys Lys 175 180 185 90 106 PRT Homo sapiens SIGNAL -71..-1 90
Met Ser Thr Asn Asn Met Ser Asp Pro Arg Arg Pro Asn Lys Val Leu -70
-65 -60 Arg Tyr Lys Pro Pro Pro Ser Glu Cys Asn Pro Ala Leu Asp Asp
Pro -55 -50 -45 -40 Thr Pro Asp Tyr Met Asn Leu Leu Gly Met Ile Phe
Ser Met Cys Gly -35 -30 -25 Leu Met Leu Lys Leu Lys Trp Cys Ala Trp
Val Ala Val Tyr Cys Ser -20 -15 -10 Phe Ile Ser Phe Ala Asn Ser Arg
Ser Ser Glu Asp Thr Lys Gln Met -5 1 5 Met Ser Ser Phe Met Leu Ser
Ile Ser Ala Val Val Met Ser Tyr Leu 10 15 20 25 Gln Asn Pro Gln Pro
Met Thr Pro Pro Trp 30 35 91 123 PRT Homo sapiens SIGNAL -84..-1 91
Met Ser Gly Gly Pro Glu Ala Arg Pro Pro Met Leu Val Glu Gly Gly -80
-75 -70 Gly Pro Glu Ser Leu Gln Lys Ala Pro Cys Thr Arg Gly Pro Pro
Ser -65 -60 -55 His Pro Val Pro Pro Ala Leu Ala Phe Thr Val Gly Asn
Gly Ser Gly -50 -45 -40 Pro Gly Val Arg Cys Pro Arg Asn Met Ala Glu
Gly His Pro Gly Pro -35 -30 -25 Glu Arg Arg Gln Ser Gln Gln Gly Leu
Phe Arg Ala Ala Trp Leu Pro -20 -15 -10 -5 Gly Ser Arg Pro Ser Pro
Leu Phe Cys Val Cys Ser Val Thr Ser Pro 1 5 10 Gly Trp Asp Val Pro
Gln Val His Arg Val Glu Val Gly His Gly Arg 15 20 25 Arg Gln Glu
Thr His Pro Val Arg Arg Arg Ala 30 35 92 75 PRT Homo sapiens SIGNAL
-49..-1 92 Met Pro Arg Gly Arg Arg Leu Gly Met Val Phe Ala Pro Pro
Arg Pro -45 -40 -35 Gly Gln Arg Gln Ala Gly Ala Pro Trp Val Pro Glu
Arg Arg Lys Arg -30 -25 -20 Arg Pro Asp Gly Asp Thr Phe Leu Leu Ser
Phe Leu Ser Thr Thr Trp -15 -10 -5 Leu Lys Thr Trp Arg Ser Gln Gln
Tyr Lys Glu Ser Lys Ser Arg Ser 1 5 10 15 Cys Ala Arg Glu Gln Met
Asn Ser Ser Ser Cys 20 25 93 80 PRT Homo sapiens SIGNAL -40..-1 93
Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met Pro Ile Ser Gln -40
-35 -30 -25 Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe
Ala Leu -20 -15 -10 Phe Val Ala Phe Leu Leu Arg Gly Lys Leu Met Glu
Thr Tyr Cys Ser -5 1 5 Gln Lys His Thr Arg Leu Asp Tyr Ile Gly Asp
Ser Lys Asn Val Leu 10 15 20 Asn Asp Val Gln His Gly Arg Glu Asp
Glu Asp Gly Leu Phe Thr Leu 25 30 35 40 94 327 PRT Homo sapiens
SIGNAL -49..-1 94 Met Phe Pro Ser Arg Arg Lys Ala Ala Gln Leu Pro
Trp Glu Asp Gly -45 -40 -35 Arg Ser Gly Leu Leu Ser Gly Gly Leu Pro
Arg Lys Cys Ser Val Phe -30 -25 -20 His Leu Phe Val Ala Cys Leu Ser
Leu Gly Phe Phe Ser Leu Leu Trp -15 -10 -5 Leu Gln Leu Ser Cys Ser
Gly Asp Val Ala Arg Ala Val Arg Gly Gln 1 5 10 15 Gly Gln Glu Thr
Ser Gly Pro Pro Arg Ala Cys Pro Pro Glu Pro Pro 20 25 30 Pro Glu
His Trp Glu Glu Asp Ala Ser Trp Gly Pro His Arg Leu Ala 35 40 45
Val Leu Val Pro Phe Arg Glu Arg Phe Glu Glu Leu Leu Val Phe Val 50
55 60 Pro His Met Arg Arg Phe Leu Ser Arg Lys Lys Ile Arg His His
Ile 65 70 75 Tyr Val Leu Asn Gln Val Asp His Phe Arg Phe Asn Arg
Ala Ala Leu 80 85 90 95 Ile Asn Val Gly Phe Leu Glu Ser Ser Asn Ser
Thr Asp Tyr Ile Ala 100 105 110 Met His Asp Val Asp Leu Leu Pro Leu
Asn Glu Glu Leu Asp Tyr Gly 115 120 125 Phe Pro Glu Ala Gly Pro Phe
His Val Ala Ser Pro Glu Leu His Pro 130 135 140 Leu Tyr His Tyr Lys
Thr Tyr Val Gly Gly Ile Leu Leu Leu Ser Lys 145 150 155 Gln His Tyr
Arg Leu Cys Asn Gly Met Ser Asn Arg Phe Trp Gly Trp 160 165 170 175
Gly Arg Glu Asp Asp Glu Phe Tyr Arg Arg Ile Lys Gly Ala Gly Leu 180
185 190 Gln Leu Phe Arg Pro Ser Gly Ile Thr Thr Gly Tyr Lys Thr Phe
Arg 195 200 205 His Leu His Asp Pro Ala Trp Arg Lys Arg Asp Gln Lys
Arg Ile Ala 210 215 220 Ala Gln Lys Gln Glu Gln Phe Lys Val Asp Arg
Glu Gly Gly Leu Asn 225 230 235 Thr Val Lys Tyr His Val Ala Ser Arg
Thr Ala Leu Ser Val Gly Gly 240 245 250 255 Ala Pro Cys Thr Val Leu
Asn Ile Met Leu Asp Cys Asp Lys Thr Ala 260 265 270 Thr Pro Trp Cys
Thr Phe Ser 275 95 235 PRT Homo sapiens SIGNAL -20..-1 95 Met Arg
Pro Leu Ala Gly Gly Leu Leu Lys Val Val Phe Val Val Phe -20 -15 -10
-5 Ala Ser Leu Cys Ala Trp Tyr Ser Gly Tyr Leu Leu Ala Glu Leu Ile
1 5 10 Pro Asp Ala Pro Leu Ser Ser Ala Ala Tyr Ser Ile Arg Ser Ile
Gly 15 20 25 Glu Arg Pro Val Leu Lys Ala Pro Val Pro Lys Arg Gln
Lys Cys Asp 30 35 40 His Trp Thr Pro Cys Pro Ser Asp Thr Tyr Ala
Tyr Arg Leu Leu Ser 45 50 55 60 Gly Gly Gly Arg Ser Lys Tyr Ala Lys
Ile Cys Phe Glu Asp Asn Leu 65 70 75 Leu Met Gly Glu Gln Leu Gly
Asn Val Ala Arg Gly Ile Asn Ile Ala 80 85 90 Ile Val Asn Tyr Val
Thr Gly Asn Val Thr Ala Thr Arg Cys Phe Asp 95 100 105 Met Tyr Glu
Gly Asp Asn Ser Gly Pro Met Thr Lys Phe Ile Gln Ser 110 115 120 Ala
Ala Pro Lys Ser Leu Leu Phe Met Val Thr Tyr Asp Asp Gly Ser 125 130
135 140 Thr Arg Leu Asn Asn Asp Ala Lys Asn Ala Ile Glu Ala Leu Gly
Ser 145 150 155 Lys Glu Ile Arg Asn Met Lys Phe Arg Ser Ser Trp Val
Phe Ile Ala 160 165 170 Ala Lys Gly Leu Glu Leu Pro Ser Glu Ile Gln
Arg Glu Lys Ile Asn 175 180 185 His Ser Asp Ala Lys Asn Asn Arg Tyr
Ser Gly Trp Pro Ala Glu Ile 190 195 200 Gln Ile Glu Gly Cys Ile Pro
Lys Glu Arg Ser 205 210 215 96 52 PRT Homo sapiens SIGNAL -31..-1
96 Met Arg Val Tyr Lys Arg Thr Gln Leu Arg Gln Glu Thr Gly Pro Lys
-30 -25 -20 Ser Tyr Val Leu Phe Ser Ala Ser Ser Phe Pro Ser Ile Ser
Gly Asn -15 -10 -5 1 Ile Arg Ser Arg Asn Tyr Phe Gln Lys Gln Asn
Asn His Trp Phe Gln 5 10 15 Thr Ser Asp Tyr 20 97 229 PRT Homo
sapiens SIGNAL -47..-1 97 Met Gln Asp Glu Asp Gly Tyr Ile Thr Leu
Asn Ile Lys Thr Arg Lys -45 -40 -35 Pro Ala Leu Val Ser Val Gly Pro
Ala Ser Ser Phe Trp Trp Arg Val -30 -25 -20 Met Ala Leu Ile Leu Leu
Ile Leu Cys Val Gly Met Val Val Gly Leu -15 -10 -5 1 Val Ala Leu
Gly Ile Trp Ser Val Met Gln Arg Asn Tyr Leu Gln Asp 5 10 15 Glu Asn
Glu Asn Arg Thr Gly Thr Leu Gln Gln Leu Ala Lys Arg Phe 20 25 30
Cys Gln Tyr Val Val Lys Gln Ser Glu Leu Lys Gly Thr Phe Lys Gly 35
40 45 His Lys Cys Ser Pro Cys Asp Thr Asn Trp Arg Tyr Tyr Gly Asp
Ser 50 55 60 65 Cys Tyr Gly Phe Phe Arg His Asn Leu Thr Trp Glu Glu
Ser Lys Gln 70 75 80 Tyr Cys Thr Asp Met Asn Ala Thr Leu Leu Lys
Ile Asp Asn Arg Asn 85 90 95 Ile Val Glu Tyr Ile Lys Ala Arg Thr
His Leu Ile Arg Trp Val Gly 100 105 110 Leu Ser Arg Gln Lys Ser Asn
Glu Val Trp Lys Trp Glu Asp Gly Ser 115 120 125 Val Ile Ser Glu Asn
Met Phe Glu Phe Leu Glu Asp Gly Lys Gly Asn 130 135 140 145 Met Asn
Cys Ala Tyr Phe His Asn Gly Lys Met His Pro Thr Phe Cys 150 155 160
Glu Asn Lys His Tyr Leu Met Cys Glu Arg Lys Ala Gly Met Thr Lys 165
170 175 Val Asp Gln Leu Pro 180 98 92 PRT Homo sapiens SIGNAL
-24..-1 98 Met Thr Lys Leu Ala Gln Trp Leu Trp Gly Leu Ala Ile Leu
Gly Ser -20 -15 -10 Thr Trp Val Ala Leu Thr Thr Gly Ala Leu Gly Leu
Glu Leu Pro Leu -5 1 5 Ser Cys Gln Glu Val Leu Trp Pro Leu Pro Ala
Tyr Leu Leu Val Ser 10 15 20 Ala Gly Cys Tyr Ala Leu Gly Thr Val
Gly Tyr Arg Val Ala Thr Phe 25 30 35 40 His Asp Cys Glu Asp Ala Ala
Arg Glu Leu Gln Ser Gln Ile Gln Glu 45 50 55 Ala Arg Ala Asp Leu
Ala Arg Arg Gly Leu Arg Phe 60 65 99 425 PRT Homo sapiens SIGNAL
-23..-1 99 Met Ala Ser Ser Ser Pro Asp Ser Pro Cys Ser Cys Asp Cys
Phe Val -20 -15 -10 Ser Val Pro Pro Ala Ser Ala Ile Pro Ala Val Ile
Phe Ala Lys Asn -5 1 5 Ser Asp Arg Pro Arg Asp Glu Val Gln Glu Val
Val Phe Val Pro Ala 10 15 20 25 Gly Thr His Thr Pro Gly Ser Arg Leu
Gln Cys Thr Tyr Ile Glu Val 30 35 40 Glu Gln Val Ser Lys Thr His
Ala Val Ile Leu Ser Arg Pro Ser Trp 45 50 55 Leu Trp Gly Ala Glu
Met Gly Ala Asn Glu His Gly Val Cys Ile Gly 60 65 70 Asn Glu Ala
Val Trp Thr Lys Glu Pro Val Gly Glu Gly Glu Ala Leu 75 80 85 Leu
Gly Met Asp Leu Leu Arg Leu Ala Leu Glu Arg Ser Ser Ser Ala 90 95
100 105 Gln Glu Ala Leu His Val Ile Thr Gly Leu Leu Glu His Tyr Gly
Gln 110 115 120 Gly Gly Asn Cys Leu Glu Asp Ala Ala Pro Phe Ser Tyr
His Ser Thr 125 130 135 Phe Leu Leu Ala Asp Arg Thr Glu Ala Trp Val
Leu Glu Thr Ala Gly 140 145 150 Arg Leu Trp Ala Ala Gln Arg Ile Gln
Glu Gly Ala Arg Asn Ile Ser 155 160 165 Asn Gln Leu Ser Ile Gly Thr
Asp Ile Ser Ala Gln His Pro Glu Leu 170 175 180 185 Arg Thr His Ala
Gln Ala Lys Gly Trp Trp Asp Gly Gln Gly Ala Phe 190 195 200 Asp Phe
Ala Gln Ile Phe Ser Leu Thr Gln Gln Pro Val Arg Met Glu 205 210 215
Ala Ala Lys Ala Arg Phe Gln Ala Gly Arg Glu Leu Leu Arg Gln Arg 220
225 230 Gln Gly Gly Ile Thr Ala Glu Val Met Met Gly Ile Leu Arg Asp
Lys 235 240 245 Glu Ser Gly Ile Cys Met Asp Ser Gly Gly Phe Arg Thr
Thr Ala Ser 250 255 260 265 Met Val Ser Val Leu Pro Gln Asp Pro Thr
Gln Pro Cys Val His Phe 270 275 280 Leu Thr Ala Thr Pro Asp Pro Ser
Arg Ser Val Phe Lys Pro Phe Ile 285 290 295 Phe Gly Val Gly Val Ala
Gln Ala Pro Gln Val Leu Ser Pro Thr Phe 300 305 310 Gly Ala Gln Asp
Pro Val Arg Thr Leu Pro Arg Phe Gln Thr Gln Val 315 320 325 Asp Arg
Arg His Thr Leu Tyr Arg Gly His Gln Ala Ala Leu Gly Leu 330 335 340
345 Met Glu Arg Asp Gln Asp Arg Gly Gln Gln Leu Gln Gln Lys Gln Gln
350 355 360 Asp Leu Glu Gln Glu Gly Leu Glu Ala Thr Gln Gly Leu Leu
Ala Gly 365 370 375 Glu Trp Ala Pro Pro Leu Trp Glu Leu Gly Ser Leu
Phe Gln Ala Phe 380 385 390 Val Lys Arg Glu Ser Gln Ala Tyr Ala 395
400 100 87 PRT Homo sapiens SIGNAL -62..-1 100 Met Ala Ile Phe Trp
Ile Val His Ala His Phe Trp Ser Pro Leu Pro -60 -55 -50 Pro Arg Leu
Pro His Gly Arg Cys Cys Cys Leu Lys Ala Pro Leu Pro -45 -40 -35 Pro
Asp Val Gly Pro Leu Gln Val Ala Pro His Leu Phe Ser Val Pro -30 -25
-20 -15 Leu His Ile Leu Thr Val Pro Leu Leu Glu Pro Ala Arg Cys Ser
Gly -10 -5 1 Ile Leu Val Phe Phe Leu His Gln Pro Val Ser Ser Leu
Ser Phe Cys 5 10 15 Tyr Phe Ile Gly Gly Trp Cys 20 25 101 149 PRT
Homo sapiens SIGNAL -100..-1 101 Met Glu Thr Leu Tyr Arg Val Pro
Phe Leu Val Leu Glu Cys Pro Asn -100 -95 -90 -85 Leu Lys Leu Lys
Lys Pro Pro Trp Leu His Met Pro Ser Ala Met Thr -80 -75 -70 Val Tyr
Ala Leu Val Val Val Ser Tyr Phe Leu Ile Thr Gly Gly Ile -65 -60 -55
Ile Tyr Asp Val Ile Val Glu Pro Pro Ser Val Gly Ser Met Thr Asp -50
-45 -40 Glu His Gly His Gln Arg Pro Val Ala Phe Leu Ala Tyr Arg Val
Asn -35 -30 -25 Gly Gln Tyr Ile Met Glu Gly Leu Ala Ser Ser Phe Leu
Phe Thr Met -20 -15 -10 -5 Gly Gly Leu Gly Phe Ile Ile Leu Asp Arg
Ser Asn Ala Pro Asn Ile 1 5 10 Pro Lys Leu Asn Arg Phe Leu Leu Leu
Phe Ile Gly Phe Val Cys Val 15 20 25 Leu Leu Ser Phe Phe Met Ala
Arg Val Phe Met Arg Met Lys Leu Pro 30 35 40 Gly Tyr Leu Met Gly 45
102 187 PRT Homo sapiens SIGNAL -35..-1 102 Met Ala Asn Asn Thr Thr
Ser Leu Gly
Ser Pro Trp Pro Glu Asn Phe -35 -30 -25 -20 Trp Glu Asp Leu Ile Met
Ser Phe Thr Val Ser Met Ala Ile Gly Leu -15 -10 -5 Val Leu Gly Gly
Phe Ile Trp Ala Val Phe Ile Cys Leu Ser Arg Arg 1 5 10 Arg Arg Ala
Ser Ala Pro Ile Ser Gln Trp Ser Ser Ser Arg Arg Ser 15 20 25 Arg
Ser Ser Tyr Thr His Gly Leu Asn Arg Thr Gly Phe Tyr Arg His 30 35
40 45 Ser Gly Cys Glu Arg Arg Ser Asn Leu Ser Leu Ala Ser Leu Thr
Phe 50 55 60 Gln Arg Gln Ala Ser Leu Glu Gln Ala Asn Ser Phe Pro
Arg Lys Ser 65 70 75 Ser Phe Arg Ala Ser Thr Phe His Pro Phe Leu
Gln Cys Pro Pro Leu 80 85 90 Pro Val Glu Thr Glu Ser Gln Leu Val
Thr Leu Pro Ser Ser Asn Ile 95 100 105 Ser Pro Thr Ile Ser Thr Ser
His Ser Leu Ser Arg Pro Asp Tyr Trp 110 115 120 125 Ser Ser Asn Ser
Leu Arg Val Gly Leu Ser Thr Pro Pro Pro Pro Ala 130 135 140 Tyr Glu
Ser Ile Ile Lys Ala Phe Pro Asp Ser 145 150 103 123 PRT Homo
sapiens SIGNAL -26..-1 103 Met Ala Thr Ala Ala Gly Ala Thr Tyr Phe
Gln Arg Gly Ser Leu Phe -25 -20 -15 Trp Phe Thr Val Ile Thr Leu Ser
Phe Gly Tyr Tyr Thr Trp Val Val -10 -5 1 5 Phe Trp Pro Gln Ser Ile
Pro Tyr Gln Asn Leu Gly Pro Leu Gly Pro 10 15 20 Phe Thr Gln Tyr
Leu Val Asp His His His Thr Leu Leu Cys Asn Gly 25 30 35 Tyr Trp
Leu Ala Trp Leu Ile His Val Gly Glu Ser Leu Tyr Ala Ile 40 45 50
Val Leu Cys Lys His Lys Gly Ile Thr Ser Gly Arg Ala Gln Leu Leu 55
60 65 70 Trp Phe Leu Gln Thr Phe Phe Phe Gly Ile Ala Ser Leu Thr
Ile Leu 75 80 85 Ile Ala Tyr Lys Arg Lys Arg Gln Lys Gln Thr 90 95
104 153 PRT Homo sapiens SIGNAL -102..-1 104 Met Ala Ala Gly Leu
Phe Gly Leu Ser Ala Arg Arg Leu Leu Ala Ala -100 -95 -90 Ala Ala
Thr Arg Gly Leu Pro Ala Ala Arg Val Arg Trp Glu Ser Ser -85 -80 -75
Phe Ser Arg Thr Val Val Ala Pro Ser Ala Val Ala Gly Lys Arg Pro -70
-65 -60 -55 Pro Glu Pro Thr Thr Pro Trp Gln Glu Asp Pro Glu Pro Glu
Asp Glu -50 -45 -40 Asn Leu Tyr Glu Lys Asn Pro Asp Ser His Gly Tyr
Asp Lys Asp Pro -35 -30 -25 Val Leu Asp Val Trp Asn Met Arg Leu Val
Phe Phe Phe Gly Val Ser -20 -15 -10 Ile Ile Leu Val Leu Gly Ser Thr
Phe Val Ala Tyr Leu Pro Asp Tyr -5 1 5 10 Arg Met Lys Glu Trp Ser
Arg Arg Glu Ala Glu Arg Leu Val Lys Tyr 15 20 25 Arg Glu Ala Asn
Gly Leu Pro Ile Met Glu Ser Asn Cys Phe Asp Pro 30 35 40 Ser Lys
Ile Gln Leu Pro Glu Asp Glu 45 50 105 72 PRT Homo sapiens 105 Leu
Pro Val Ser Thr Arg Ile Ile Asn His Ile Tyr Ser Phe Pro Ser 1 5 10
15 Val Asp Leu Trp Ile Val Cys Ile Phe Thr Val Ser Val Ser His Leu
20 25 30 Phe Glu Lys Gly Thr Leu Tyr Gly Tyr Phe Tyr Val Ile Asn
Ser Ser 35 40 45 Ile Asn Leu Cys Val Asn Asp Cys Leu Pro Val Met
Asp Ser Ile Ser 50 55 60 Leu Ser Pro Leu Phe Leu Ser His 65 70 106
175 PRT Homo sapiens SIGNAL -20..-1 106 Met Glu Lys Ile Pro Val Ser
Ala Phe Leu Leu Leu Val Ala Leu Ser -20 -15 -10 -5 Tyr Thr Leu Ala
Arg Asp Thr Thr Val Lys Pro Gly Ala Lys Lys Asp 1 5 10 Thr Lys Asp
Ser Arg Pro Lys Leu Pro Gln Thr Leu Ser Arg Gly Trp 15 20 25 Gly
Asp Gln Leu Ile Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys 30 35
40 Ser Lys Thr Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu
45 50 55 60 Cys Pro His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn
Lys Glu 65 70 75 Ile Gln Lys Leu Ala Glu Gln Phe Val Leu Leu Asn
Leu Val Tyr Glu 80 85 90 Thr Thr Asp Lys His Leu Ser Pro Asp Gly
Gln Tyr Val Pro Arg Ile 95 100 105 Met Phe Val Asp Pro Ser Leu Thr
Val Arg Ala Asp Ile Thr Gly Arg 110 115 120 Tyr Ser Asn Arg Leu Tyr
Ala Tyr Glu Pro Ala Asp Thr Ala Leu Leu 125 130 135 140 Leu Asp Asn
Met Lys Lys Ala Leu Lys Leu Leu Lys Thr Glu Leu 145 150 155 107 303
PRT Homo sapiens SIGNAL -20..-1 107 Met Ala Asp Ala Ala Ser Gln Val
Leu Leu Gly Ser Gly Leu Thr Ile -20 -15 -10 -5 Leu Ser Gln Pro Leu
Met Tyr Val Lys Val Leu Ile Gln Val Gly Tyr 1 5 10 Glu Pro Leu Pro
Pro Thr Ile Gly Arg Asn Ile Phe Gly Arg Gln Val 15 20 25 Cys Gln
Leu Pro Gly Leu Phe Ser Tyr Ala Gln His Ile Ala Ser Ile 30 35 40
Asp Gly Arg Arg Gly Leu Phe Thr Gly Leu Thr Pro Arg Leu Cys Ser 45
50 55 60 Gly Val Leu Gly Thr Val Val His Gly Lys Val Leu Gln His
Tyr Gln 65 70 75 Glu Ser Asp Lys Gly Glu Glu Leu Gly Pro Gly Asn
Val Gln Lys Glu 80 85 90 Val Ser Ser Ser Phe Asp His Val Ile Lys
Glu Thr Thr Arg Glu Met 95 100 105 Ile Ala Arg Ser Ala Ala Thr Leu
Ile Thr His Pro Phe His Val Ile 110 115 120 Thr Leu Arg Ser Met Val
Gln Phe Ile Gly Arg Glu Ser Lys Tyr Cys 125 130 135 140 Gly Leu Cys
Asp Ser Ile Ile Thr Ile Tyr Arg Glu Glu Gly Ile Leu 145 150 155 Gly
Phe Phe Ala Gly Leu Val Pro Arg Leu Leu Gly Asp Ile Leu Ser 160 165
170 Leu Trp Leu Cys Asn Ser Leu Ala Tyr Leu Val Asn Thr Tyr Ala Leu
175 180 185 Asp Ser Gly Val Ser Thr Met Asn Glu Met Lys Ser Tyr Ser
Gln Ala 190 195 200 Val Thr Gly Phe Phe Ala Ser Met Leu Thr Tyr Pro
Phe Val Leu Val 205 210 215 220 Ser Asn Leu Met Ala Val Asn Asn Cys
Gly Leu Ala Gly Gly Cys Pro 225 230 235 Pro Tyr Ser Pro Ile Tyr Thr
Ser Trp Ile Asp Cys Trp Cys Met Leu 240 245 250 Gln Lys Glu Gly Asn
Met Ser Arg Gly Asn Ser Leu Phe Phe Arg Lys 255 260 265 Val Pro Phe
Gly Lys Thr Tyr Cys Cys Asp Leu Lys Met Leu Ile 270 275 280 108 65
PRT Homo sapiens SIGNAL -39..-1 108 Met Ser Thr Gly Ile Met Glu Tyr
Lys Lys Thr Thr Lys Ala Met Lys -35 -30 -25 Lys Lys Lys Asp Val Leu
Phe Thr Ser Tyr Phe Lys Thr Ile Ala Phe -20 -15 -10 Leu Leu Leu Tyr
Val Ser Ala Gly Pro Ile Ser Arg Ile Phe Ile Arg -5 1 5 Ser Leu Glu
Leu Phe Leu Met Phe Pro Ser Asn Lys His Trp Tyr Ile 10 15 20 25 Ser
109 137 PRT Homo sapiens SIGNAL -17..-1 109 Met Gly Phe Gly Ala Thr
Leu Ala Val Gly Leu Thr Ile Phe Val Leu -15 -10 -5 Ser Val Val Thr
Ile Ile Ile Cys Phe Thr Cys Ser Cys Cys Cys Leu 1 5 10 15 Tyr Lys
Thr Cys Arg Arg Pro Arg Pro Val Val Thr Thr Thr Thr Ser 20 25 30
Thr Thr Val Val His Ala Pro Tyr Pro Gln Pro Pro Ser Val Pro Pro 35
40 45 Ser Tyr Pro Gly Pro Ser Tyr Gln Gly Tyr His Thr Met Pro Pro
Gln 50 55 60 Pro Gly Met Pro Ala Ala Pro Tyr Pro Met Gln Tyr Pro
Pro Pro Tyr 65 70 75 Pro Ala Gln Pro Met Gly Pro Pro Ala Tyr His
Glu Thr Leu Ala Gly 80 85 90 95 Gly Ala Ala Ala Pro Tyr Pro Ala Ser
Gln Pro Pro Tyr Asn Pro Ala 100 105 110 Tyr Met Asp Ala Pro Lys Ala
Ala Leu 115 120 110 154 PRT Homo sapiens SIGNAL -13..-1 110 Met Ala
Leu Leu Leu Ser Val Leu Arg Val Leu Leu Gly Gly Phe Phe -10 -5 1
Ala Leu Val Gly Leu Ala Lys Leu Ser Glu Glu Ile Ser Ala Pro Val 5
10 15 Ser Glu Arg Met Asn Ala Leu Phe Val Gln Phe Ala Glu Val Phe
Pro 20 25 30 35 Leu Lys Val Phe Gly Tyr Gln Pro Asp Pro Leu Asn Tyr
Gln Ile Ala 40 45 50 Val Gly Phe Leu Glu Leu Leu Ala Gly Leu Leu
Leu Val Met Gly Pro 55 60 65 Pro Met Leu Gln Glu Ile Ser Asn Leu
Phe Leu Ile Leu Leu Met Met 70 75 80 Gly Ala Ile Phe Thr Leu Ala
Ala Leu Lys Glu Ser Leu Ser Thr Cys 85 90 95 Ile Pro Ala Ile Val
Cys Leu Gly Phe Leu Leu Leu Leu Asn Val Gly 100 105 110 115 Gln Leu
Leu Ala Gln Thr Lys Lys Val Val Arg Pro Thr Arg Lys Lys 120 125 130
Thr Leu Ser Thr Phe Lys Glu Ser Trp Lys 135 140 111 103 PRT Homo
sapiens SIGNAL -36..-1 111 Met Ala Asn Leu Phe Ile Arg Lys Met Val
Asn Pro Leu Leu Tyr Leu -35 -30 -25 Ser Arg His Thr Val Lys Pro Arg
Ala Leu Ser Thr Phe Leu Phe Gly -20 -15 -10 -5 Ser Ile Arg Gly Ala
Ala Pro Val Ala Val Glu Pro Gly Ala Ala Val 1 5 10 Arg Ser Leu Leu
Ser Pro Gly Leu Leu Pro His Leu Leu Pro Ala Leu 15 20 25 Gly Phe
Lys Asn Lys Thr Val Leu Asn Lys Arg Cys Lys Asp Cys Tyr 30 35 40
Leu Val Lys Arg Arg Gly Arg Trp Tyr Val Tyr Cys Lys Thr His Pro 45
50 55 60 Arg His Lys Gln Arg Gln Met 65 112 86 PRT Homo sapiens
SIGNAL -74..-1 112 Met Pro Tyr Ala Phe Thr Ser Pro Cys Pro Cys Ser
Phe Val Ser Leu -70 -65 -60 Pro Glu Ile Ser Phe Tyr Phe Thr Lys Leu
Leu Leu Ile Leu Lys Ala -55 -50 -45 Leu Pro Glu Ser Pro Phe Leu Leu
Ala Ser Ser Pro Leu Pro Pro Leu -40 -35 -30 Pro Thr Thr Leu Arg Lys
Phe Ile Pro Pro Pro Ser Leu Ile Ser Cys -25 -20 -15 Thr Cys Leu Leu
Leu Tyr Leu Thr His Cys Ile Leu Gly Ile Cys Phe -10 -5 1 5 Ala Tyr
Pro Phe Ile Leu 10 113 395 PRT Homo sapiens SIGNAL -310..-1 113 Met
Asp Leu Gly Ile Pro Asp Leu Leu Asp Ala Trp Leu Glu Pro Pro -310
-305 -300 -295 Glu Asp Ile Phe Ser Thr Gly Ser Val Leu Glu Leu Gly
Leu His Cys -290 -285 -280 Pro Pro Pro Glu Val Pro Val Thr Arg Leu
Gln Glu Gln Gly Leu Gln -275 -270 -265 Gly Trp Lys Ser Gly Gly Asp
Arg Gly Cys Gly Leu Gln Glu Ser Glu -260 -255 -250 Pro Glu Asp Phe
Leu Lys Leu Phe Ile Asp Pro Asn Glu Val Tyr Cys -245 -240 -235 Ser
Glu Ala Ser Pro Gly Ser Asp Ser Gly Ile Ser Glu Asp Ser Cys -230
-225 -220 -215 His Pro Asp Ser Pro Pro Ala Pro Arg Ala Thr Ser Ser
Pro Met Leu -210 -205 -200 Tyr Glu Val Val Tyr Glu Ala Gly Ala Leu
Glu Arg Met Gln Gly Glu -195 -190 -185 Thr Gly Pro Asn Val Gly Leu
Ile Ser Ile Gln Leu Asp Gln Trp Ser -180 -175 -170 Pro Ala Phe Met
Val Pro Asp Ser Cys Met Val Ser Glu Leu Pro Phe -165 -160 -155 Asp
Ala His Ala His Ile Leu Pro Arg Ala Gly Thr Val Ala Pro Val -150
-145 -140 -135 Pro Cys Thr Thr Leu Leu Pro Cys Gln Thr Leu Phe Leu
Thr Asp Glu -130 -125 -120 Glu Lys Arg Leu Leu Gly Gln Glu Gly Val
Ser Leu Pro Ser His Leu -115 -110 -105 Pro Leu Thr Lys Ala Glu Glu
Arg Val Leu Lys Lys Val Arg Arg Lys -100 -95 -90 Ile Arg Asn Lys
Gln Ser Ala Gln Asp Ser Arg Arg Arg Lys Lys Glu -85 -80 -75 Tyr Ile
Asp Gly Leu Glu Ser Arg Val Ala Ala Cys Ser Ala Gln Asn -70 -65 -60
-55 Gln Glu Leu Gln Lys Lys Val Gln Glu Leu Glu Arg His Asn Ile Ser
-50 -45 -40 Leu Val Ala Gln Leu Arg Gln Leu Gln Thr Leu Ile Ala Gln
Thr Ser -35 -30 -25 Asn Lys Ala Ala Gln Thr Ser Thr Cys Val Leu Ile
Leu Leu Phe Ser -20 -15 -10 Leu Ala Leu Ile Ile Leu Pro Ser Phe Ser
Pro Phe Gln Ser Arg Pro -5 1 5 10 Glu Ala Gly Ser Glu Asp Tyr Gln
Pro His Gly Val Thr Ser Arg Asn 15 20 25 Ile Leu Thr His Lys Asp
Val Thr Glu Asn Leu Glu Thr Gln Val Val 30 35 40 Glu Ser Arg Leu
Arg Glu Pro Pro Gly Ala Lys Asp Ala Asn Gly Ser 45 50 55 Thr Arg
Thr Leu Leu Glu Lys Met Gly Gly Lys Pro Arg Pro Ser Gly 60 65 70
Arg Ile Arg Ser Val Leu His Ala Asp Glu Met 75 80 85 114 93 PRT
Homo sapiens SIGNAL -18..-1 114 Met Ile His Leu Gly His Ile Leu Phe
Leu Leu Leu Leu Pro Val Ala -15 -10 -5 Ala Ala Gln Thr Thr Pro Gly
Glu Arg Ser Ser Leu Pro Ala Phe Tyr 1 5 10 Pro Gly Thr Ser Gly Ser
Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 15 20 25 30 Leu Leu Ala Gly
Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 35 40 45 Val Gly
Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 50 55 60
Glu Tyr Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 65 70 75 115 61
PRT Homo sapiens SIGNAL -21..-1 115 Met Arg Glu Met Pro Val Pro Ser
Leu Ile Asn Leu Ala Ala Ser Arg -20 -15 -10 Thr Leu Ser Phe Cys Ile
Ser Asp Asn His Val Ser Ser Pro Gly Pro -5 1 5 10 Ala Asn Pro Ser
Cys Gly Leu His Pro His Trp Leu Arg Pro Leu Lys 15 20 25 Leu Leu
Thr Tyr Thr Cys Arg Glu Leu Lys Leu Gln Gly 30 35 40 116 331 PRT
Homo sapiens SIGNAL -31..-1 116 Met Trp Leu Trp Glu Asp Gln Gly Gly
Leu Leu Gly Pro Phe Ser Phe -30 -25 -20 Leu Leu Leu Val Leu Leu Leu
Val Thr Arg Ser Pro Val Asn Ala Cys -15 -10 -5 1 Leu Leu Thr Gly
Ser Leu Phe Val Leu Leu Arg Val Phe Ser Phe Glu 5 10 15 Pro Val Pro
Ser Cys Arg Ala Leu Gln Val Leu Lys Pro Arg Asp Arg 20 25 30 Ile
Ser Ala Ile Ala His Arg Gly Gly Ser His Asp Ala Pro Glu Asn 35 40
45 Thr Leu Ala Ala Ile Arg Gln Ala Ala Lys Asn Gly Ala Thr Gly Val
50 55 60 65 Glu Leu Asp Ile Glu Phe Thr Ser Asp Gly Ile Pro Val Leu
Met His 70 75 80 Asp Asn Thr Val Asp Arg Thr Thr Asp Gly Thr Gly
Arg Leu Cys Asp 85 90 95 Leu Thr Phe Glu Gln Ile Arg Lys Leu Asn
Pro Ala Ala Asn His Arg 100 105 110 Leu Arg Asn Asp Phe Pro Asp Glu
Lys Ile Pro Thr Leu Met Glu Ala 115 120 125 Val Ala Glu Cys Leu Asn
His Asn Leu Thr Ile Phe Phe Asp Val Lys 130 135 140 145 Gly His Ala
His Lys Ala Thr Glu Ala Leu Lys Lys Met Tyr Met Glu 150 155 160 Phe
Pro Gln Leu Tyr Asn Asn Ser Val Val Cys Ser Phe Leu Pro Glu 165 170
175 Val Ile Tyr Lys Met Arg Gln Thr Asp Arg Asp Val Ile Thr Ala Leu
180 185 190 Thr His Arg Pro Trp Ser Leu Ser His Thr Gly Asp Gly Lys
Pro Arg 195 200 205 Tyr Asp Thr Phe Trp Lys His Phe Ile Phe Val Met
Met Asp Ile Leu 210 215 220 225 Leu Asp Trp Ser Met His Asn Ile
Leu
Trp Tyr Leu Cys Gly Ile Ser 230 235 240 Ala Phe Leu Met Gln Lys Asp
Phe Val Ser Pro Ala Tyr Leu Lys Lys 245 250 255 Trp Ser Ala Lys Gly
Ile Gln Val Val Gly Trp Thr Val Asn Thr Phe 260 265 270 Asp Glu Lys
Ser Tyr Tyr Glu Ser His Leu Gly Ser Ser Tyr Ile Thr 275 280 285 Asp
Ser Met Val Glu Asp Cys Glu Pro His Phe 290 295 300 117 210 PRT
Homo sapiens SIGNAL -99..-1 117 Met Ala Ala Ser Val Glu Gln Arg Glu
Gly Thr Ile Gln Val Gln Gly -95 -90 -85 Gln Ala Leu Phe Phe Arg Glu
Ala Leu Pro Gly Ser Gly Gln Ala Arg -80 -75 -70 Phe Ser Val Leu Leu
Leu His Gly Ile Arg Phe Ser Ser Glu Thr Trp -65 -60 -55 Gln Asn Leu
Gly Thr Leu His Arg Leu Ala Gln Ala Gly Tyr Arg Ala -50 -45 -40 Val
Ala Ile Asp Leu Pro Gly Leu Gly His Ser Lys Glu Ala Ala Ala -35 -30
-25 -20 Pro Ala Pro Ile Gly Glu Leu Ala Pro Gly Ser Phe Leu Ala Ala
Val -15 -10 -5 Val Asp Ala Leu Glu Leu Gly Pro Pro Val Val Ile Ser
Pro Ser Leu 1 5 10 Ser Gly Met Tyr Ser Leu Pro Phe Leu Thr Ala Pro
Gly Ser Gln Leu 15 20 25 Pro Gly Phe Val Pro Val Ala Pro Ile Cys
Thr Asp Lys Ile Asn Ala 30 35 40 45 Ala Asn Tyr Ala Ser Val Lys Thr
Pro Ala Leu Ile Val Tyr Gly Asp 50 55 60 Gln Asp Pro Met Gly Gln
Thr Ser Phe Glu His Leu Lys Gln Leu Pro 65 70 75 Asn His Arg Val
Leu Ile Met Lys Gly Ala Gly His Pro Cys Tyr Leu 80 85 90 Asp Lys
Pro Glu Glu Trp His Thr Gly Leu Leu Asp Phe Leu Gln Gly 95 100 105
Leu Gln 110 118 79 PRT Homo sapiens SIGNAL -67..-1 118 Met Glu Leu
Glu Ala Met Ser Arg Tyr Thr Ser Pro Val Asn Pro Ala -65 -60 -55 Val
Phe Pro His Leu Thr Val Val Leu Leu Ala Ile Gly Met Phe Phe -50 -45
-40 Thr Ala Trp Phe Phe Val Tyr Glu Val Thr Ser Thr Lys Tyr Thr Arg
-35 -30 -25 -20 Asp Ile Tyr Lys Glu Leu Leu Ile Ser Leu Val Ala Ser
Leu Phe Met -15 -10 -5 Gly Phe Gly Val Leu Phe Leu Leu Leu Trp Val
Gly Ile Tyr Val 1 5 10 119 84 PRT Homo sapiens 119 Met Ala Val Trp
Pro Glu Val Ser Gln Asn Arg Leu Thr Arg Gly Leu 1 5 10 15 Leu Leu
Pro Asn Tyr Gln Leu Arg Gly Ser Val Pro Lys Arg Glu Lys 20 25 30
Arg Pro Lys Arg Lys His Gln His Leu Phe Thr Pro Ser Glu Arg His 35
40 45 Ser Val Cys Leu Asp Cys Leu Leu Glu Ile Ser Leu Ser Gly Lys
Gln 50 55 60 Trp Arg Asn Val Ile Ser Phe Asn Cys Phe Cys Thr Thr
Lys Thr Leu 65 70 75 80 Phe Trp Val Asn 120 92 PRT Homo sapiens
SIGNAL -20..-1 120 Met Ala Ser Leu Gly His Ile Leu Val Phe Cys Val
Gly Leu Leu Thr -20 -15 -10 -5 Met Ala Lys Ala Glu Ser Pro Lys Glu
His Asp Pro Phe Thr Tyr Asp 1 5 10 Tyr Gln Ser Leu Gln Ile Gly Gly
Leu Val Ile Ala Gly Ile Leu Phe 15 20 25 Ile Leu Gly Ile Leu Ile
Val Leu Ser Arg Arg Cys Arg Cys Lys Phe 30 35 40 Asn Gln Gln Gln
Arg Thr Gly Glu Pro Asp Glu Glu Glu Gly Thr Phe 45 50 55 60 Arg Ser
Ser Ile Arg Arg Leu Ser Thr Arg Arg Arg 65 70 121 210 PRT Homo
sapiens SIGNAL -14..-1 121 Met Leu Thr Leu Leu Gly Leu Ser Leu Ile
Leu Ala Gly Leu Ile Val -10 -5 1 Gly Gly Ala Cys Ile Tyr Lys His
Phe Met Pro Lys Ser Thr Ile Tyr 5 10 15 Arg Gly Glu Met Cys Phe Phe
Asp Ser Glu Asp Pro Ala Asn Ser Leu 20 25 30 Arg Gly Gly Glu Pro
Asn Phe Leu Pro Val Thr Glu Glu Ala Asp Ile 35 40 45 50 Arg Glu Asp
Asp Asn Ile Ala Ile Ile Asp Val Pro Val Pro Ser Phe 55 60 65 Ser
Asp Ser Asp Pro Ala Ala Ile Ile His Asp Phe Glu Lys Gly Met 70 75
80 Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys Tyr Leu Met Pro Leu
85 90 95 Asn Thr Ser Ile Val Met Pro Pro Glu Asn Leu Val Glu Leu
Phe Gly 100 105 110 Lys Leu Ala Ser Gly Arg Tyr Leu Pro Gln Thr Tyr
Val Val Arg Glu 115 120 125 130 Asp Leu Val Ala Val Glu Glu Ile Arg
Asp Val Ser Asn Leu Gly Ile 135 140 145 Phe Ile Tyr Gln Leu Cys Asn
Asn Arg Lys Ser Phe Arg Leu Arg Arg 150 155 160 Arg Asp Leu Leu Leu
Gly Phe Asn Lys Arg Ala Ile Asp Lys Cys Trp 165 170 175 Lys Ile Arg
His Phe Pro Asn Glu Phe Ile Val Glu Thr Lys Ile Cys 180 185 190 Gln
Glu 195 122 205 PRT Homo sapiens SIGNAL -139..-1 122 Met Ala Pro
Thr Arg Lys Asp Lys Leu Leu Gln Phe Tyr Pro Ser Leu -135 -130 -125
Glu Asp Pro Ala Ser Ser Arg Tyr Gln Asn Phe Ser Lys Gly Ser Arg
-120 -115 -110 His Gly Ser Glu Glu Ala Tyr Ile Asp Pro Ile Ala Met
Glu Tyr Tyr -105 -100 -95 Asn Trp Gly Arg Phe Ser Lys Pro Pro Glu
Gly Glu Ala Lys Asp Lys -90 -85 -80 Ala Gly Gly Gly Gly Ser Gly Val
Gly Ala Gln Gly Arg Ser His Thr -75 -70 -65 -60 Ser Arg Gln Glu Arg
Arg Leu Gly Leu Gly Ser Asp Asp Asp Ala Asn -55 -50 -45 Ser Tyr Glu
Asn Val Leu Ile Cys Lys Gln Lys Thr Thr Glu Thr Gly -40 -35 -30 Ala
Gln Gln Glu Asp Val Gly Gly Leu Cys Arg Gly Asp Leu Ser Leu -25 -20
-15 Ser Leu Ala Leu Lys Thr Gly Pro Thr Ser Gly Leu Cys Pro Ser Ala
-10 -5 1 5 Ser Pro Glu Glu Asp Gly Glu Ser Glu Asp Tyr Gln Asn Ser
Ala Ser 10 15 20 Ile His Gln Trp Arg Glu Ser Arg Lys Val Met Gly
Gln Leu Gln Arg 25 30 35 Glu Ala Ser Pro Gly Pro Val Gly Ser Pro
Asp Glu Glu Asp Gly Glu 40 45 50 Pro Asp Tyr Val Asn Gly Glu Val
Ala Ala Thr Glu Ala 55 60 65 123 85 PRT Homo sapiens SIGNAL -17..-1
123 Met Lys Lys Val Leu Leu Leu Ile Thr Ala Ile Leu Ala Val Ala Val
-15 -10 -5 Gly Phe Pro Val Ser Gln Asp Gln Glu Arg Glu Lys Arg Ser
Ile Ser 1 5 10 15 Asp Ser Asp Glu Leu Ala Ser Gly Phe Phe Val Phe
Pro Tyr Pro Tyr 20 25 30 Pro Phe Arg Pro Leu Pro Pro Ile Pro Phe
Pro Arg Phe Pro Trp Phe 35 40 45 Arg Arg Asn Phe Pro Ile Pro Ile
Pro Glu Ser Ala Pro Thr Thr Pro 50 55 60 Leu Pro Ser Glu Lys 65 124
115 PRT Homo sapiens SIGNAL -51..-1 124 Met Gln Ala Gln Ala Pro Val
Val Val Val Thr Gln Pro Gly Val Gly -50 -45 -40 Pro Gly Pro Ala Pro
Gln Asn Ser Asn Trp Gln Thr Gly Met Cys Asp -35 -30 -25 -20 Cys Phe
Ser Asp Cys Gly Val Cys Leu Cys Gly Thr Phe Cys Phe Pro -15 -10 -5
Cys Leu Gly Cys Gln Val Ala Ala Asp Met Asn Glu Cys Cys Leu Cys 1 5
10 Gly Thr Ser Val Ala Met Arg Thr Leu Tyr Arg Thr Arg Tyr Gly Ile
15 20 25 Pro Gly Pro Ile Cys Asp Asp Tyr Met Ala Thr Leu Cys Cys
Pro His 30 35 40 45 Cys Thr Leu Cys Gln Ile Lys Arg Asp Ile Asn Arg
Arg Arg Ala Met 50 55 60 Arg Thr Phe 125 81 PRT Homo sapiens SIGNAL
-31..-1 125 Met Ser Asn Thr His Thr Val Leu Val Ser Leu Pro His Pro
His Pro -30 -25 -20 Ala Leu Thr Cys Cys His Leu Gly Leu Pro His Pro
Val Arg Ala Pro -15 -10 -5 1 Arg Pro Leu Pro Arg Val Glu Pro Trp
Asp Pro Arg Trp Gln Asp Ser 5 10 15 Glu Leu Arg Tyr Pro Gln Ala Met
Asn Ser Phe Leu Asn Glu Arg Ser 20 25 30 Ser Pro Cys Arg Thr Leu
Arg Gln Glu Ala Ser Ala Asp Arg Cys Asp 35 40 45 Leu 50 126 235 PRT
Homo sapiens SIGNAL -39..-1 126 Met Gly Thr Ala Asp Ser Asp Glu Met
Ala Pro Glu Ala Pro Gln His -35 -30 -25 Thr His Ile Asp Val His Ile
His Gln Glu Ser Ala Leu Ala Lys Leu -20 -15 -10 Leu Leu Thr Cys Cys
Ser Ala Leu Arg Pro Arg Ala Thr Gln Ala Arg -5 1 5 Gly Ser Ser Arg
Leu Leu Val Ala Ser Trp Val Met Gln Ile Val Leu 10 15 20 25 Gly Ile
Leu Ser Ala Val Leu Gly Gly Phe Phe Tyr Ile Arg Asp Tyr 30 35 40
Thr Leu Leu Val Thr Ser Gly Ala Ala Ile Trp Thr Gly Ala Val Ala 45
50 55 Val Leu Ala Gly Ala Ala Ala Phe Ile Tyr Glu Lys Arg Gly Gly
Thr 60 65 70 Tyr Trp Ala Leu Leu Arg Thr Leu Leu Ala Leu Ala Ala
Phe Ser Thr 75 80 85 Ala Ile Ala Ala Leu Lys Leu Trp Asn Glu Asp
Phe Arg Tyr Gly Tyr 90 95 100 105 Ser Tyr Tyr Asn Ser Ala Cys Arg
Ile Ser Ser Ser Ser Asp Trp Asn 110 115 120 Thr Pro Ala Pro Thr Gln
Ser Pro Glu Glu Val Arg Arg Leu His Leu 125 130 135 Cys Thr Ser Phe
Met Asp Met Leu Lys Ala Leu Phe Arg Thr Leu Gln 140 145 150 Ala Met
Leu Leu Gly Val Trp Ile Leu Leu Leu Leu Ala Ser Leu Ala 155 160 165
Pro Leu Trp Leu Tyr Cys Trp Arg Met Phe Pro Thr Lys Gly Lys Arg 170
175 180 185 Asp Gln Lys Glu Met Leu Glu Val Ser Gly Ile 190 195 127
62 PRT Homo sapiens SIGNAL -21..-1 127 Met Glu Ser Arg Val Leu Leu
Arg Thr Phe Cys Leu Ile Phe Gly Leu -20 -15 -10 Gly Ala Val Trp Gly
Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val -5 1 5 10 Leu Thr Glu
Leu Glu Leu Gly Glu Ser Thr Thr Gly Val Arg Gln Val 15 20 25 Pro
Gly Leu His Asn Gly Thr Lys Ala Phe Leu Phe Gln Ala 30 35 40 128 11
PRT Homo sapiens 128 Met Gly Leu Ser Ser Ser Glu Gly Asp Ile Pro 1
5 10 129 56 PRT Homo sapiens SIGNAL -34..-1 129 Met Glu Arg Gly Leu
Lys Ser Ala Asp Pro Arg Asp Gly Thr Gly Tyr -30 -25 -20 Thr Gly Trp
Ala Gly Ile Ala Val Leu Tyr Leu His Leu Tyr Asp Val -15 -10 -5 Phe
Gly Asp Pro Ala Ser Met Phe Cys Lys Val Phe Asp Leu Leu Val 1 5 10
Leu Asn Lys Ile Leu Leu Gly Leu 15 20 130 542 DNA Homo sapiens CDS
15..311 sig_peptide 15..110 Von Heijne matrix score 3.5 seq
RIHLCQRSXGSQG/VR polyA_signal 507..512 polyA_site 531..542 130
agatattaac aagg atg gcg gcg gcc gca gca agt cga gga gtc ggg gca 50
Met Ala Ala Ala Ala Ala Ser Arg Gly Val Gly Ala -30 -25 aag ctg ggc
ctg cgt gag att cgc atc cac tta tgt cag cgc tcg scc 98 Lys Leu Gly
Leu Arg Glu Ile Arg Ile His Leu Cys Gln Arg Ser Xaa -20 -15 -10 -5
ggc agc cag ggc gtc agg gac ttc att gag aaa cgc tac gtg gag ctg 146
Gly Ser Gln Gly Val Arg Asp Phe Ile Glu Lys Arg Tyr Val Glu Leu 1 5
10 aag aag gcg aat ccc gac cta ccc atc cta atc cgc gaa tgc tcc gat
194 Lys Lys Ala Asn Pro Asp Leu Pro Ile Leu Ile Arg Glu Cys Ser Asp
15 20 25 gtg cag ccc aag ctc tgg gcc cgc tac gca ttt ggc caa rag
acg aat 242 Val Gln Pro Lys Leu Trp Ala Arg Tyr Ala Phe Gly Gln Xaa
Thr Asn 30 35 40 gtc cct ttg aac aac ttc agt gct gat cag gta acc
aga rcc ctg gag 290 Val Pro Leu Asn Asn Phe Ser Ala Asp Gln Val Thr
Arg Xaa Leu Glu 45 50 55 60 aac gtt cta agt ggt aaa gcc tgaagcctcc
actgaggatt aagagcaaca 341 Asn Val Leu Ser Gly Lys Ala 65 gccccagagc
ctgggctctg ctggacttar tataatgtga aaaaaatgtg ttctcctatt 401
cctcataaag cttgtgctgt aaaatacttt ctcagggtgt tcttgtcctc atctaccctc
461 taccccttac tgtgcaacca ctgaggcaaa gtagcttaat ataaaaataa
aactttattc 521 tgtctcatca aaaaaaaaaa a 542 131 909 DNA Homo sapiens
CDS 50..529 sig_peptide 50..130 Von Heijne matrix score
7.19999980926514 seq VLWLSGLSEPGAA/RQ polyA_signal 877..882
polyA_site 899..909 131 aagacggtgg cgcgattggg acagtcgcca gggatggctg
agcgtgaag atg cag cgg 58 Met Gln Arg -25 gtg tcc ggg ctg ctc tcc
tgg acg ctg agc aga gtc ctg tgg ctc tcc 106 Val Ser Gly Leu Leu Ser
Trp Thr Leu Ser Arg Val Leu Trp Leu Ser -20 -15 -10 ggc ctc tct gag
ccg gga gct gcc cgg cag ccc cgg atc atg gaa gag 154 Gly Leu Ser Glu
Pro Gly Ala Ala Arg Gln Pro Arg Ile Met Glu Glu -5 1 5 aaa gcg cta
gag gtt tat gat ttg att aga act atc cgg gac cca gaa 202 Lys Ala Leu
Glu Val Tyr Asp Leu Ile Arg Thr Ile Arg Asp Pro Glu 10 15 20 aag
ccc aat act tta gaa gaa ctg gaa gtg gtc tcg gaa agt tgt gtg 250 Lys
Pro Asn Thr Leu Glu Glu Leu Glu Val Val Ser Glu Ser Cys Val 25 30
35 40 gaa gtt cag gag ata aat gaa gaa raa tat ctg gtt att atc agg
ttc 298 Glu Val Gln Glu Ile Asn Glu Glu Xaa Tyr Leu Val Ile Ile Arg
Phe 45 50 55 acg cca aca gta cct cat tgc tct ttg gcg act ctt att
ggg ctg tgc 346 Thr Pro Thr Val Pro His Cys Ser Leu Ala Thr Leu Ile
Gly Leu Cys 60 65 70 yta arw kta aaa ctt cag cga tgt tta cca ttt
aaa cat aag ttg gma 394 Leu Xaa Xaa Lys Leu Gln Arg Cys Leu Pro Phe
Lys His Lys Leu Xaa 75 80 85 atc tac att tct gaa gga acc cac tca
rsa gar gaa gac atc aat wwk 442 Ile Tyr Ile Ser Glu Gly Thr His Ser
Xaa Glu Glu Asp Ile Asn Xaa 90 95 100 cag ata aat gac aaa gag cgw
ktg gca kct gca atg gaa aac ccc awc 490 Gln Ile Asn Asp Lys Glu Arg
Xaa Ala Xaa Ala Met Glu Asn Pro Xaa 105 110 115 120 tta cgg gaa att
gtg gaa cag tgt gtc ctt gaa cct gac tgawakctgt 539 Leu Arg Glu Ile
Val Glu Gln Cys Val Leu Glu Pro Asp 125 130 tttaaragcc actggcctgt
aattgtttga tatatttgtt taaactcttt gtataatgtc 599 agaggactca
tgtttaatac ataggtgatt tgtacctcag agcatttttt aaaggattct 659
ttccaagcga gatttaatta taaggtagta cctaatttgt tcaatgtata acattctcag
719 gatttgtaac acttaaatga tcagacagaa taatattttc tagttattat
gtgtaagatg 779 agttgctatt tttctgatgc tcattctgat acaactattt
ttcgtgtcaa atatctactg 839 tgcccaaatg tactcaattt aaatcattac
tctgtaaaat aaataagcag atgattctta 899 aaaaaaaaaa 909 132 1149 DNA
Homo sapiens CDS 240..416 sig_peptide 240..305 Von Heijne matrix
score 3.70000004768372 seq AVLDCAFYDPTHA/WS polyA_signal 1117..1122
polyA_site 1139..1149 132 actagcctgc gagtgttctg agggaagcaa
ggaggcggcg gcggccgcag cgagtggcga 60 gtagtggaaa cgttgcttct
gaggggtgtc caagatgacc ggttctaacg gagttcaagc 120 tgaaccagcc
acccgaggat ggcatctcct ccgtgaagtt cagccccaac acctcccagt 180
tcctgcttgt ctcctcctgg gacacgtccg tgcgtctcta cgatgtgccg gccaactcc
239 atg cgg ctc aag tac cag cac acc ggc gcc gtc ctg gac tgc gcc ttc
287 Met Arg Leu Lys Tyr Gln His Thr Gly Ala Val Leu Asp Cys Ala Phe
-20 -15 -10 tac gat cca acg cat gcc tgg agt gga gga cta gat cat caa
ttg aaa 335 Tyr Asp Pro Thr His Ala Trp Ser Gly Gly Leu Asp His Gln
Leu Lys -5 1 5 10 atg cat gat ttg aac act gat caa gaa aat ctt gtt
ggg acc atg atg 383 Met His Asp Leu Asn Thr Asp Gln Glu Asn Leu Val
Gly Thr Met Met 15 20 25 ccc cta tca gat gtg ttg aat act gtc cac
aaa tgaatgtgat ggtcmctgga 436 Pro Leu Ser Asp Val Leu Asn Thr Val
His Lys 30 35 akttgggatc aaacagttaa actgtgggat cccamaactc
cttgtaatgc tgggaccttc 496 tctcmkcctg aaaaggtata taccctctca
gtgtctggag accggctgat tgtgggaaca 556 gcaggccgca gagtgttggt
gtgggactta
cggaacatgg gttacgtgca gcagcgcagg 616 gagtccagcc tgaaatacca
gactcgctgc atacgagcgt ttccaaacaa gcagggttat 676 gtattaagct
ctattgaagg ccgagtggca gttgagtatt tggacccaag ccctgaggta 736
cagaagaaga agtatgcctt caaatgtcac agactaaaag aaaataatat tgagcagatt
796 tacccagtca atgccatttc ttttcacaat atccacaata catttgccac
aggtggttct 856 gatggctttg taaatatttg ggatccattt aacaaaaagc
gactgtgcca attccatcgg 916 taccccacga gcatcgcatc acttgccttc
agtaatgatg ggactacgct tgcaatagcg 976 tcatcatata tgtatgaaat
ggatgacaca gaacatcctg aagatggtat cttcattcgc 1036 caagtgacag
atgcagaaac aaaacccaag tcaccatgta cttgacaaga tttcatttac 1096
ttaagtgcca tgttgatgat aataaaacaa ttcgtactcc ccaaaaaaaa aaa 1149 133
921 DNA Homo sapiens CDS 111..446 sig_peptide 111..254 Von Heijne
matrix score 4.90000009536743 seq PSLAAGLLFGSLA/GL polyA_signal
890..895 polyA_site 909..921 133 agacacctcg cagtcattcc tgcggcttgc
gcgcccttgt agacagccgg ggccttcgtg 60 agaccggtgc aggcctgggg
tagtctccag tctggacaga gaagagaaaa atg cag 116 Met Gln gac act ggc
tca gta gtg cct ttg cat tgg ttt ggc ttt ggc tac gca 164 Asp Thr Gly
Ser Val Val Pro Leu His Trp Phe Gly Phe Gly Tyr Ala -45 -40 -35 gca
ctg gtt gct tct ggt ggg atc att ggc tat gta aaa gca ggb agc 212 Ala
Leu Val Ala Ser Gly Gly Ile Ile Gly Tyr Val Lys Ala Gly Ser -30 -25
-20 -15 gtg ccg tcc ctg gct gca ggg ctg ctc ttt ggc agt cta gcc ggc
ctg 260 Val Pro Ser Leu Ala Ala Gly Leu Leu Phe Gly Ser Leu Ala Gly
Leu -10 -5 1 ggt gct tac cag ctg tct cag gat cca agg aac gtt tgg
gtt ttc cta 308 Gly Ala Tyr Gln Leu Ser Gln Asp Pro Arg Asn Val Trp
Val Phe Leu 5 10 15 gct aca tct ggt acc ttg gct ggc att atg gga atg
agg ttc tac cac 356 Ala Thr Ser Gly Thr Leu Ala Gly Ile Met Gly Met
Arg Phe Tyr His 20 25 30 tct gga aaa ttc atg cct gca ggt tta att
gca ggt gcc akt ttg ctg 404 Ser Gly Lys Phe Met Pro Ala Gly Leu Ile
Ala Gly Ala Xaa Leu Leu 35 40 45 50 atg gtc gcc aaa att gga gtt agt
atg ttc aac aga ccc cat 446 Met Val Ala Lys Ile Gly Val Ser Met Phe
Asn Arg Pro His 55 60 tagcagaakt catgttccag cttagactga tgaagaatta
aaaatctgca tcttccacta 506 ttttcaatat attaagagaa ataagtgcag
catttttgca tctgacattt tacctaaaaa 566 aaaagacacc aaacttggma
raraggtgga aaatcagtca tgattacaaa cctacagagg 626 tggcgagtat
gtaacacaag agcttaataa gaccctcata ragcttgatt cttgtawatt 686
gatgttgtct tttctttckg tatctgtagg taaatctcaa gggtaaaatg ttaggtgtca
746 gctttcaggg ctctgaaacc chattccctg ctctgaggaa cagtgtgaaa
aaaagtcttt 806 taggagattt acaatatctg ttcttttgct catcttagac
cacagactga ctttgaaatt 866 atgttaagtg aaatatcaat gaaaataaag
tttactataa ataataaaaa aaaaa 921 134 916 DNA Homo sapiens CDS
123..455 sig_peptide 123..290 Von Heijne matrix score 4.5 seq
FCAGVLLTLLLIA/FI polyA_signal 886..891 polyA_site 904..916 134
aaagtaatct ttatttcgtc atttttgara catagaagcc gtaacggaag caagtgaaat
60 gctcagtctt agacgactgc gtcgtgctat gaccggactt tttcttgaaa
ggggatgaca 120 gc atg gga ggc aat ggc tcc aca tgt aaa ccc gac act
gaa aga caa 167 Met Gly Gly Asn Gly Ser Thr Cys Lys Pro Asp Thr Glu
Arg Gln -55 -50 -45 ggc act ctc tcc aca gca gcc cca aca act agc cct
gca ccc tgt ctc 215 Gly Thr Leu Ser Thr Ala Ala Pro Thr Thr Ser Pro
Ala Pro Cys Leu -40 -35 -30 tct aac cac cac aac aaa aaa cat tta atc
ctt gcc ttt tgt gct ggg 263 Ser Asn His His Asn Lys Lys His Leu Ile
Leu Ala Phe Cys Ala Gly -25 -20 -15 -10 gtt cta ctg aca ctg ctg ctg
ata gcc ttt atc ttc ctc atc ata aag 311 Val Leu Leu Thr Leu Leu Leu
Ile Ala Phe Ile Phe Leu Ile Ile Lys -5 1 5 agc tac aga aaa tat cac
tcc aag ccc cag gcc cca gat cct cac tca 359 Ser Tyr Arg Lys Tyr His
Ser Lys Pro Gln Ala Pro Asp Pro His Ser 10 15 20 gat cct cca kcc
rrg ctt tca tcc atc cca ggg gaa tca ctt acc tat 407 Asp Pro Pro Xaa
Xaa Leu Ser Ser Ile Pro Gly Glu Ser Leu Thr Tyr 25 30 35 gcc agc
aca ags ktt caa act ctc aga aka ama gag cam yca ctt ggc 455 Ala Ser
Thr Xaa Xaa Gln Thr Leu Arg Xaa Xaa Glu Xaa Xaa Leu Gly 40 45 50 55
tgagaaccat tctgcagact ttgaccccak kgtctatgct caaattaaag taacaaacta
515 actcagcttt tccaatgagg cttgaatcca tttcctcksa tctcagccct
atcttcacas 575 atcactttca cttttttaca wattttggac caccacctgt
gtgaaactgc agtcggagtt 635 gtttasatgt gatctggcaa tgctatccag
catctttgga gaccaatggt cagtcttttc 695 ctggccakag gaaasattga
tggccctccc asttggaact gacagcctgt gagccccttg 755 ggggcataga
ctgccttcct tggacccttc caaagtgtgt ggtacrgagc tcagtgcaca 815
gagtattcac ccagcatcat gaatcaactt gggaggagtc aaccaaatga acaatctacc
875 aaaaatttca aataaagtca aaccccccac aaaaaaaaaa a 916 135 520 DNA
Homo sapiens CDS 2..433 sig_peptide 2..232 Von Heijne matrix score
4.40000009536743 seq FEARIALLPLLQA/ET polyA_signal 488..493
polyA_site 510..520 135 a atg gcg gcg tca aag gtg aag cag gac atg
cct ccr mcg ggg ggc tat 49 Met Ala Ala Ser Lys Val Lys Gln Asp Met
Pro Pro Xaa Gly Gly Tyr -75 -70 -65 ggg ccc atc gac tac aaa cgg aac
ttg ccg cgt cga gga ctg tcg ggc 97 Gly Pro Ile Asp Tyr Lys Arg Asn
Leu Pro Arg Arg Gly Leu Ser Gly -60 -55 -50 tac agc atg ctg gcc ata
ggg att gga acc ctg atc tac ggg cac tgg 145 Tyr Ser Met Leu Ala Ile
Gly Ile Gly Thr Leu Ile Tyr Gly His Trp -45 -40 -35 -30 agc ata atg
aag tgg aac cgt gag cgc agg cgc cta caa atc gag gac 193 Ser Ile Met
Lys Trp Asn Arg Glu Arg Arg Arg Leu Gln Ile Glu Asp -25 -20 -15 ttc
gag gct cgc atc gcg ctg ttg cca ctg tta cag gca gaa acc gac 241 Phe
Glu Ala Arg Ile Ala Leu Leu Pro Leu Leu Gln Ala Glu Thr Asp -10 -5
1 cgg agg acc ttg cag atg ctt cgg gag aac ctg gag gag gag gcc atc
289 Arg Arg Thr Leu Gln Met Leu Arg Glu Asn Leu Glu Glu Glu Ala Ile
5 10 15 atc atg aag gac gtg ccc gac tgg aag gtg ggg gak tct gtg tyc
cac 337 Ile Met Lys Asp Val Pro Asp Trp Lys Val Gly Xaa Ser Val Xaa
His 20 25 30 35 aca acc cgc tgg gtg ccc ccc ttg atc ggg gag ctg tac
ggg ctg cgc 385 Thr Thr Arg Trp Val Pro Pro Leu Ile Gly Glu Leu Tyr
Gly Leu Arg 40 45 50 acc aca aag gag gct ctc cat gcc agc cac ggc
ttc atg tgg tac acg 433 Thr Thr Lys Glu Ala Leu His Ala Ser His Gly
Phe Met Trp Tyr Thr 55 60 65 taggccctgt gccctccggc cacctggatc
cctgcccctc cccactgggg acggaataaa 493 tgctctgcag acctggaaaa aaaaaaa
520 136 568 DNA Homo sapiens CDS 34..363 sig_peptide 34..87 Von
Heijne matrix score 8.30000019073486 seq LLSLSSLPLVLLG/WE
polyA_signal 536..541 polyA_site 558..568 136 aaccagactt ctgacccctt
gggcaacagc cag atg gag act ggt cgc ctt ttg 54 Met Glu Thr Gly Arg
Leu Leu -15 agc ctc agc tct ctt cct ctt gtt ctc cta ggg tgg gag tac
agc agc 102 Ser Leu Ser Ser Leu Pro Leu Val Leu Leu Gly Trp Glu Tyr
Ser Ser -10 -5 1 5 caa acg ctg aac tta gtc cca tcc act tcc atc tta
tcc ttt gtg ccc 150 Gln Thr Leu Asn Leu Val Pro Ser Thr Ser Ile Leu
Ser Phe Val Pro 10 15 20 ttc atc ccc ctg cat ctt gtc ctt ttt gcc
ctc tgg tac ctc cca gtg 198 Phe Ile Pro Leu His Leu Val Leu Phe Ala
Leu Trp Tyr Leu Pro Val 25 30 35 ccc cat cat ctc tac ccc cag gga
ctc gga rat cat gca gca raa gca 246 Pro His His Leu Tyr Pro Gln Gly
Leu Gly Xaa His Ala Ala Xaa Ala 40 45 50 gaa raa ggc aaa cga raa
gaa gga gga acc caa kta gct ttg tgg ctt 294 Glu Xaa Gly Lys Arg Xaa
Glu Gly Gly Thr Gln Xaa Ala Leu Trp Leu 55 60 65 cgt gtc caa ccc
tct tgc cct tcg cct gtg tgc ctg gag cca gtc cca 342 Arg Val Gln Pro
Ser Cys Pro Ser Pro Val Cys Leu Glu Pro Val Pro 70 75 80 85 cca cgc
tcg cgt ttc ctc ctg tagtgctcac aggtcccagc accgatggca 393 Pro Arg
Ser Arg Phe Leu Leu 90 ttccctttgc cctgagtctg carcgggtcc cttttgtgct
tccttcccct caggtagcct 453 ctctccccct gggccactcc cgggggtgag
ggggtttacc ccttcccagt gttttttatt 513 cctgtggggc tcaccccaaa
gtattaaaag tagctttgta attcaaaaaa aaaaa 568 137 419 DNA Homo sapiens
CDS 50..286 sig_peptide 50..157 Von Heijne matrix score
4.80000019073486 seq VLLAIGMFFTAWF/FV polyA_signal 385..390
polyA_site 405..416 137 agacgtgttc ttccggtggc ggasggcgga ttagccttcg
cggggcaaa atg gag ctc 58 Met Glu Leu -35 gag gcc atg agc aga tat
acc agc cca gtg aac cca gct gtc ttc ccc 106 Glu Ala Met Ser Arg Tyr
Thr Ser Pro Val Asn Pro Ala Val Phe Pro -30 -25 -20 cat ctg acc gtg
gtg ctt ttg gcc att ggc atg ttc ttc acc gcc tgg 154 His Leu Thr Val
Val Leu Leu Ala Ile Gly Met Phe Phe Thr Ala Trp -15 -10 -5 ttc ttc
gtt tac gag gtc acc tct acc aag tac act cgt gat atc tat 202 Phe Phe
Val Tyr Glu Val Thr Ser Thr Lys Tyr Thr Arg Asp Ile Tyr 1 5 10 15
aaa gag ctc ctc atc tcc tta gtg gcc tca ctc ttc atg ggc ttt gga 250
Lys Glu Leu Leu Ile Ser Leu Val Ala Ser Leu Phe Met Gly Phe Gly 20
25 30 gtc ctc ttc ctg ctg ctc tgg gtt ggc atc tac gtg tgagcaccca
296 Val Leu Phe Leu Leu Leu Trp Val Gly Ile Tyr Val 35 40
agggtaacaa ccagatggct tcactgaaac ctgcttttgt aaattacttt tttttactgt
356 tgctggaagt gtcccacctg ctgctcataa taaatgcaga agtatagcaa
aaaaaaaaaa 416 ccc 419 138 1289 DNA Homo sapiens CDS 50..637
sig_peptide 50..151 Von Heijne matrix score 5.90000009536743 seq
LGAAALALLLANT/DV polyA_site 1277..1289 138 aatatacttc tttgtcaaga
gaagcagagg tgtggacgct gtgtatgaa atg tct ttc 58 Met Ser Phe ctc cag
gac cca agt ttc ttc acc atg ggg atg tgg tcc att ggt gca 106 Leu Gln
Asp Pro Ser Phe Phe Thr Met Gly Met Trp Ser Ile Gly Ala -30 -25 -20
gga gcc ctg ggg gct gct gcc ttg gca ttg ctg ctt gcc aac aca gac 154
Gly Ala Leu Gly Ala Ala Ala Leu Ala Leu Leu Leu Ala Asn Thr Asp -15
-10 -5 1 gtg ttt ctg tcc aag ccc cag aaa gcg gcc ctg gag tac ctg
gag gat 202 Val Phe Leu Ser Lys Pro Gln Lys Ala Ala Leu Glu Tyr Leu
Glu Asp 5 10 15 ata gac ctg aaa aca ctg gag aag gaa cca agg act ttc
aaa gca aag 250 Ile Asp Leu Lys Thr Leu Glu Lys Glu Pro Arg Thr Phe
Lys Ala Lys 20 25 30 gag cta tgg gaa aaa aat gga gct gtg att atg
gcc gtg cgg agg cca 298 Glu Leu Trp Glu Lys Asn Gly Ala Val Ile Met
Ala Val Arg Arg Pro 35 40 45 ggc tgt ttc ctc tgt cga gag gaa gct
gcg gat ctg tcc tcc ctg aaa 346 Gly Cys Phe Leu Cys Arg Glu Glu Ala
Ala Asp Leu Ser Ser Leu Lys 50 55 60 65 agc atg ttg gac cag ctg ggc
gtc ccc ctc tat gca gtg gta aag gas 394 Ser Met Leu Asp Gln Leu Gly
Val Pro Leu Tyr Ala Val Val Lys Xaa 70 75 80 cac atc rgg act gaa
ktg aag gat ttc cag cct tat ttc aaa gga gaa 442 His Ile Xaa Thr Glu
Xaa Lys Asp Phe Gln Pro Tyr Phe Lys Gly Glu 85 90 95 atc ttc ctg
gat gaa aar aaa aag ttc tat ggt cca caa agg cgg aag 490 Ile Phe Leu
Asp Glu Lys Lys Lys Phe Tyr Gly Pro Gln Arg Arg Lys 100 105 110 atg
atg ttt atg gga ttt atc cgt ctg gga atg tgg tac aac ttc ttc 538 Met
Met Phe Met Gly Phe Ile Arg Leu Gly Met Trp Tyr Asn Phe Phe 115 120
125 cga rcc tgg aac gga rgc ttc tct gga aac ctg gaa gga raa ggc ttc
586 Arg Xaa Trp Asn Gly Xaa Phe Ser Gly Asn Leu Glu Gly Xaa Gly Phe
130 135 140 145 atc ctt ggg gga att ttc gtg gtg gga tca asg aaa gca
ggg cat tct 634 Ile Leu Gly Gly Ile Phe Val Val Gly Ser Xaa Lys Ala
Gly His Ser 150 155 160 tct tgarcmccga gaaaaagaat ttggagacaa
agtaaaccta ctttctgttc 687 Ser tggaagctgc taagatgatc aaaccacaga
ctttggcctc agagaaaaaa tgattgtgtg 747 aaactgccca gctcagggat
aaccagggac attcacctgt gttcatggga tgtattgttt 807 ccactcgtgt
ccctaaggag tgagaaaccc atttatactc tactctcagt atggattatt 867
aatgtatttt aatattctgt ttaggcccac taaggcaaaa tasccccaaa acaagactga
927 caaaaatctg aaaaactaat gaggattatt aagctaaaac ctgggaaata
ggaggcttaa 987 aattgactgc caggctgggt gcagtggctc acacctgtaa
tcccagcact ttgggaggcc 1047 aaggtgagca agtcacttga ggtcgggagt
tcgagaccag cctgagcaac atggcgaaac 1107 cccgtctcta ckaaaaatac
araaatcacc cgggtgtggt ggcaggcacc tgtagtccca 1167 gctacccggg
aggctgaggc aggagaatca cttgaacctg ggaggtggag gttgcggtga 1227
gctgagatca caccactgta ttccagcctg ggtgactgag actctaacca aaaaaaaaaa
1287 aa 1289 139 715 DNA Homo sapiens CDS 72..602 sig_peptide
72..125 Von Heijne matrix score 5.59999990463257 seq
LTPLFFMFPTGFS/SP polyA_site 704..715 139 acttcccttc cccctctagc
attgctacct tctctcctac acgcacgcag gcatataaac 60 gtaggttttt g atg ctc
ctc tgc ctg ttg acc ccg cta ttt ttc atg ttt 110 Met Leu Leu Cys Leu
Leu Thr Pro Leu Phe Phe Met Phe -15 -10 cca aca ggt ttt tct tcc ccc
agt ccc tca gct gct gct gct gct cag 158 Pro Thr Gly Phe Ser Ser Pro
Ser Pro Ser Ala Ala Ala Ala Ala Gln -5 1 5 10 gag gtc aga tct gcc
act gat ggt aat acc agc acc act ccg ccc acc 206 Glu Val Arg Ser Ala
Thr Asp Gly Asn Thr Ser Thr Thr Pro Pro Thr 15 20 25 tct gcc aar
aar aka aag tta aac agc agc agc agt agc agc agt aac 254 Ser Ala Lys
Lys Xaa Lys Leu Asn Ser Ser Ser Ser Ser Ser Ser Asn 30 35 40 agt
agt aac gag aga gaa gac ttt gat tcs acc tct tcc tcc tct tcc 302 Ser
Ser Asn Glu Arg Glu Asp Phe Asp Ser Thr Ser Ser Ser Ser Ser 45 50
55 act cct cct tta caa ccc agg gat tcg gca tcc cct tca acc tcg tcc
350 Thr Pro Pro Leu Gln Pro Arg Asp Ser Ala Ser Pro Ser Thr Ser Ser
60 65 70 75 ttc tgc ctg ggg gtt tca gtg gct gct tcc agc cac gta ccg
ata swg 398 Phe Cys Leu Gly Val Ser Val Ala Ala Ser Ser His Val Pro
Ile Xaa 80 85 90 aar aag ctg cgt ttt gaa rac acc ctg gag ttt gta
ggg ttt gat gcg 446 Lys Lys Leu Arg Phe Glu Xaa Thr Leu Glu Phe Val
Gly Phe Asp Ala 95 100 105 aar atg gct gar gaa tcc tcc tcc tcc tcc
tcc tca tct tca cca ack 494 Lys Met Ala Glu Glu Ser Ser Ser Ser Ser
Ser Ser Ser Ser Pro Thr 110 115 120 gct gca aca tct cag cag cag caa
ctt aaa aat aag agt ata ttg aat 542 Ala Ala Thr Ser Gln Gln Gln Gln
Leu Lys Asn Lys Ser Ile Leu Asn 125 130 135 ctc ttc tgt ggc ttc ggt
gca tca tgc aaa cgg cct agc caa atc ttc 590 Leu Phe Cys Gly Phe Gly
Ala Ser Cys Lys Arg Pro Ser Gln Ile Phe 140 145 150 155 tac cac cgt
ctc tagctttgct aacagcaaac ctggctctgc taagaagtta 642 Tyr His Arg Leu
gtgatcaaga actttaaaga taagcctaaa ttaccagaaa actacacaga tgaaacctgg
702 caaaaaaaaa aaa 715 140 931 DNA Homo sapiens CDS 120..434
sig_peptide 120..185 Von Heijne matrix score 6.30000019073486 seq
FALVWLWLRSTGC/FW polyA_signal 899..904 polyA_site 918..931 140
aatttccggc gacacctcgc agtcattcct gcggcttgcg cgcccttgta gacagccggg
60 gccttcgtga gaccggtgca ggcctggggt agtctcctgt ctggacagag aagagaaaa
119 atg cag gga cac tgg ctc agt agt gcc ttt gca ttg gtt tgg ctt tgg
167 Met Gln Gly His Trp Leu Ser Ser Ala Phe Ala Leu Val Trp Leu Trp
-20 -15 -10 cta cgc agc act ggt tgc ttc tgg tgg gat cat tgg cta tgt
aaa agc 215 Leu Arg Ser Thr Gly Cys Phe Trp Trp Asp His Trp Leu Cys
Lys Ser -5 1 5 10 agg cag cgt gcc gtc cct ggc tgc agg gct gct ctt
tgg cag tct agc 263 Arg Gln Arg Ala Val Pro Gly Cys Arg Ala Ala Leu
Trp Gln Ser Ser 15 20 25 cgg cct ggg tgc tta cca gct gtc tca gga
tcc aag gaa cgt ttg ggt 311 Arg Pro Gly Cys Leu Pro Ala Val Ser Gly
Ser Lys Glu Arg Leu Gly 30 35 40 ttt cct agc tac atc tgg tac ctt
ggc tgg cat tat ggg aat gag gtt 359 Phe Pro Ser Tyr Ile Trp Tyr Leu
Gly Trp His Tyr Gly Asn Glu Val 45 50 55 cta cca ctc tgg aaa att
cat gcc tgc agg ttt aat tgc agg tgc cag 407 Leu Pro Leu Trp Lys Ile
His Ala Cys Arg Phe Asn Cys Arg Cys Gln 60
65 70 ttt gct gat ggt cgc caa agt tgg agt tagtatgtkc aacagacccc 454
Phe Ala Asp Gly Arg Gln Ser Trp Ser 75 80 attagcagaa gtcatgttcc
agcttagatg atgaaraatt aaaaatctgc atcttccact 514 attttcaata
tattaagaga aataagtgca gcatttttgc atctgacatt ttacctaaaa 574
aaaaaaacmc caaacttggc aaaaaggtgg aaaatcagtc atgattacaa acctacagag
634 gtggcgagta tgtaacacaa gagcttaata agaccctcat agagcttgat
tcttgtatat 694 tgatgttgtc ttttctttct gtatctgtag gtaaatctca
agggtaaaat gttaggtgtc 754 agctttcagg gctctgaaac cchattccct
gctctgagga acagtgtgaa aaaaagtctt 814 ttaggaratt tacaatatct
gttcttttgc tcatcttara ccacagactg actttgaaat 874 takgttaagt
gaaatatcaa tgaaaataaa gtttactata aataawaaaa aaaaaaa 931 141 891 DNA
Homo sapiens CDS 4..447 sig_peptide 4..147 Von Heijne matrix score
5.69999980926514 seq LLLFFGKLLVVGG/VG polyA_signal 858..863
polyA_site 880..891 141 atc atg atc gcc atc tac ggg aag aat ttc tgt
gtc tca gcc aaa aat 48 Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val
Ser Ala Lys Asn -45 -40 -35 gcg ttc atg cta ctc atg cga aac att gtc
agg gtg gtc gtc ctg gac 96 Ala Phe Met Leu Leu Met Arg Asn Ile Val
Arg Val Val Val Leu Asp -30 -25 -20 aaa gtc aca gac ctg ctg ctg ttc
ttt ggg aag ctg ctg gtg gtc gga 144 Lys Val Thr Asp Leu Leu Leu Phe
Phe Gly Lys Leu Leu Val Val Gly -15 -10 -5 ggc gtg ggg gtc ctg tcc
ttc ttt ttt ttc tcc ggt cgc atc ccg ggg 192 Gly Val Gly Val Leu Ser
Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 1 5 10 15 ctg ggt aaa gac
ttt aag agc ccc cac ctc aac tat tac tgg ctg ccc 240 Leu Gly Lys Asp
Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu Pro 20 25 30 ayc atg
acc tcc atc ctg ggg gcc tat gtc atc gcc agy ggc ttc ttc 288 Xaa Met
Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe Phe 35 40 45
agc gtt ttc ggc atg tgt gtg gac acg ctc ttc ctc tgc ttc ctg gaa 336
Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu 50
55 60 gac ctg gag cgg aca acg gct ccc tgg acg gcc cta cta cat gtc
caa 384 Asp Leu Glu Arg Thr Thr Ala Pro Trp Thr Ala Leu Leu His Val
Gln 65 70 75 gag ctt cta aag att ctg ggc aag aag aac gag gcg ccc
ccg gac aac 432 Glu Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro
Pro Asp Asn 80 85 90 95 aag aaa agg aaa aak tgacagctcc ggccctgatc
caggactgca ccccaccccc 487 Lys Lys Arg Lys Xaa 100 accgtccagc
catccaacct cacttcgcct tacaggtctc cattttgtgg taaaaaaagg 547
ttttaggcca ggcgccgtgg ctcacgcctg twatccaaca ctttgaragg ctgaggcggg
607 cggatcacct kaktcaggak tycgagacca kcctggccaa catggtgaaa
cctccgtctc 667 tattaaaaat acaaaaatta gccgagagtg gtggcatgca
cctgtcatcc cagctactcg 727 ggaggctgag gcaggagaat cgcttgaacc
cgggaggcag aggttgcagt gagccgagat 787 cgcgccactg cactccaacc
tgggtgacag actctgtctc caaaacaaaa caaacaaaca 847 aaaagatttt
attaaagata ttttgttaac tcaraaaaaa aaaa 891 142 817 DNA Homo sapiens
CDS 28..804 sig_peptide 28..96 Von Heijne matrix score 10 seq
PLLGLLLSLPAGA/DV polyA_site 806..817 142 aaccgagctg gatttgtatg
ttgcacc atg cct tct tgg atc ggg gct gtg att 54 Met Pro Ser Trp Ile
Gly Ala Val Ile -20 -15 ctt ccc ctc ttg ggg ctg ctg ctc tcc ctc ccc
gcc ggg gcg gat gtg 102 Leu Pro Leu Leu Gly Leu Leu Leu Ser Leu Pro
Ala Gly Ala Asp Val -10 -5 1 aag gct cgg agc tgc gga gag gtc cgc
cag gcg tac ggt gcc aag gga 150 Lys Ala Arg Ser Cys Gly Glu Val Arg
Gln Ala Tyr Gly Ala Lys Gly 5 10 15 ttc agc ctg gcg gac atc ccc tac
cag gag atc gca kgg gaa cac tta 198 Phe Ser Leu Ala Asp Ile Pro Tyr
Gln Glu Ile Ala Xaa Glu His Leu 20 25 30 aga atc tgt cct cag gaa
tat aca tgc tgc acc aca gaa atg gar gac 246 Arg Ile Cys Pro Gln Glu
Tyr Thr Cys Cys Thr Thr Glu Met Glu Asp 35 40 45 50 aag tta agc caa
caa agc aaa ctc gaa ttt gaa aac ctt gtg gaa gag 294 Lys Leu Ser Gln
Gln Ser Lys Leu Glu Phe Glu Asn Leu Val Glu Glu 55 60 65 aca agc
cat ttt gtg cgc acc act ttt gtg tcc agg cat aag aaa ttt 342 Thr Ser
His Phe Val Arg Thr Thr Phe Val Ser Arg His Lys Lys Phe 70 75 80
gac gaw ttt ttc cga rag ctc ckg gag aat gca raa aag tca cta aat 390
Asp Xaa Phe Phe Arg Xaa Leu Xaa Glu Asn Ala Xaa Lys Ser Leu Asn 85
90 95 gat rtg ttt gtm cgg acc tat ggc atg ctg tac wtg car aat kca
gaa 438 Asp Xaa Phe Val Arg Thr Tyr Gly Met Leu Tyr Xaa Gln Asn Xaa
Glu 100 105 110 gtc ttc crg gac ctc ttc aca rag ctg aaa agg tac tac
act ggg ggt 486 Val Phe Xaa Asp Leu Phe Thr Xaa Leu Lys Arg Tyr Tyr
Thr Gly Gly 115 120 125 130 aat gtg aat ctg gag gaa atg ctc aat gac
ttt tgg gct cgg ctc ctg 534 Asn Val Asn Leu Glu Glu Met Leu Asn Asp
Phe Trp Ala Arg Leu Leu 135 140 145 gaa cgg atg ttt cag cwr awa aac
cct cag tat cac ttc agt gaa gac 582 Glu Arg Met Phe Gln Xaa Xaa Asn
Pro Gln Tyr His Phe Ser Glu Asp 150 155 160 tac ctg gaa tgt gtg agc
aaa tac act gac cak ctc aag cca ttt gga 630 Tyr Leu Glu Cys Val Ser
Lys Tyr Thr Asp Xaa Leu Lys Pro Phe Gly 165 170 175 gac gtg ccc cgg
aaa ctg aag att cag gtk acc cgc gcc ttc atk gsk 678 Asp Val Pro Arg
Lys Leu Lys Ile Gln Val Thr Arg Ala Phe Xaa Xaa 180 185 190 gcc agg
acc ttt gtc cag ggg ctg act gtg ggc aga gaa gtt gca aac 726 Ala Arg
Thr Phe Val Gln Gly Leu Thr Val Gly Arg Glu Val Ala Asn 195 200 205
210 cga gtt tcc aag gta att gaa aac gtg ctt tct ttc tca ttg gtg ttc
774 Arg Val Ser Lys Val Ile Glu Asn Val Leu Ser Phe Ser Leu Val Phe
215 220 225 ctt gtt tat tct gtt ttt aaa acc aat gtt taaaaaaaaa aaa
817 Leu Val Tyr Ser Val Phe Lys Thr Asn Val 230 235 143 1020 DNA
Homo sapiens CDS 27..359 sig_peptide 27..212 Von Heijne matrix
score 3.59999990463257 seq SWLSLLAALAHLA/AA polyA_signal 988..993
polyA_site 1009..1020 143 agtgggtcga kctggggcgc agtcgc atg ggg gag
tct atc ccg ctg gcc gcc 53 Met Gly Glu Ser Ile Pro Leu Ala Ala -60
-55 ccg gtc ccg gtg gaa cag gcg gtg ctg gag acg ttc ttc tct cac ctg
101 Pro Val Pro Val Glu Gln Ala Val Leu Glu Thr Phe Phe Ser His Leu
-50 -45 -40 ggt atc ttc tct tac gac aag gct aag gac aat gtg gag aag
gaa cga 149 Gly Ile Phe Ser Tyr Asp Lys Ala Lys Asp Asn Val Glu Lys
Glu Arg -35 -30 -25 gag gcc aac aag agc gcg ggg ggc agc tgg ctg tcg
ctg ctg gcg gcc 197 Glu Ala Asn Lys Ser Ala Gly Gly Ser Trp Leu Ser
Leu Leu Ala Ala -20 -15 -10 ttg gcg cac ctg gcc gcg gcc gag aag gtc
tat cac agc ctc acc tac 245 Leu Ala His Leu Ala Ala Ala Glu Lys Val
Tyr His Ser Leu Thr Tyr -5 1 5 10 ctg ggg cag aaa cta ggt acc tcc
gcc ccg ccc ccc gag ccc ctt gag 293 Leu Gly Gln Lys Leu Gly Thr Ser
Ala Pro Pro Pro Glu Pro Leu Glu 15 20 25 gag gaa gta aag ggg gta
tat tcc cca dtc ggc agt ggc ttg ggt btc 341 Glu Glu Val Lys Gly Val
Tyr Ser Pro Xaa Gly Ser Gly Leu Gly Xaa 30 35 40 ccg tct ctg tgt
cac ttc tagtcgcagg ctcgactcgg cattcccaga 389 Pro Ser Leu Cys His
Phe 45 tctcctccca ccgttccttt ccttccctgg gcttccacaa gccccgccca
ccrgcctgcr 449 ctgctgatag attggcgaac tgggtagatg ctctttgcaa
ggctgtgacc caaaccgaaw 509 ggtttgccct tttgcctcgt gcatggattg
atgccataaa tgagaagtta accaaaaaaa 569 aaaaacmcwd tycckktttm
ccccccccgg grmcagaaga gcaaaacttt gcaaaacaac 629 ctagttctat
tactgaacac tgttgtgtgg cctcttaagg ttaaggcccg agagtcacat 689
ttagagtcct accccgtctt catagtcccc caatacatat ttaatgacta aagtwataaa
749 tgaatattgg gcaggaaagg caagaaatat gcctaacact agcaagaaga
gacttaaggg 809 gaaaatggta aacactctta gcacttcatg tacatcttgc
ctctgaaata agattcaaga 869 gctgattcaa ctgattttta ctagtagaag
caataagtat aagtagatga gaaggaaata 929 atagatgtaa aaggcatgga
atatgcatac aaaataatat tactgcttaa ttatgacaaa 989 taaatatatt
ttgaatccta aaaaaaaaaa a 1020 144 1399 DNA Homo sapiens CDS 25..957
sig_peptide 25..93 Von Heijne matrix score 4.09999990463257 seq
LEAFSQAISAIQA/LR polyA_signal 1368..1373 polyA_site 1388..1399 144
aakagctgct gtggcggcgg caac atg gcg gac gtg ata aat gtc agt gtg 51
Met Ala Asp Val Ile Asn Val Ser Val -20 -15 aac ctg gag gcc ttt tcc
cag gcc att agt gcc atc cag gcg ctg cga 99 Asn Leu Glu Ala Phe Ser
Gln Ala Ile Ser Ala Ile Gln Ala Leu Arg -10 -5 1 tcc agc gtg agc
agg gtg ttc gac tgc ctg aag gat ggg atg cgg aac 147 Ser Ser Val Ser
Arg Val Phe Asp Cys Leu Lys Asp Gly Met Arg Asn 5 10 15 aag gag acg
ctg gag ggc cgg gag aag gcc ttt att gcg cac ttc cag 195 Lys Glu Thr
Leu Glu Gly Arg Glu Lys Ala Phe Ile Ala His Phe Gln 20 25 30 gac
aac tta cat tcg gtc aac cgg gac ctc aat gag ctg gaa cgt ctg 243 Asp
Asn Leu His Ser Val Asn Arg Asp Leu Asn Glu Leu Glu Arg Leu 35 40
45 50 agc aat ctg gta ggc arg cca tct gar aac cat cct ctt cat aac
agt 291 Ser Asn Leu Val Gly Xaa Pro Ser Glu Asn His Pro Leu His Asn
Ser 55 60 65 ggg ctg tta asc ctg gat cct gtg car gac aaa act cct
ctc tat agt 339 Gly Leu Leu Xaa Leu Asp Pro Val Gln Asp Lys Thr Pro
Leu Tyr Ser 70 75 80 caa ctc ctt caa gca tat aag tgg tca aac aag
ttg cag tac cat gca 387 Gln Leu Leu Gln Ala Tyr Lys Trp Ser Asn Lys
Leu Gln Tyr His Ala 85 90 95 gga cta gca tct ggc ctt tta aat cas
car tca ktg aag cgt ycc gct 435 Gly Leu Ala Ser Gly Leu Leu Asn Xaa
Gln Ser Xaa Lys Arg Xaa Ala 100 105 110 aat cag atg gga gta tct gcc
aaa cgt aga cca aag gct cag ccc aca 483 Asn Gln Met Gly Val Ser Ala
Lys Arg Arg Pro Lys Ala Gln Pro Thr 115 120 125 130 act ctt gtc cta
cca cct caa tat gtt gat gat gtg atc agc cgc att 531 Thr Leu Val Leu
Pro Pro Gln Tyr Val Asp Asp Val Ile Ser Arg Ile 135 140 145 gac agg
atg ttt cct gaa atg tcc atc cac tta tcc aga ccc aat gga 579 Asp Arg
Met Phe Pro Glu Met Ser Ile His Leu Ser Arg Pro Asn Gly 150 155 160
aca tca gca atg ctt ctg gtg acc ttg gga aar gtg ttg aaa gtg awc 627
Thr Ser Ala Met Leu Leu Val Thr Leu Gly Lys Val Leu Lys Val Xaa 165
170 175 gtc gtc rtr cgg arm ctg ttc att gat cga aca ata gtw aag gga
tat 675 Val Val Xaa Arg Xaa Leu Phe Ile Asp Arg Thr Ile Val Lys Gly
Tyr 180 185 190 wac gag aat gtc tac rca gaa kat ggc mag ctt gat ata
tgg tcc aaa 723 Xaa Glu Asn Val Tyr Xaa Glu Xaa Gly Xaa Leu Asp Ile
Trp Ser Lys 195 200 205 210 tcc aac tat caa gta ttc cag aag gtg aca
gac cat gcc acc act gcc 771 Ser Asn Tyr Gln Val Phe Gln Lys Val Thr
Asp His Ala Thr Thr Ala 215 220 225 ctg ctc cac taw mag ctg ccc cag
atg ccg gat gtc gtg gtc cga tcc 819 Leu Leu His Xaa Xaa Leu Pro Gln
Met Pro Asp Val Val Val Arg Ser 230 235 240 ttc awg acc tgg tta aga
agt tac ata aag ctg ttc cag gcc ccg tgc 867 Phe Xaa Thr Trp Leu Arg
Ser Tyr Ile Lys Leu Phe Gln Ala Pro Cys 245 250 255 cag cgc tgc ggg
aag ttt ctg cag gac ggc ctt ccc ccg aca tgg agg 915 Gln Arg Cys Gly
Lys Phe Leu Gln Asp Gly Leu Pro Pro Thr Trp Arg 260 265 270 gat ttc
cga acc ctc gaa gcc ttc cat gac acc tgc cgg cag 957 Asp Phe Arg Thr
Leu Glu Ala Phe His Asp Thr Cys Arg Gln 275 280 285 tagcccccac
gctggcccca gcctcagacc ccacccagca ccttcccaga cacgcaggaa 1017
gcccacagaa ggctcagctg gttcctcact gcccagatgt gtacagctgc tcctcccttt
1077 cataaagcag cgccatgtgt gcagaggcca ctcttgaaga gcagactccc
tctgtggctg 1137 atgggactaa ttattcccac tagccagcgg actgaaggca
aagaagacct ttctagaacc 1197 tggtagaagg aagctgtgca gcatgctcct
cgtccatgtg tgtcggcagt gctggtgtct 1257 gtcgtctccg cgagctgtta
ctggaatgag cccttgtgtt catgggtatc gtcatgcggg 1317 gttcttgtgt
tttgtggggc ttgggttttg gttaacttat ttttataagc aataaacctt 1377
ttgtatcctg aaaaaaaaaa aa 1399 145 666 DNA Homo sapiens CDS 47..319
sig_peptide 47..226 Von Heijne matrix score 3.90000009536743 seq
SSLVPFFLFTCFG/HF polyA_site 656..666 145 acttttgcct agcatttgac
tttggtgttt taagttctgt agttcc atg aca tca 55 Met Thr Ser -60 ttg ttt
gct gtt gtg tta cag aga gag aag gaa cct cac ctg tgg ctc 103 Leu Phe
Ala Val Val Leu Gln Arg Glu Lys Glu Pro His Leu Trp Leu -55 -50 -45
agc tca ccc cac atc cgt ttc tca tta cgt gta aat aaa ctg tca gag 151
Ser Ser Pro His Ile Arg Phe Ser Leu Arg Val Asn Lys Leu Ser Glu -40
-35 -30 ctg atg tta cag ctt tta cag ttt aaa gca ttc ccc tcg tct cta
gtt 199 Leu Met Leu Gln Leu Leu Gln Phe Lys Ala Phe Pro Ser Ser Leu
Val -25 -20 -15 -10 cct ttt ttc ttg ttt aca tgt ttt ggg cac ttt ccc
tca ttc acc acc 247 Pro Phe Phe Leu Phe Thr Cys Phe Gly His Phe Pro
Ser Phe Thr Thr -5 1 5 ttc cag ggc ttc ata gaa aat aac ttg tta caa
aat cag ttc aat tct 295 Phe Gln Gly Phe Ile Glu Asn Asn Leu Leu Gln
Asn Gln Phe Asn Ser 10 15 20 aat gtg gac ata gtg gca tgt tca
taattagacc catatagggg acactgagct 349 Asn Val Asp Ile Val Ala Cys
Ser 25 30 ttaaatcgtt gattctaaac tctatacatt aaaaaaattc agcccaggcc
cctcaaagcc 409 tgaraaaatt taatttgctc ttaatttaat gttccaaaac
tcactcttgg aaaaatgcct 469 gttggaaaac tacaggtggg tcacatgtkg
gggctgtctc cgtgacactc aggattccag 529 tcaraaccta atcctcatat
ctattgccta caaaaataga ccaagaatgt tgctgctctt 589 ttataatcct
ttaaatattt aacattcaag ttttctttgt cttaaattca gcctcttcct 649
aaaagcaaaa aaaaaaa 666 146 1131 DNA Homo sapiens CDS 80..940
sig_peptide 80..130 Von Heijne matrix score 3.70000004768372 seq
RIVSAALLAFVQT/HL polyA_signal 1101..1106 polyA_site 1119..1130 146
agttggtggg gctgggggat gagagctgca ccgcgcggga yaagtcgccg gcggcgcccg
60 amggagcaga acagagagc atg gag ctg gag agg atc gtc agt gca gcc ctc
112 Met Glu Leu Glu Arg Ile Val Ser Ala Ala Leu -15 -10 ctt gcc ttt
gtc cag aca cac ctc ccg gag gcc gac ctc agt ggc ttg 160 Leu Ala Phe
Val Gln Thr His Leu Pro Glu Ala Asp Leu Ser Gly Leu -5 1 5 10 gat
gag gtc atc ttc tcc tat gtg ctt ggg gtc ctg gag gac ctg ggc 208 Asp
Glu Val Ile Phe Ser Tyr Val Leu Gly Val Leu Glu Asp Leu Gly 15 20
25 ccc tcg ggc cca tca gag gag aac ttc gat atg gag gct ttc act gag
256 Pro Ser Gly Pro Ser Glu Glu Asn Phe Asp Met Glu Ala Phe Thr Glu
30 35 40 atg atg gag gcc tat gtg cct ggc ttc gcc cac atc ccc agg
ggc aca 304 Met Met Glu Ala Tyr Val Pro Gly Phe Ala His Ile Pro Arg
Gly Thr 45 50 55 ata ggg gac atg atg cag aar ctc tca ggg cag ctg
agc gat gcc vgg 352 Ile Gly Asp Met Met Gln Lys Leu Ser Gly Gln Leu
Ser Asp Ala Xaa 60 65 70 aac aaa gag aac ctg caa ccg cag aac tct
ggt gtc caa ggt cag gtg 400 Asn Lys Glu Asn Leu Gln Pro Gln Asn Ser
Gly Val Gln Gly Gln Val 75 80 85 90 ccc atc tcc cca gag ccc ctg cag
cgg ccc gaa atg ctc aaa gaa gag 448 Pro Ile Ser Pro Glu Pro Leu Gln
Arg Pro Glu Met Leu Lys Glu Glu 95 100 105 act agg tct tcg gct gct
gct gct gca gac acc caa gat gag gca act 496 Thr Arg Ser Ser Ala Ala
Ala Ala Ala Asp Thr Gln Asp Glu Ala Thr 110 115 120 ggc gct gag gag
gag ctt ctg cca ggg gtg gat gta ctc ctg gag gtg 544 Gly Ala Glu Glu
Glu Leu Leu Pro Gly Val Asp Val Leu Leu Glu Val 125 130 135 ttc cct
acc tgt tcg gtg gag cag gcc cag tgg gtg ctg gcc aaa gct 592 Phe Pro
Thr Cys Ser Val Glu Gln Ala Gln Trp Val Leu Ala Lys Ala 140 145 150
cgg ggg gac ttg gaa gaa gct gtg cag atg ctg gta gag gga aag gaa 640
Arg Gly Asp Leu Glu Glu Ala Val Gln Met Leu Val Glu Gly Lys Glu 155
160 165 170 gag ggg cct gca gcc tgg gag ggc ccc aac cag gac ctg ccc
aga cgc 688 Glu Gly Pro Ala Ala Trp Glu Gly Pro Asn Gln Asp Leu Pro
Arg Arg
175 180 185 ctc aga ggc ccc caa aag gat gag ctg aag tcc ttc atc ctg
cag aag 736 Leu Arg Gly Pro Gln Lys Asp Glu Leu Lys Ser Phe Ile Leu
Gln Lys 190 195 200 tac atg atg gtg gat agc gca gag gat cag aag att
cac cgg ccc atg 784 Tyr Met Met Val Asp Ser Ala Glu Asp Gln Lys Ile
His Arg Pro Met 205 210 215 gct ccc aag gag gcc ccc aag aag ctg atc
cga tac atc gac aac cag 832 Ala Pro Lys Glu Ala Pro Lys Lys Leu Ile
Arg Tyr Ile Asp Asn Gln 220 225 230 gta gtg agc acc aaa ggg gag cga
ttc aaa gat gtg cgg aac cct gag 880 Val Val Ser Thr Lys Gly Glu Arg
Phe Lys Asp Val Arg Asn Pro Glu 235 240 245 250 gcc gag gag atg aag
gcc aca tac atc aac ctc aag cca gcc aga aag 928 Ala Glu Glu Met Lys
Ala Thr Tyr Ile Asn Leu Lys Pro Ala Arg Lys 255 260 265 tac cgc ttc
cat tgaggcactc gccggactct gcccgagcct tctaggctca 980 Tyr Arg Phe His
270 gatcccagag ggatgcagga gccctatacc cctacacagg ggccccctaa
ctcctgtccc 1040 ccttctctac tcctttgctc catagtgtta acctactctc
ggagctgcct ccatgggcac 1100 agtaaaggtg gcccaaggaa aaaaaaaaaw t 1131
147 475 DNA Homo sapiens CDS 146..457 sig_peptide 146..292 Von
Heijne matrix score 5.19999980926514 seq CFLCLYPIPLCTS/HP
polyA_signal 442..447 polyA_site 465..475 147 attgtaacaa acagtaccaa
tttattttgg ccgtgggttt ttgctttttt tccagttgat 60 gactttgtga
acattcccag gtattggagc ctctgtggcc ttaaatgtgg ctcagtggag 120
ggagacccag catagccagg ccagt atg gag cac ctc acg cac agc tct cag 172
Met Glu His Leu Thr His Ser Ser Gln -45 aag ctg cag gcg gac gaa cat
ctg acc aaa gag gtg tgg tcg agg ctc 220 Lys Leu Gln Ala Asp Glu His
Leu Thr Lys Glu Val Trp Ser Arg Leu -40 -35 -30 -25 ctg aaa gag aaa
ggg cct gct ggt ctc atc ctc tgc ttc ctt tgc ctt 268 Leu Lys Glu Lys
Gly Pro Ala Gly Leu Ile Leu Cys Phe Leu Cys Leu -20 -15 -10 tac cct
ata cct ctc tgc acg tcc cac ccc gtt tkg ctg tgt gcy cac 316 Tyr Pro
Ile Pro Leu Cys Thr Ser His Pro Val Xaa Leu Cys Ala His -5 1 5 ccc
cag gat gtg tac ccg gtt gta gta aga gct gaa atc cat gct gag 364 Pro
Gln Asp Val Tyr Pro Val Val Val Arg Ala Glu Ile His Ala Glu 10 15
20 ctg tac cag gaa ctt gca tat cta aaa aca gaa act gag tca ctg gcc
412 Leu Tyr Gln Glu Leu Ala Tyr Leu Lys Thr Glu Thr Glu Ser Leu Ala
25 30 35 40 cat ctc ttt gct ctt gtg ccc cag gcc aaa ata aag aat aga
gtg 457 His Leu Phe Ala Leu Val Pro Gln Ala Lys Ile Lys Asn Arg Val
45 50 55 taragtgaaa aaaaaaaa 475 148 949 DNA Homo sapiens CDS
100..351 sig_peptide 100..207 Von Heijne matrix score
4.19999980926514 seq CLAVSWEAAGCHG/AG polyA_site 940..949
misc_feature 745 n=a, g, c or t 148 aaaggaatac tgacagataa
ggccggaaac aaaactgatg gcttgaaaaa catttttatg 60 gaatgtattt
actatcattt tgttttacta tagaggtag atg gga ctc tta act 114 Met Gly Leu
Leu Thr -35 ttt ggg tac att gaa amc akg ckg aaa act gaa cac aat cct
gat cat 162 Phe Gly Tyr Ile Glu Xaa Xaa Xaa Lys Thr Glu His Asn Pro
Asp His -30 -25 -20 cac tcc tgc ctg gct gtc tcc tgg gag gct gcc ggg
tgc cac gga gct 210 His Ser Cys Leu Ala Val Ser Trp Glu Ala Ala Gly
Cys His Gly Ala -15 -10 -5 1 ggg aca cag cag agc ccg cta ggt gtt
gca ggg ccc tgg agg cca agg 258 Gly Thr Gln Gln Ser Pro Leu Gly Val
Ala Gly Pro Trp Arg Pro Arg 5 10 15 cca ccc tgt gtg ggg tcc ctg ttg
gca gcc agg tcc cta cac aaa caa 306 Pro Pro Cys Val Gly Ser Leu Leu
Ala Ala Arg Ser Leu His Lys Gln 20 25 30 gta atc ctg ttt ggc ctc
cta ggt ttt gca tat gac cac gca gcc 351 Val Ile Leu Phe Gly Leu Leu
Gly Phe Ala Tyr Asp His Ala Ala 35 40 45 taatttgggg tgtaggggaa
cctctgctgg cccttgctcc tttgtatgtt gggtgacttt 411 aatggctggc
cacatacccc tttctcccag ctactcattc actgacttgg gtaagttcta 471
gcacaatgcg cacttagaaa cagaatgtga cacatcaaca ttaacttttc ctgaaaagaa
531 cagtttgcct aacatggacc cmaaagaagc ttggaattta taagactttc
ctttataaga 591 tatagtgggg gtttttttgg gtggaggggg gttgtttttt
gttttttgtt ttcaagacag 651 agtctcgctc agttgtccag gctggartgt
aktggcatga tctcggctca ctgcarcctc 711 tgcctcccag gttcatgcca
ttctcctgcc tcancctccc gagtagctgg gactacaggt 771 gtctgccgcc
acgcctggct aatttttttg tatttttagt agagacgggg tttcaccatg 831
ttggtcagga tggtctcgat ttcctgacct cgtgatccgc ctgtctcggc ctcccaaagt
891 gctgggatta caggcgtgag ccaccacgcc tggcctataa gatacggyaa aaaaaaaa
949 149 940 DNA Homo sapiens CDS 177..569 sig_peptide 177..236 Von
Heijne matrix score 11.1999998092651 seq AFLLLVALSYTLA/RD
polyA_site 931..939 misc_feature 482 n=a, g, c or t 149 agaagataat
cacttgggga aaggaaggtt cgtttctgag ttagcaacaa gtaaatgcag 60
cactagtggg tgggattgag gtatgccctg gtgcataaat agagactcag ctgtgctggc
120 acactcagaa gcttggaccg catcctagcc gccgactcac acaaggcaga gttgcc
atg 179 Met -20 gaa aaa att cca gtg tca gca ttc ttg ctc ctt gtg gcc
ctc tcc tac 227 Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala
Leu Ser Tyr -15 -10 -5 act ctg gcc aga gat acc aca gtc aaa cct gga
gcc aaa aag gac aca 275 Thr Leu Ala Arg Asp Thr Thr Val Lys Pro Gly
Ala Lys Lys Asp Thr 1 5 10 aag gac tct cga ccc aaa ctg ccc cag acc
ctc tcc aga ggt tgg ggt 323 Lys Asp Ser Arg Pro Lys Leu Pro Gln Thr
Leu Ser Arg Gly Trp Gly 15 20 25 gac caa ctc atc tgg aca car aca
tat gaa raa rct cta twt aaa tcc 371 Asp Gln Leu Ile Trp Thr Gln Thr
Tyr Glu Xaa Xaa Leu Xaa Lys Ser 30 35 40 45 aar aca agc aac aaa ccc
ttg atg att att cat cac ttg gat gad tgc 419 Lys Thr Ser Asn Lys Pro
Leu Met Ile Ile His His Leu Asp Xaa Cys 50 55 60 cca cac agt caa
gct tta aaa aaa ktg ttt gct gaa aat aaa raa atc 467 Pro His Ser Gln
Ala Leu Lys Lys Xaa Phe Ala Glu Asn Lys Xaa Ile 65 70 75 cag aaa
ttg gca ran cag ttt gtc cyc ctc aat ctg gtt tat gaa aca 515 Gln Lys
Leu Ala Xaa Gln Phe Val Xaa Leu Asn Leu Val Tyr Glu Thr 80 85 90
act gac aaa cac ctt tct cct gat ggc caa tat ktc ccc cmg gat tat 563
Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Xaa Pro Xaa Asp Tyr 95
100 105 gtt tgt tgacccatct ctgacagtta gagccgatat cactggaaga
tattcaaayc 619 Val Cys 110 gtctctatgc ttacgaacct gcagatacag
ctctgttgct tgacaacatg aagaaagctc 679 tcaagttgct gaagactgaa
ttgtaaagaa aaaaaatctc caagcccttc tgtctgtcag 739 gccttgagac
ttgaaaccag aagaagtgtg agaagactgg ctagtgtgga agcatagtga 799
acacactgat taggttatgg tttaatgtta caacaactat tttttaagaa aaacaagttt
859 tagaaatttg gtttcaagtg tacatgtgtg aaaacaatat tgtatactac
catagtgagc 919 catgattttc taaaaaaaaa a 940 150 887 DNA Homo sapiens
CDS 67..459 sig_peptide 67..135 Von Heijne matrix score
5.19999980926514 seq IGVGLYLLASAAA/FY polyA_signal 856..861
polyA_site 875..887 150 agcggcggca tccgggacgg cgggcgggct ggccaccacg
ggacaggaag gcacagagca 60 tggaga atg atg aac ttc cgt cag cgg atg gga
tgg att gga gtg gga 108 Met Met Asn Phe Arg Gln Arg Met Gly Trp Ile
Gly Val Gly -20 -15 -10 ttg tat ctg tta gcc agt gca gca gca ttt tac
tat gtt ttt gaa atc 156 Leu Tyr Leu Leu Ala Ser Ala Ala Ala Phe Tyr
Tyr Val Phe Glu Ile -5 1 5 agt gag act tac aac agg ctg gcc ttg gaa
cac att caa cag cac cct 204 Ser Glu Thr Tyr Asn Arg Leu Ala Leu Glu
His Ile Gln Gln His Pro 10 15 20 ggg gag ccc ctt gaa gga acc aca
tgg aca cac tcc ttg aaa gct caa 252 Gly Glu Pro Leu Glu Gly Thr Thr
Trp Thr His Ser Leu Lys Ala Gln 25 30 35 tta ctc tcc ttg cct ttt
tgg gtg tgg aca gtt att ttt ctg gta cct 300 Leu Leu Ser Leu Pro Phe
Trp Val Trp Thr Val Ile Phe Leu Val Pro 40 45 50 55 tac tta car atk
ttt ttg ttc cta tac tct tgt aca aaa vct gat ccc 348 Tyr Leu Gln Xaa
Phe Leu Phe Leu Tyr Ser Cys Thr Lys Xaa Asp Pro 60 65 70 aaa aca
gtg ggc tac tgt wtc atc cct ata tgc ttg gca rtt att tsc 396 Lys Thr
Val Gly Tyr Cys Xaa Ile Pro Ile Cys Leu Ala Xaa Ile Xaa 75 80 85
aat cgc cac cag gat ttt gtc aag gct tct aat caa atc agc aaa cta 444
Asn Arg His Gln Asp Phe Val Lys Ala Ser Asn Gln Ile Ser Lys Leu 90
95 100 caa ctg att gac acg taaaatcagt caccgttttt tccctacgat
tacaaaactg 499 Gln Leu Ile Asp Thr 105 ccagtcctat atggagtctg
atcacaagac tgcagtttct tcacagatct caggaagttg 559 tcgtggggca
gaggcttttt aaaaacatgt gattagggag ctatctttat ctgaataata 619
acgaattttt aggtaaaacc tgagatagag tactacaaaa tcatgttgat gacttcagat
679 tttggaagtt aaatcatgtc tgttatttgc attctttaga aacttgacta
agtacctgaa 739 ttcatatttc tattctactg tgcaacatag tgatgattca
gaaatttttc ctttggggaa 799 aaaaatgaat atgaacattt ccattgtgtt
aagtgtaaaa aggtccagka catgatcata 859 aaatttaaat tttatacaaa aaaaaaaa
887 151 2010 DNA Homo sapiens CDS 65..1069 sig_peptide 65..112 Von
Heijne matrix score 12.5 seq FVVLLALVAGVLG/NE polyA_signal
1978..1983 polyA_site 1999..2010 151 atgtcgcccg tgtcccgccg
gcccgttccg tgtcgccccg cagtgytgcg gccgccgckk 60 cacc atg gct gtg ttt
gtc gtg ctc ctg gcg ttg gtg gcg ggt gtt ttg 109 Met Ala Val Phe Val
Val Leu Leu Ala Leu Val Ala Gly Val Leu -15 -10 -5 ggg aac gag ttt
agt ata tta aaa tca cca ggg tct gtt gtt ttc cga 157 Gly Asn Glu Phe
Ser Ile Leu Lys Ser Pro Gly Ser Val Val Phe Arg 1 5 10 15 aat gga
aat tgg cct ata cca gga gag cgg atc cca gac gtg gct gca 205 Asn Gly
Asn Trp Pro Ile Pro Gly Glu Arg Ile Pro Asp Val Ala Ala 20 25 30
ttg tcc atg ggc ttc tct gtg aaa gaa gac ctt tct tgg cca gga ctc 253
Leu Ser Met Gly Phe Ser Val Lys Glu Asp Leu Ser Trp Pro Gly Leu 35
40 45 gca gtg ggt aac ctg ttt cat cgt cct cgg gct agc gtc atg gtg
atg 301 Ala Val Gly Asn Leu Phe His Arg Pro Arg Ala Ser Val Met Val
Met 50 55 60 gtg aag gga gtt aac aac tmc cct cta ccc cca ggc tgt
gtc att tcg 349 Val Lys Gly Val Asn Asn Xaa Pro Leu Pro Pro Gly Cys
Val Ile Ser 65 70 75 tac cct ttg gag aat gca gtt cct ttt agt ctt
gac agt gtt gca aat 397 Tyr Pro Leu Glu Asn Ala Val Pro Phe Ser Leu
Asp Ser Val Ala Asn 80 85 90 95 tcc att cac tcc tta ttt tct gag gaa
act cct gtt gtt ttg cag ttg 445 Ser Ile His Ser Leu Phe Ser Glu Glu
Thr Pro Val Val Leu Gln Leu 100 105 110 gct ccc agt gag gaa aga gtg
tat atg gta ggg aag gcm aac tca gtg 493 Ala Pro Ser Glu Glu Arg Val
Tyr Met Val Gly Lys Ala Asn Ser Val 115 120 125 tgg aar acc ttt cag
tca ctt gcg cca gct ccg kta atc rcc tgt ttc 541 Trp Lys Thr Phe Gln
Ser Leu Ala Pro Ala Pro Xaa Ile Xaa Cys Phe 130 135 140 aag aaa act
ctg ttc tca gtt cac tcc ccc ycc att cma ctg agt agg 589 Lys Lys Thr
Leu Phe Ser Val His Ser Pro Xaa Ile Xaa Leu Ser Arg 145 150 155 aac
aat gaa gtt gac cyg ctc ttt ctt tct gaa ctg caa gtg cta cat 637 Asn
Asn Glu Val Asp Xaa Leu Phe Leu Ser Glu Leu Gln Val Leu His 160 165
170 175 gat att tca agc ttg ctg tct cgt cat aag cat cta gcc aag gat
cat 685 Asp Ile Ser Ser Leu Leu Ser Arg His Lys His Leu Ala Lys Asp
His 180 185 190 tct cct gat tta tat tca ctg gag ctg gca ggt ttg gat
gaa att ggg 733 Ser Pro Asp Leu Tyr Ser Leu Glu Leu Ala Gly Leu Asp
Glu Ile Gly 195 200 205 aag cgt tat ggg gaa gac tct gaa caa ttc aga
gat gct tct aag atc 781 Lys Arg Tyr Gly Glu Asp Ser Glu Gln Phe Arg
Asp Ala Ser Lys Ile 210 215 220 ctt gtt gac gct ctg caa aag ttt gca
gat gac atg tac agt ctt tat 829 Leu Val Asp Ala Leu Gln Lys Phe Ala
Asp Asp Met Tyr Ser Leu Tyr 225 230 235 ggt ggg aat gca gtg gta gag
tta gtc act gtc aag tca ttt gac acc 877 Gly Gly Asn Ala Val Val Glu
Leu Val Thr Val Lys Ser Phe Asp Thr 240 245 250 255 tcc ctc att agg
aag aca agg act atc ctt gag gca aaa caa gcg aag 925 Ser Leu Ile Arg
Lys Thr Arg Thr Ile Leu Glu Ala Lys Gln Ala Lys 260 265 270 aac cca
gca agt ccc tat aac ctt gca tat aag tat aat ttt gaa tat 973 Asn Pro
Ala Ser Pro Tyr Asn Leu Ala Tyr Lys Tyr Asn Phe Glu Tyr 275 280 285
tcc gtg gtt ttc aac atg gta ctt tgg ata atg atc gcc ttg gcc ttg
1021 Ser Val Val Phe Asn Met Val Leu Trp Ile Met Ile Ala Leu Ala
Leu 290 295 300 gct gtg att atc acc tct tac aat att tgg aac atg gaa
tcc tgg ata 1069 Ala Val Ile Ile Thr Ser Tyr Asn Ile Trp Asn Met
Glu Ser Trp Ile 305 310 315 tgatagcatc atttatagga tgacaaacca
gaagattcgg aatggattga atgttacctg 1129 tgccagaatt akaaaagggg
gttggaaatt ggctgttttg ttaaaatata tcttttagtg 1189 tgctttaaag
tagatagtat actttacatt tataaaaaaa aatcaaattt tgttctttat 1249
tttgtgtgtg cctgtgatgt ttttctagag tgaattatag tattgacgtg aatcccactg
1309 tggtatagat tccataatat gcttgaatat tatgatatag ccatttaata
acattgattt 1369 cattctgttt aatgaatttg gaaatatgca ctgaaagaaa
tgtaaaacat ttagaatagc 1429 tcgtgttatg gaaaaaagtg cactgaattt
attagacaaa cttacgaatg cttaacttct 1489 ttacacagca taggtgaaaa
tcatatttgg gctattgtat actatgaaca atttgtaaat 1549 gtcttaattt
gatgtaaata actctgaaac aagagaaaag gtttttaact tagagtagcc 1609
ctaaaatatg gatgtgctta tataatcgct tagttttgga actgtatctg agtaacagag
1669 gacagctgtt ttttaaccct cttctgcaag tttgttgacc tacatgggct
aatatggata 1729 ctaaaaatac tacattgatc taagaagaaa ctagccttgt
ggagtatata gatgcttttc 1789 attatacaca caaaaatccc tgagggacat
tttgaggcat gaatataaaa catttttatt 1849 tcagtaactt ttccccctgt
gtaagttact atggtttgtg gtacaacttc attctataga 1909 atattaagtg
gaagtgggtg aattctactt tttatgttgg agtggaccaa tgtctatcaa 1969
gagtgacaaa taaagttaat gatgattcca aaaaaaaaaa a 2010 152 387 DNA Homo
sapiens CDS 70..321 sig_peptide 70..234 Von Heijne matrix score
4.09999990463257 seq AVCAALLASHPTA/EV polyA_signal 364..369
polyA_site 375..387 152 agaaatcgta ggacttccga aagcagcggc ggcgtttgct
tcactgcttg gaagtgtgag 60 tgcgcgaag atg cga aag gtg gtt ttr att acc
ggg gct agc agt ggc att 111 Met Arg Lys Val Val Leu Ile Thr Gly Ala
Ser Ser Gly Ile -55 -50 -45 ggc ctg gcc ctc tgc aag cgg ctg ctg gcg
gaa gat gat gag ctt cat 159 Gly Leu Ala Leu Cys Lys Arg Leu Leu Ala
Glu Asp Asp Glu Leu His -40 -35 -30 ctg tgt ttg gcg tgc agg aat atg
agc aag gca gaa gct gtc tgt gct 207 Leu Cys Leu Ala Cys Arg Asn Met
Ser Lys Ala Glu Ala Val Cys Ala -25 -20 -15 -10 gct ctg ctg gcc tct
cac ccc act gct gag gtc acc att gtc cag gtg 255 Ala Leu Leu Ala Ser
His Pro Thr Ala Glu Val Thr Ile Val Gln Val -5 1 5 gat gtc agc aac
ctg cag tca ttc ttc cgg gcc tcc aag gaa ctt aag 303 Asp Val Ser Asn
Leu Gln Ser Phe Phe Arg Ala Ser Lys Glu Leu Lys 10 15 20 caa agg
atg atc tct tgc tgatggattt tttttctcat gtgattgtgc 351 Gln Arg Met
Ile Ser Cys 25 ascataacac ttaataaaat aagaaaaaaa aaaaaa 387 153 983
DNA Homo sapiens CDS 38..877 sig_peptide 38..91 Von Heijne matrix
score 7.40000009536743 seq GWLVLCVLAISLA/SM polyA_signal 947..952
polyA_site 974..983 153 aatccagtyg gasttgacaa caggaggcag aggcatc
atg gag ggt ccc cgg gga 55 Met Glu Gly Pro Arg Gly -15 tgg ctg gtg
ctc tgt gtg ctg gcc ata tcg ctg gcc tct atg gtg acc 103 Trp Leu Val
Leu Cys Val Leu Ala Ile Ser Leu Ala Ser Met Val Thr -10 -5 1 gag
gac ttg tgc cga gca cca gac ggg aag aaa ggg gag gca gga aga 151 Glu
Asp Leu Cys Arg Ala Pro Asp Gly Lys Lys Gly Glu Ala Gly Arg 5 10 15
20 cct ggc aga cgg ggg cgg cca ggc ctc aag ggg gag caa ggg gag ccg
199 Pro Gly Arg Arg Gly Arg Pro Gly Leu Lys Gly Glu Gln Gly Glu Pro
25 30 35 ggg gcc cct ggc atc cgg aca ggc atc caa ggc ctt aaa gga
gac cag 247 Gly Ala Pro Gly Ile Arg Thr Gly Ile Gln Gly Leu Lys Gly
Asp Gln 40 45 50 ggg gaa cct ggg ccc
tct gga aac ccc ggc aag gtg ggc tac cca ggg 295 Gly Glu Pro Gly Pro
Ser Gly Asn Pro Gly Lys Val Gly Tyr Pro Gly 55 60 65 ccc agc ggc
ccc ctc gga gcc cgt ggc atc ccg gga att aaa ggc acc 343 Pro Ser Gly
Pro Leu Gly Ala Arg Gly Ile Pro Gly Ile Lys Gly Thr 70 75 80 aag
ggc agc cca gga aac atc aag gac cag ccg agg cca gcc ttc tcc 391 Lys
Gly Ser Pro Gly Asn Ile Lys Asp Gln Pro Arg Pro Ala Phe Ser 85 90
95 100 gcc att cgg cgg aac ccc cca atg ggg ggc aac gtg gtc atc ttc
gac 439 Ala Ile Arg Arg Asn Pro Pro Met Gly Gly Asn Val Val Ile Phe
Asp 105 110 115 acg gtc atc acc aac cag gaa gaa ccg tac cag aac cac
tcc ggc cga 487 Thr Val Ile Thr Asn Gln Glu Glu Pro Tyr Gln Asn His
Ser Gly Arg 120 125 130 ttc gtc tgc act gta ccc gct act act act tca
cct tcc agg tgc tgt 535 Phe Val Cys Thr Val Pro Ala Thr Thr Thr Ser
Pro Ser Arg Cys Cys 135 140 145 ccc agt ggg aaa tct gcc tgt cca tcg
tct cct cct caa ggg gcc agg 583 Pro Ser Gly Lys Ser Ala Cys Pro Ser
Ser Pro Pro Gln Gly Ala Arg 150 155 160 tcc gac gct ccc tgg gct tct
gtg aca cca cca aca agg ggc tct tcc 631 Ser Asp Ala Pro Trp Ala Ser
Val Thr Pro Pro Thr Arg Gly Ser Ser 165 170 175 180 agg tgg tgt cag
ggg gca tgg tgc ttc agc tgc agc agg gtg acc agg 679 Arg Trp Cys Gln
Gly Ala Trp Cys Phe Ser Cys Ser Arg Val Thr Arg 185 190 195 tct ggg
ttg aaa aag acc cca aaa agg gtc aca ttt acc agg gct ctg 727 Ser Gly
Leu Lys Lys Thr Pro Lys Arg Val Thr Phe Thr Arg Ala Leu 200 205 210
agg ccg aca gcg tct tca gcg gct tcc tca tct tcc cat ctg cct gag 775
Arg Pro Thr Ala Ser Ser Ala Ala Ser Ser Ser Ser His Leu Pro Glu 215
220 225 cca ggg aag gac ccc ctc ccc cac cca cct ctc tgg ctt cca tgc
tcc 823 Pro Gly Lys Asp Pro Leu Pro His Pro Pro Leu Trp Leu Pro Cys
Ser 230 235 240 gcc tgt aaa atg ggg gcg cta ttg ctt cag ctg ctg aag
gga ggg ggc 871 Ala Cys Lys Met Gly Ala Leu Leu Leu Gln Leu Leu Lys
Gly Gly Gly 245 250 255 260 tgg ctc tgagagcccc aggactggct
gccccgtgac acatgctcta agaagctcgt 927 Trp Leu ttcttagacc tcttcctgga
ataaacatct gtgtctgtgt ctgctgaaaa aaaaaa 983 154 1614 DNA Homo
sapiens CDS 51..470 sig_peptide 51..203 Von Heijne matrix score
5.80000019073486 seq AVGLFPAPTECFA/RV polyA_signal 1585..1590
polyA_site 1604..1614 154 ataagcctgt ggttgatgga aattcacaaa
gtgaggcatt atcactggaa atg aga 56 Met Arg -50 aag gat ccg agc ggg
gct ggc ctc tgg ctt cac agt ggc ggc cca gtg 104 Lys Asp Pro Ser Gly
Ala Gly Leu Trp Leu His Ser Gly Gly Pro Val -45 -40 -35 ctt cca tat
gtg aga gaa tca gta aga aga aat cca gcc tca gca gcc 152 Leu Pro Tyr
Val Arg Glu Ser Val Arg Arg Asn Pro Ala Ser Ala Ala -30 -25 -20 act
ccg agc aca gcc gtg ggt ttg ttc cct gct cca aca gag tgt ttt 200 Thr
Pro Ser Thr Ala Val Gly Leu Phe Pro Ala Pro Thr Glu Cys Phe -15 -10
-5 gct cgg gtg tcc tgc agt ggt gtt gaa gct ctg ggg cgg cga gac tgg
248 Ala Arg Val Ser Cys Ser Gly Val Glu Ala Leu Gly Arg Arg Asp Trp
1 5 10 15 ctg gga gga ggg ccc agg gcc cac tgr msg gcv aca gag gmc
agt gcc 296 Leu Gly Gly Gly Pro Arg Ala His Xaa Xaa Ala Thr Glu Xaa
Ser Ala 20 25 30 cca aag gag agc ctc ggg tgt cac gac tgc cac gcc
atc aaa aag tgc 344 Pro Lys Glu Ser Leu Gly Cys His Asp Cys His Ala
Ile Lys Lys Cys 35 40 45 cgg aaa tgg gaa gtt ttc agg atg acc cac
caa gtg ctt ttc cca agg 392 Arg Lys Trp Glu Val Phe Arg Met Thr His
Gln Val Leu Phe Pro Arg 50 55 60 gtc tgg gct ctg agt tgg aac ccg
ctt gcc tgc act cca tcc tgt ctg 440 Val Trp Ala Leu Ser Trp Asn Pro
Leu Ala Cys Thr Pro Ser Cys Leu 65 70 75 caa cgc tgc aca tgt atc
ccg aak tgc tcc tgagtgagga racaaaacgc 490 Gln Arg Cys Thr Cys Ile
Pro Xaa Cys Ser 80 85 atktyccttg accgtytaaa gcccatgttt ycaaagcaaa
caatavaatt caaraarrtg 550 cttaaaagca cctcaratgg tckgcaaata
acactggggt tactggctct gcaacctttt 610 gaattavcaa atacattatg
ccatagttaa ggtacaagca gaacaatacc aatagattaa 670 ttttaagagt
tgtcttagaa tgatttcttt cgcataaagt ctggatgcaa actgtgcagc 730
ccttaggtmc ctgctgtagt tttgtacgac ctggcagact taaagtaaat tgagtttaaa
790 ttcaaagcca gttgatgcgg aaggaacttt tttggcatgt gttaaattgt
gctttaaaag 850 acatataaag aattgggaaa catttcagga gacgatcata
gcctgtataa ataccagatt 910 agaacatacg gatttaccat gaagttctgt
cttcaacatc cattctaaag ggctactgtc 970 ccaaatcctg tgtgtccttt
tgacttgtct gatcacccaa tggaagtgga tacttgtaaa 1030 gtctacacca
ctgtacttgg cgttaaatct tgctgaattc gtggtaagct gttaccatgt 1090
ctacattttg tagaatgatt ttggtctgca gcaaaattcg atttcacttc tcatacccct
1150 ttccttccac ttgaaatgca atttagacag akgccctgtg gtgaaagttg
caatattaag 1210 tttaccttta gaagatccct tctcaaactc agaaccctag
cagtgttacc ttaaacaaaa 1270 atgakctcga gaaaaaagta gctcagttac
agagaagcaa atcgagttat ttcccacata 1330 aaaagtttcc cagattctaa
gaattgcagt atcctgtacc ctaaaatttt tcaaggtgac 1390 tcctgttgtc
gtctgttgat aactttaata aaggtcattt aaggacataa gtttttaaag 1450
actcccaaag tgaaacttaa acattttcgg gattatcgat tgcatatatc agtttatgct
1510 gtgtgctgaa ttactatgcc atgtgctatt ttagtgtttg gggaaaatga
aaaataaaat 1570 ttgttcttta gcttaataaa tawgtcttat tttaaaaaaa aaaa
1614 155 99 PRT Homo sapiens SIGNAL -32..-1 UNSURE -5,42,58 Xaa =
any one of the twenty amino acids 155 Met Ala Ala Ala Ala Ala Ser
Arg Gly Val Gly Ala Lys Leu Gly Leu -30 -25 -20 Arg Glu Ile Arg Ile
His Leu Cys Gln Arg Ser Xaa Gly Ser Gln Gly -15 -10 -5 Val Arg Asp
Phe Ile Glu Lys Arg Tyr Val Glu Leu Lys Lys Ala Asn 1 5 10 15 Pro
Asp Leu Pro Ile Leu Ile Arg Glu Cys Ser Asp Val Gln Pro Lys 20 25
30 Leu Trp Ala Arg Tyr Ala Phe Gly Gln Xaa Thr Asn Val Pro Leu Asn
35 40 45 Asn Phe Ser Ala Asp Gln Val Thr Arg Xaa Leu Glu Asn Val
Leu Ser 50 55 60 Gly Lys Ala 65 156 160 PRT Homo sapiens SIGNAL
-27..-1 UNSURE 49,74,75,88,98,104,112,114,120 Xaa = any one of the
twenty amino acids 156 Met Gln Arg Val Ser Gly Leu Leu Ser Trp Thr
Leu Ser Arg Val Leu -25 -20 -15 Trp Leu Ser Gly Leu Ser Glu Pro Gly
Ala Ala Arg Gln Pro Arg Ile -10 -5 1 5 Met Glu Glu Lys Ala Leu Glu
Val Tyr Asp Leu Ile Arg Thr Ile Arg 10 15 20 Asp Pro Glu Lys Pro
Asn Thr Leu Glu Glu Leu Glu Val Val Ser Glu 25 30 35 Ser Cys Val
Glu Val Gln Glu Ile Asn Glu Glu Xaa Tyr Leu Val Ile 40 45 50 Ile
Arg Phe Thr Pro Thr Val Pro His Cys Ser Leu Ala Thr Leu Ile 55 60
65 Gly Leu Cys Leu Xaa Xaa Lys Leu Gln Arg Cys Leu Pro Phe Lys His
70 75 80 85 Lys Leu Xaa Ile Tyr Ile Ser Glu Gly Thr His Ser Xaa Glu
Glu Asp 90 95 100 Ile Asn Xaa Gln Ile Asn Asp Lys Glu Arg Xaa Ala
Xaa Ala Met Glu 105 110 115 Asn Pro Xaa Leu Arg Glu Ile Val Glu Gln
Cys Val Leu Glu Pro Asp 120 125 130 157 59 PRT Homo sapiens SIGNAL
-22..-1 157 Met Arg Leu Lys Tyr Gln His Thr Gly Ala Val Leu Asp Cys
Ala Phe -20 -15 -10 Tyr Asp Pro Thr His Ala Trp Ser Gly Gly Leu Asp
His Gln Leu Lys -5 1 5 10 Met His Asp Leu Asn Thr Asp Gln Glu Asn
Leu Val Gly Thr Met Met 15 20 25 Pro Leu Ser Asp Val Leu Asn Thr
Val His Lys 30 35 158 112 PRT Homo sapiens SIGNAL -48..-1 UNSURE 48
Xaa = any one of the twenty amino acids 158 Met Gln Asp Thr Gly Ser
Val Val Pro Leu His Trp Phe Gly Phe Gly -45 -40 -35 Tyr Ala Ala Leu
Val Ala Ser Gly Gly Ile Ile Gly Tyr Val Lys Ala -30 -25 -20 Gly Ser
Val Pro Ser Leu Ala Ala Gly Leu Leu Phe Gly Ser Leu Ala -15 -10 -5
Gly Leu Gly Ala Tyr Gln Leu Ser Gln Asp Pro Arg Asn Val Trp Val 1 5
10 15 Phe Leu Ala Thr Ser Gly Thr Leu Ala Gly Ile Met Gly Met Arg
Phe 20 25 30 Tyr His Ser Gly Lys Phe Met Pro Ala Gly Leu Ile Ala
Gly Ala Xaa 35 40 45 Leu Leu Met Val Ala Lys Ile Gly Val Ser Met
Phe Asn Arg Pro His 50 55 60 159 111 PRT Homo sapiens SIGNAL
-56..-1 UNSURE 27,28,43,44,49,50,52,53 Xaa = any one of the twenty
amino acids 159 Met Gly Gly Asn Gly Ser Thr Cys Lys Pro Asp Thr Glu
Arg Gln Gly -55 -50 -45 Thr Leu Ser Thr Ala Ala Pro Thr Thr Ser Pro
Ala Pro Cys Leu Ser -40 -35 -30 -25 Asn His His Asn Lys Lys His Leu
Ile Leu Ala Phe Cys Ala Gly Val -20 -15 -10 Leu Leu Thr Leu Leu Leu
Ile Ala Phe Ile Phe Leu Ile Ile Lys Ser -5 1 5 Tyr Arg Lys Tyr His
Ser Lys Pro Gln Ala Pro Asp Pro His Ser Asp 10 15 20 Pro Pro Xaa
Xaa Leu Ser Ser Ile Pro Gly Glu Ser Leu Thr Tyr Ala 25 30 35 40 Ser
Thr Xaa Xaa Gln Thr Leu Arg Xaa Xaa Glu Xaa Xaa Leu Gly 45 50 55
160 144 PRT Homo sapiens SIGNAL -77..-1 UNSURE -65,31,34 Xaa = any
one of the twenty amino acids 160 Met Ala Ala Ser Lys Val Lys Gln
Asp Met Pro Pro Xaa Gly Gly Tyr -75 -70 -65 Gly Pro Ile Asp Tyr Lys
Arg Asn Leu Pro Arg Arg Gly Leu Ser Gly -60 -55 -50 Tyr Ser Met Leu
Ala Ile Gly Ile Gly Thr Leu Ile Tyr Gly His Trp -45 -40 -35 -30 Ser
Ile Met Lys Trp Asn Arg Glu Arg Arg Arg Leu Gln Ile Glu Asp -25 -20
-15 Phe Glu Ala Arg Ile Ala Leu Leu Pro Leu Leu Gln Ala Glu Thr Asp
-10 -5 1 Arg Arg Thr Leu Gln Met Leu Arg Glu Asn Leu Glu Glu Glu
Ala Ile 5 10 15 Ile Met Lys Asp Val Pro Asp Trp Lys Val Gly Xaa Ser
Val Xaa His 20 25 30 35 Thr Thr Arg Trp Val Pro Pro Leu Ile Gly Glu
Leu Tyr Gly Leu Arg 40 45 50 Thr Thr Lys Glu Ala Leu His Ala Ser
His Gly Phe Met Trp Tyr Thr 55 60 65 161 110 PRT Homo sapiens
SIGNAL -18..-1 UNSURE 48,52,55,59,65 Xaa = any one of the twenty
amino acids 161 Met Glu Thr Gly Arg Leu Leu Ser Leu Ser Ser Leu Pro
Leu Val Leu -15 -10 -5 Leu Gly Trp Glu Tyr Ser Ser Gln Thr Leu Asn
Leu Val Pro Ser Thr 1 5 10 Ser Ile Leu Ser Phe Val Pro Phe Ile Pro
Leu His Leu Val Leu Phe 15 20 25 30 Ala Leu Trp Tyr Leu Pro Val Pro
His His Leu Tyr Pro Gln Gly Leu 35 40 45 Gly Xaa His Ala Ala Xaa
Ala Glu Xaa Gly Lys Arg Xaa Glu Gly Gly 50 55 60 Thr Gln Xaa Ala
Leu Trp Leu Arg Val Gln Pro Ser Cys Pro Ser Pro 65 70 75 Val Cys
Leu Glu Pro Val Pro Pro Arg Ser Arg Phe Leu Leu 80 85 90 162 79 PRT
Homo sapiens SIGNAL -36..-1 162 Met Glu Leu Glu Ala Met Ser Arg Tyr
Thr Ser Pro Val Asn Pro Ala -35 -30 -25 Val Phe Pro His Leu Thr Val
Val Leu Leu Ala Ile Gly Met Phe Phe -20 -15 -10 -5 Thr Ala Trp Phe
Phe Val Tyr Glu Val Thr Ser Thr Lys Tyr Thr Arg 1 5 10 Asp Ile Tyr
Lys Glu Leu Leu Ile Ser Leu Val Ala Ser Leu Phe Met 15 20 25 Gly
Phe Gly Val Leu Phe Leu Leu Leu Trp Val Gly Ile Tyr Val 30 35 40
163 196 PRT Homo sapiens SIGNAL -34..-1 UNSURE
81,84,87,131,135,143,156 Xaa = any one of the twenty amino acids
163 Met Ser Phe Leu Gln Asp Pro Ser Phe Phe Thr Met Gly Met Trp Ser
-30 -25 -20 Ile Gly Ala Gly Ala Leu Gly Ala Ala Ala Leu Ala Leu Leu
Leu Ala -15 -10 -5 Asn Thr Asp Val Phe Leu Ser Lys Pro Gln Lys Ala
Ala Leu Glu Tyr 1 5 10 Leu Glu Asp Ile Asp Leu Lys Thr Leu Glu Lys
Glu Pro Arg Thr Phe 15 20 25 30 Lys Ala Lys Glu Leu Trp Glu Lys Asn
Gly Ala Val Ile Met Ala Val 35 40 45 Arg Arg Pro Gly Cys Phe Leu
Cys Arg Glu Glu Ala Ala Asp Leu Ser 50 55 60 Ser Leu Lys Ser Met
Leu Asp Gln Leu Gly Val Pro Leu Tyr Ala Val 65 70 75 Val Lys Xaa
His Ile Xaa Thr Glu Xaa Lys Asp Phe Gln Pro Tyr Phe 80 85 90 Lys
Gly Glu Ile Phe Leu Asp Glu Lys Lys Lys Phe Tyr Gly Pro Gln 95 100
105 110 Arg Arg Lys Met Met Phe Met Gly Phe Ile Arg Leu Gly Met Trp
Tyr 115 120 125 Asn Phe Phe Arg Xaa Trp Asn Gly Xaa Phe Ser Gly Asn
Leu Glu Gly 130 135 140 Xaa Gly Phe Ile Leu Gly Gly Ile Phe Val Val
Gly Ser Xaa Lys Ala 145 150 155 Gly His Ser Ser 160 164 177 PRT
Homo sapiens SIGNAL -18..-1 UNSURE 32,91,98 Xaa = any one of the
twenty amino acids 164 Met Leu Leu Cys Leu Leu Thr Pro Leu Phe Phe
Met Phe Pro Thr Gly -15 -10 -5 Phe Ser Ser Pro Ser Pro Ser Ala Ala
Ala Ala Ala Gln Glu Val Arg 1 5 10 Ser Ala Thr Asp Gly Asn Thr Ser
Thr Thr Pro Pro Thr Ser Ala Lys 15 20 25 30 Lys Xaa Lys Leu Asn Ser
Ser Ser Ser Ser Ser Ser Asn Ser Ser Asn 35 40 45 Glu Arg Glu Asp
Phe Asp Ser Thr Ser Ser Ser Ser Ser Thr Pro Pro 50 55 60 Leu Gln
Pro Arg Asp Ser Ala Ser Pro Ser Thr Ser Ser Phe Cys Leu 65 70 75
Gly Val Ser Val Ala Ala Ser Ser His Val Pro Ile Xaa Lys Lys Leu 80
85 90 Arg Phe Glu Xaa Thr Leu Glu Phe Val Gly Phe Asp Ala Lys Met
Ala 95 100 105 110 Glu Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Pro
Thr Ala Ala Thr 115 120 125 Ser Gln Gln Gln Gln Leu Lys Asn Lys Ser
Ile Leu Asn Leu Phe Cys 130 135 140 Gly Phe Gly Ala Ser Cys Lys Arg
Pro Ser Gln Ile Phe Tyr His Arg 145 150 155 Leu 165 105 PRT Homo
sapiens SIGNAL -22..-1 165 Met Gln Gly His Trp Leu Ser Ser Ala Phe
Ala Leu Val Trp Leu Trp -20 -15 -10 Leu Arg Ser Thr Gly Cys Phe Trp
Trp Asp His Trp Leu Cys Lys Ser -5 1 5 10 Arg Gln Arg Ala Val Pro
Gly Cys Arg Ala Ala Leu Trp Gln Ser Ser 15 20 25 Arg Pro Gly Cys
Leu Pro Ala Val Ser Gly Ser Lys Glu Arg Leu Gly 30 35 40 Phe Pro
Ser Tyr Ile Trp Tyr Leu Gly Trp His Tyr Gly Asn Glu Val 45 50 55
Leu Pro Leu Trp Lys Ile His Ala Cys Arg Phe Asn Cys Arg Cys Gln 60
65 70 Phe Ala Asp Gly Arg Gln Ser Trp Ser 75 80 166 148 PRT Homo
sapiens SIGNAL -48..-1 UNSURE 32,100 Xaa = any one of the twenty
amino acids 166 Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala
Lys Asn Ala -45 -40 -35 Phe Met Leu Leu Met Arg Asn Ile Val Arg Val
Val Val Leu Asp Lys -30 -25 -20 Val Thr Asp Leu Leu Leu Phe Phe Gly
Lys Leu Leu Val Val Gly Gly -15 -10 -5 Val Gly Val Leu Ser Phe Phe
Phe Phe Ser Gly Arg Ile Pro Gly Leu 1 5 10 15 Gly Lys Asp Phe Lys
Ser Pro His Leu Asn Tyr Tyr Trp Leu Pro Xaa 20 25 30 Met Thr Ser
Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe Phe Ser 35 40 45 Val
Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu Asp 50 55
60 Leu Glu Arg Thr Thr Ala Pro Trp Thr Ala Leu Leu His Val Gln Glu
65 70 75 80 Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp
Asn Lys 85 90
95 Lys Arg Lys Xaa 100 167 259 PRT Homo sapiens SIGNAL -23..-1
UNSURE 31,84,88,90,94,100,110,113,117,122,152,153,173,193,194 Xaa =
any one of the twenty amino acids 167 Met Pro Ser Trp Ile Gly Ala
Val Ile Leu Pro Leu Leu Gly Leu Leu -20 -15 -10 Leu Ser Leu Pro Ala
Gly Ala Asp Val Lys Ala Arg Ser Cys Gly Glu -5 1 5 Val Arg Gln Ala
Tyr Gly Ala Lys Gly Phe Ser Leu Ala Asp Ile Pro 10 15 20 25 Tyr Gln
Glu Ile Ala Xaa Glu His Leu Arg Ile Cys Pro Gln Glu Tyr 30 35 40
Thr Cys Cys Thr Thr Glu Met Glu Asp Lys Leu Ser Gln Gln Ser Lys 45
50 55 Leu Glu Phe Glu Asn Leu Val Glu Glu Thr Ser His Phe Val Arg
Thr 60 65 70 Thr Phe Val Ser Arg His Lys Lys Phe Asp Xaa Phe Phe
Arg Xaa Leu 75 80 85 Xaa Glu Asn Ala Xaa Lys Ser Leu Asn Asp Xaa
Phe Val Arg Thr Tyr 90 95 100 105 Gly Met Leu Tyr Xaa Gln Asn Xaa
Glu Val Phe Xaa Asp Leu Phe Thr 110 115 120 Xaa Leu Lys Arg Tyr Tyr
Thr Gly Gly Asn Val Asn Leu Glu Glu Met 125 130 135 Leu Asn Asp Phe
Trp Ala Arg Leu Leu Glu Arg Met Phe Gln Xaa Xaa 140 145 150 Asn Pro
Gln Tyr His Phe Ser Glu Asp Tyr Leu Glu Cys Val Ser Lys 155 160 165
Tyr Thr Asp Xaa Leu Lys Pro Phe Gly Asp Val Pro Arg Lys Leu Lys 170
175 180 185 Ile Gln Val Thr Arg Ala Phe Xaa Xaa Ala Arg Thr Phe Val
Gln Gly 190 195 200 Leu Thr Val Gly Arg Glu Val Ala Asn Arg Val Ser
Lys Val Ile Glu 205 210 215 Asn Val Leu Ser Phe Ser Leu Val Phe Leu
Val Tyr Ser Val Phe Lys 220 225 230 Thr Asn Val 235 168 111 PRT
Homo sapiens SIGNAL -62..-1 UNSURE 37,43 Xaa = any one of the
twenty amino acids 168 Met Gly Glu Ser Ile Pro Leu Ala Ala Pro Val
Pro Val Glu Gln Ala -60 -55 -50 Val Leu Glu Thr Phe Phe Ser His Leu
Gly Ile Phe Ser Tyr Asp Lys -45 -40 -35 Ala Lys Asp Asn Val Glu Lys
Glu Arg Glu Ala Asn Lys Ser Ala Gly -30 -25 -20 -15 Gly Ser Trp Leu
Ser Leu Leu Ala Ala Leu Ala His Leu Ala Ala Ala -10 -5 1 Glu Lys
Val Tyr His Ser Leu Thr Tyr Leu Gly Gln Lys Leu Gly Thr 5 10 15 Ser
Ala Pro Pro Pro Glu Pro Leu Glu Glu Glu Val Lys Gly Val Tyr 20 25
30 Ser Pro Xaa Gly Ser Gly Leu Gly Xaa Pro Ser Leu Cys His Phe 35
40 45 169 311 PRT Homo sapiens SIGNAL -23..-1 UNSURE
56,70,107,110,113,178,181,183,195,200,202,204,230,231, 244 Xaa =
any one of the twenty amino acids 169 Met Ala Asp Val Ile Asn Val
Ser Val Asn Leu Glu Ala Phe Ser Gln -20 -15 -10 Ala Ile Ser Ala Ile
Gln Ala Leu Arg Ser Ser Val Ser Arg Val Phe -5 1 5 Asp Cys Leu Lys
Asp Gly Met Arg Asn Lys Glu Thr Leu Glu Gly Arg 10 15 20 25 Glu Lys
Ala Phe Ile Ala His Phe Gln Asp Asn Leu His Ser Val Asn 30 35 40
Arg Asp Leu Asn Glu Leu Glu Arg Leu Ser Asn Leu Val Gly Xaa Pro 45
50 55 Ser Glu Asn His Pro Leu His Asn Ser Gly Leu Leu Xaa Leu Asp
Pro 60 65 70 Val Gln Asp Lys Thr Pro Leu Tyr Ser Gln Leu Leu Gln
Ala Tyr Lys 75 80 85 Trp Ser Asn Lys Leu Gln Tyr His Ala Gly Leu
Ala Ser Gly Leu Leu 90 95 100 105 Asn Xaa Gln Ser Xaa Lys Arg Xaa
Ala Asn Gln Met Gly Val Ser Ala 110 115 120 Lys Arg Arg Pro Lys Ala
Gln Pro Thr Thr Leu Val Leu Pro Pro Gln 125 130 135 Tyr Val Asp Asp
Val Ile Ser Arg Ile Asp Arg Met Phe Pro Glu Met 140 145 150 Ser Ile
His Leu Ser Arg Pro Asn Gly Thr Ser Ala Met Leu Leu Val 155 160 165
Thr Leu Gly Lys Val Leu Lys Val Xaa Val Val Xaa Arg Xaa Leu Phe 170
175 180 185 Ile Asp Arg Thr Ile Val Lys Gly Tyr Xaa Glu Asn Val Tyr
Xaa Glu 190 195 200 Xaa Gly Xaa Leu Asp Ile Trp Ser Lys Ser Asn Tyr
Gln Val Phe Gln 205 210 215 Lys Val Thr Asp His Ala Thr Thr Ala Leu
Leu His Xaa Xaa Leu Pro 220 225 230 Gln Met Pro Asp Val Val Val Arg
Ser Phe Xaa Thr Trp Leu Arg Ser 235 240 245 Tyr Ile Lys Leu Phe Gln
Ala Pro Cys Gln Arg Cys Gly Lys Phe Leu 250 255 260 265 Gln Asp Gly
Leu Pro Pro Thr Trp Arg Asp Phe Arg Thr Leu Glu Ala 270 275 280 Phe
His Asp Thr Cys Arg Gln 285 170 91 PRT Homo sapiens SIGNAL -60..-1
170 Met Thr Ser Leu Phe Ala Val Val Leu Gln Arg Glu Lys Glu Pro His
-60 -55 -50 -45 Leu Trp Leu Ser Ser Pro His Ile Arg Phe Ser Leu Arg
Val Asn Lys -40 -35 -30 Leu Ser Glu Leu Met Leu Gln Leu Leu Gln Phe
Lys Ala Phe Pro Ser -25 -20 -15 Ser Leu Val Pro Phe Phe Leu Phe Thr
Cys Phe Gly His Phe Pro Ser -10 -5 1 Phe Thr Thr Phe Gln Gly Phe
Ile Glu Asn Asn Leu Leu Gln Asn Gln 5 10 15 20 Phe Asn Ser Asn Val
Asp Ile Val Ala Cys Ser 25 30 171 287 PRT Homo sapiens SIGNAL
-17..-1 UNSURE 74 Xaa = any one of the twenty amino acids 171 Met
Glu Leu Glu Arg Ile Val Ser Ala Ala Leu Leu Ala Phe Val Gln -15 -10
-5 Thr His Leu Pro Glu Ala Asp Leu Ser Gly Leu Asp Glu Val Ile Phe
1 5 10 15 Ser Tyr Val Leu Gly Val Leu Glu Asp Leu Gly Pro Ser Gly
Pro Ser 20 25 30 Glu Glu Asn Phe Asp Met Glu Ala Phe Thr Glu Met
Met Glu Ala Tyr 35 40 45 Val Pro Gly Phe Ala His Ile Pro Arg Gly
Thr Ile Gly Asp Met Met 50 55 60 Gln Lys Leu Ser Gly Gln Leu Ser
Asp Ala Xaa Asn Lys Glu Asn Leu 65 70 75 Gln Pro Gln Asn Ser Gly
Val Gln Gly Gln Val Pro Ile Ser Pro Glu 80 85 90 95 Pro Leu Gln Arg
Pro Glu Met Leu Lys Glu Glu Thr Arg Ser Ser Ala 100 105 110 Ala Ala
Ala Ala Asp Thr Gln Asp Glu Ala Thr Gly Ala Glu Glu Glu 115 120 125
Leu Leu Pro Gly Val Asp Val Leu Leu Glu Val Phe Pro Thr Cys Ser 130
135 140 Val Glu Gln Ala Gln Trp Val Leu Ala Lys Ala Arg Gly Asp Leu
Glu 145 150 155 Glu Ala Val Gln Met Leu Val Glu Gly Lys Glu Glu Gly
Pro Ala Ala 160 165 170 175 Trp Glu Gly Pro Asn Gln Asp Leu Pro Arg
Arg Leu Arg Gly Pro Gln 180 185 190 Lys Asp Glu Leu Lys Ser Phe Ile
Leu Gln Lys Tyr Met Met Val Asp 195 200 205 Ser Ala Glu Asp Gln Lys
Ile His Arg Pro Met Ala Pro Lys Glu Ala 210 215 220 Pro Lys Lys Leu
Ile Arg Tyr Ile Asp Asn Gln Val Val Ser Thr Lys 225 230 235 Gly Glu
Arg Phe Lys Asp Val Arg Asn Pro Glu Ala Glu Glu Met Lys 240 245 250
255 Ala Thr Tyr Ile Asn Leu Lys Pro Ala Arg Lys Tyr Arg Phe His 260
265 270 172 104 PRT Homo sapiens SIGNAL -49..-1 UNSURE 4 Xaa = any
one of the twenty amino acids 172 Met Glu His Leu Thr His Ser Ser
Gln Lys Leu Gln Ala Asp Glu His -45 -40 -35 Leu Thr Lys Glu Val Trp
Ser Arg Leu Leu Lys Glu Lys Gly Pro Ala -30 -25 -20 Gly Leu Ile Leu
Cys Phe Leu Cys Leu Tyr Pro Ile Pro Leu Cys Thr -15 -10 -5 Ser His
Pro Val Xaa Leu Cys Ala His Pro Gln Asp Val Tyr Pro Val 1 5 10 15
Val Val Arg Ala Glu Ile His Ala Glu Leu Tyr Gln Glu Leu Ala Tyr 20
25 30 Leu Lys Thr Glu Thr Glu Ser Leu Ala His Leu Phe Ala Leu Val
Pro 35 40 45 Gln Ala Lys Ile Lys Asn Arg Val 50 55 173 84 PRT Homo
sapiens SIGNAL -36..-1 UNSURE -26,-25,-24 Xaa = any one of the
twenty amino acids 173 Met Gly Leu Leu Thr Phe Gly Tyr Ile Glu Xaa
Xaa Xaa Lys Thr Glu -35 -30 -25 His Asn Pro Asp His His Ser Cys Leu
Ala Val Ser Trp Glu Ala Ala -20 -15 -10 -5 Gly Cys His Gly Ala Gly
Thr Gln Gln Ser Pro Leu Gly Val Ala Gly 1 5 10 Pro Trp Arg Pro Arg
Pro Pro Cys Val Gly Ser Leu Leu Ala Ala Arg 15 20 25 Ser Leu His
Lys Gln Val Ile Leu Phe Gly Leu Leu Gly Phe Ala Tyr 30 35 40 Asp
His Ala Ala 45 174 131 PRT Homo sapiens SIGNAL -20..-1 UNSURE
40,41,43,60,70,76,82,86,105,107 Xaa = any one of the twenty amino
acids 174 Met Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala
Leu Ser -20 -15 -10 -5 Tyr Thr Leu Ala Arg Asp Thr Thr Val Lys Pro
Gly Ala Lys Lys Asp 1 5 10 Thr Lys Asp Ser Arg Pro Lys Leu Pro Gln
Thr Leu Ser Arg Gly Trp 15 20 25 Gly Asp Gln Leu Ile Trp Thr Gln
Thr Tyr Glu Xaa Xaa Leu Xaa Lys 30 35 40 Ser Lys Thr Ser Asn Lys
Pro Leu Met Ile Ile His His Leu Asp Xaa 45 50 55 60 Cys Pro His Ser
Gln Ala Leu Lys Lys Xaa Phe Ala Glu Asn Lys Xaa 65 70 75 Ile Gln
Lys Leu Ala Xaa Gln Phe Val Xaa Leu Asn Leu Val Tyr Glu 80 85 90
Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Xaa Pro Xaa Asp 95
100 105 Tyr Val Cys 110 175 131 PRT Homo sapiens SIGNAL -23..-1
UNSURE 59,69,78,85,87 Xaa = any one of the twenty amino acids 175
Met Met Asn Phe Arg Gln Arg Met Gly Trp Ile Gly Val Gly Leu Tyr -20
-15 -10 Leu Leu Ala Ser Ala Ala Ala Phe Tyr Tyr Val Phe Glu Ile Ser
Glu -5 1 5 Thr Tyr Asn Arg Leu Ala Leu Glu His Ile Gln Gln His Pro
Gly Glu 10 15 20 25 Pro Leu Glu Gly Thr Thr Trp Thr His Ser Leu Lys
Ala Gln Leu Leu 30 35 40 Ser Leu Pro Phe Trp Val Trp Thr Val Ile
Phe Leu Val Pro Tyr Leu 45 50 55 Gln Xaa Phe Leu Phe Leu Tyr Ser
Cys Thr Lys Xaa Asp Pro Lys Thr 60 65 70 Val Gly Tyr Cys Xaa Ile
Pro Ile Cys Leu Ala Xaa Ile Xaa Asn Arg 75 80 85 His Gln Asp Phe
Val Lys Ala Ser Asn Gln Ile Ser Lys Leu Gln Leu 90 95 100 105 Ile
Asp Thr 176 335 PRT Homo sapiens SIGNAL -16..-1 UNSURE
70,139,141,154,156,165 Xaa = any one of the twenty amino acids 176
Met Ala Val Phe Val Val Leu Leu Ala Leu Val Ala Gly Val Leu Gly -15
-10 -5 Asn Glu Phe Ser Ile Leu Lys Ser Pro Gly Ser Val Val Phe Arg
Asn 1 5 10 15 Gly Asn Trp Pro Ile Pro Gly Glu Arg Ile Pro Asp Val
Ala Ala Leu 20 25 30 Ser Met Gly Phe Ser Val Lys Glu Asp Leu Ser
Trp Pro Gly Leu Ala 35 40 45 Val Gly Asn Leu Phe His Arg Pro Arg
Ala Ser Val Met Val Met Val 50 55 60 Lys Gly Val Asn Asn Xaa Pro
Leu Pro Pro Gly Cys Val Ile Ser Tyr 65 70 75 80 Pro Leu Glu Asn Ala
Val Pro Phe Ser Leu Asp Ser Val Ala Asn Ser 85 90 95 Ile His Ser
Leu Phe Ser Glu Glu Thr Pro Val Val Leu Gln Leu Ala 100 105 110 Pro
Ser Glu Glu Arg Val Tyr Met Val Gly Lys Ala Asn Ser Val Trp 115 120
125 Lys Thr Phe Gln Ser Leu Ala Pro Ala Pro Xaa Ile Xaa Cys Phe Lys
130 135 140 Lys Thr Leu Phe Ser Val His Ser Pro Xaa Ile Xaa Leu Ser
Arg Asn 145 150 155 160 Asn Glu Val Asp Xaa Leu Phe Leu Ser Glu Leu
Gln Val Leu His Asp 165 170 175 Ile Ser Ser Leu Leu Ser Arg His Lys
His Leu Ala Lys Asp His Ser 180 185 190 Pro Asp Leu Tyr Ser Leu Glu
Leu Ala Gly Leu Asp Glu Ile Gly Lys 195 200 205 Arg Tyr Gly Glu Asp
Ser Glu Gln Phe Arg Asp Ala Ser Lys Ile Leu 210 215 220 Val Asp Ala
Leu Gln Lys Phe Ala Asp Asp Met Tyr Ser Leu Tyr Gly 225 230 235 240
Gly Asn Ala Val Val Glu Leu Val Thr Val Lys Ser Phe Asp Thr Ser 245
250 255 Leu Ile Arg Lys Thr Arg Thr Ile Leu Glu Ala Lys Gln Ala Lys
Asn 260 265 270 Pro Ala Ser Pro Tyr Asn Leu Ala Tyr Lys Tyr Asn Phe
Glu Tyr Ser 275 280 285 Val Val Phe Asn Met Val Leu Trp Ile Met Ile
Ala Leu Ala Leu Ala 290 295 300 Val Ile Ile Thr Ser Tyr Asn Ile Trp
Asn Met Glu Ser Trp Ile 305 310 315 177 84 PRT Homo sapiens SIGNAL
-55..-1 177 Met Arg Lys Val Val Leu Ile Thr Gly Ala Ser Ser Gly Ile
Gly Leu -55 -50 -45 -40 Ala Leu Cys Lys Arg Leu Leu Ala Glu Asp Asp
Glu Leu His Leu Cys -35 -30 -25 Leu Ala Cys Arg Asn Met Ser Lys Ala
Glu Ala Val Cys Ala Ala Leu -20 -15 -10 Leu Ala Ser His Pro Thr Ala
Glu Val Thr Ile Val Gln Val Asp Val -5 1 5 Ser Asn Leu Gln Ser Phe
Phe Arg Ala Ser Lys Glu Leu Lys Gln Arg 10 15 20 25 Met Ile Ser Cys
178 280 PRT Homo sapiens SIGNAL -18..-1 178 Met Glu Gly Pro Arg Gly
Trp Leu Val Leu Cys Val Leu Ala Ile Ser -15 -10 -5 Leu Ala Ser Met
Val Thr Glu Asp Leu Cys Arg Ala Pro Asp Gly Lys 1 5 10 Lys Gly Glu
Ala Gly Arg Pro Gly Arg Arg Gly Arg Pro Gly Leu Lys 15 20 25 30 Gly
Glu Gln Gly Glu Pro Gly Ala Pro Gly Ile Arg Thr Gly Ile Gln 35 40
45 Gly Leu Lys Gly Asp Gln Gly Glu Pro Gly Pro Ser Gly Asn Pro Gly
50 55 60 Lys Val Gly Tyr Pro Gly Pro Ser Gly Pro Leu Gly Ala Arg
Gly Ile 65 70 75 Pro Gly Ile Lys Gly Thr Lys Gly Ser Pro Gly Asn
Ile Lys Asp Gln 80 85 90 Pro Arg Pro Ala Phe Ser Ala Ile Arg Arg
Asn Pro Pro Met Gly Gly 95 100 105 110 Asn Val Val Ile Phe Asp Thr
Val Ile Thr Asn Gln Glu Glu Pro Tyr 115 120 125 Gln Asn His Ser Gly
Arg Phe Val Cys Thr Val Pro Ala Thr Thr Thr 130 135 140 Ser Pro Ser
Arg Cys Cys Pro Ser Gly Lys Ser Ala Cys Pro Ser Ser 145 150 155 Pro
Pro Gln Gly Ala Arg Ser Asp Ala Pro Trp Ala Ser Val Thr Pro 160 165
170 Pro Thr Arg Gly Ser Ser Arg Trp Cys Gln Gly Ala Trp Cys Phe Ser
175 180 185 190 Cys Ser Arg Val Thr Arg Ser Gly Leu Lys Lys Thr Pro
Lys Arg Val 195 200 205 Thr Phe Thr Arg Ala Leu Arg Pro Thr Ala Ser
Ser Ala Ala Ser Ser 210 215 220 Ser Ser His Leu Pro Glu Pro Gly Lys
Asp Pro Leu Pro His Pro Pro 225 230 235 Leu Trp Leu Pro Cys Ser Ala
Cys Lys Met Gly Ala Leu Leu Leu Gln 240 245 250 Leu Leu Lys Gly Gly
Gly Trp Leu 255 260 179 140 PRT Homo sapiens SIGNAL -51..-1 UNSURE
24,25,29,87 Xaa = any one of the twenty amino acids 179 Met Arg Lys
Asp Pro Ser Gly Ala Gly Leu Trp Leu His Ser Gly Gly -50 -45 -40 Pro
Val Leu Pro Tyr Val Arg Glu Ser Val Arg Arg Asn Pro Ala Ser -35 -30
-25 -20 Ala Ala Thr Pro Ser Thr Ala Val Gly Leu Phe Pro Ala Pro Thr
Glu -15 -10 -5 Cys Phe Ala Arg Val Ser Cys Ser Gly Val Glu Ala Leu
Gly Arg Arg 1 5 10 Asp Trp Leu Gly Gly Gly Pro Arg Ala His Xaa Xaa
Ala Thr Glu Xaa 15 20 25 Ser Ala
Pro Lys Glu Ser Leu Gly Cys His Asp Cys His Ala Ile Lys 30 35 40 45
Lys Cys Arg Lys Trp Glu Val Phe Arg Met Thr His Gln Val Leu Phe 50
55 60 Pro Arg Val Trp Ala Leu Ser Trp Asn Pro Leu Ala Cys Thr Pro
Ser 65 70 75 Cys Leu Gln Arg Cys Thr Cys Ile Pro Xaa Cys Ser 80 85
180 279 DNA Homo sapiens 180 atggcacctc tccaccacat cttggttttc
tgtgtgggtc tcctcaccat ggccaaggca 60 gaaagtccaa aggaacacga
cccgttcact tacgactacc agtccctgca gatcggaggc 120 ctcgtcatcg
ccgggatcct cttcatcctg ggcatcctca tcgtgctgag cagaagatgc 180
cggtgcaagt tcaaccagca gcagaggact ggggaacccg atgaagagga gggaactttc
240 cgcagctcca tccgccgtct gtccacccgc aggcggtag 279 181 1082 DNA
Homo sapiens 181 gatcccagac ctcggcttgc agtagtgtta gactgaagat
aaagtaagtg ctgtttgggc 60 taacaggatc tcctcttgca gtctgcagcc
caggacgctg attccagcag cgccttaccg 120 cgcagcccga agattcacta
tggtgaaaat cgccttcaat acccctaccg ccgtgcaaaa 180 ggaggaggcg
cggcaagacg tggaggccct cctgagccgc acggtcagaa ctcagatact 240
gaccggcaag gagctccgag ttgccaccca ggaaaaagag ggctcctctg ggagatgtat
300 gcttactctc ttaggccttt cattcatctt ggcaggactt attgttggtg
gagcctgcat 360 ttacaagtac ttcatgccca agagcaccat ttaccgtgga
gagatgtgct tttttgattc 420 tgaggatcct gcaaattccc ttcgtggagg
agagcctaac ttcctgcctg tgactgagga 480 ggctgacatt cgtgaggatg
acaacattgc aatcattgat gtgcctgtcc ccagtttctc 540 tgatagtgac
cctgcagcaa ttattcatga ctttgaaaag ggaatgactg cttacctgga 600
cttgttgctg gggaactgct atctgatgcc cctcaatact tctattgtta tgcctccaaa
660 aaatctggta gagctctttg gcaaactggc gagtggcaga tatctgcctc
aaacttatgt 720 ggttcgagaa gacctagttg ctgtggagga aattcgtgat
gttagtaacc ttggcatctt 780 tatttaccaa ctttgcaata acagaaagtc
cttccgcctt cgtcgcagag acctcttgct 840 gggtttcaac aaacgtgcca
ttgataaatg ctggaagatt agacacttcc ccaacgaatt 900 tattgttgag
accaagatct gtcaagagta agaggcaaca gatagagtgt ccttggtaat 960
aagaagtcag agatttacaa tatgacttta acattaaggt ttatgggata ctcaagatat
1020 ttactcatgc atttactcta ttgcttatgc cgtaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1080 aa 1082 182 1292 DNA Mus musculus 182 ctctccccgg
ccgggagctc cggccgcgga gtgatggtgg caccggtggc gatgggccgg 60
gcagggacca tggaagtggc ggcagaggtg gcaggggagg ggcagctggc ggtggaggag
120 gctgtggtct tcaggggtct gtaggtggag gcatggctcg ggccagcagc
aggaacagca 180 gcgaagaggc ctgggggtca cttcaggcgc cgcaacagca
gcagagtccg gcagcatctt 240 ctcttgaggg agcaatttgg agacgagctg
gaacccagac tcgcgccctg gataccatcc 300 tttaccatcc acagcaatcc
catctgcttc gagagctgtg cccaggagtg aatacccagc 360 cctacctctg
tgagactggt cattgctgtg gggagactgg ctgctgcacc tactactatg 420
aactctggtg gttctggctg ctttggactg tcctcatcct ctttagctgc tgttgtgcct
480 tccgccaccg aagggctaaa ctcaggctgc aacagcaaca gcggcagcgt
gaaatcaact 540 tgttggctta ccatggggca tgccacgggg ctggccctgt
tccaaccggt tcactgcttg 600 accttcgcct cctcagcgcc ttcaaacccc
cagcctacga ggatgtggtt caccacccag 660 gcacaccgcc acctccttac
actgtgggcc caggctaccc ttggactact tccagtgaat 720 gcacccgctg
ctcttccgaa tccagctgct ctgcccactt ggaggggaca aatgtagaag 780
gtgtttcctc ccagcagagt gctctccctc accaggaggg tgagcccagg gcaggattga
840 gcccagttca cataccccct tcctgccgct atcgtcgcct aactggtgac
tcgggtattg 900 agctctgccc ttgtcctgac tccagtgaag gtgagccact
caaggaagcg agggctagtg 960 cctcccagcc agatctggaa gaccattccc
cttgtgcact gcccccagat tctgtgtccc 1020 aagttcctcc catggggctg
gcttctagtt gtgggacatc ccataagtag tttcaagagg 1080 gaaactgggt
attacttggc caccaggatt cagccctggt ttcaactgca gtcctccatg 1140
tgggaccgtc cccaccctcc tagaacacgc ctgaaaggct ggagccctga agaggggcag
1200 caccgaggac tgtgctatct ttactcactc ccaagacata cacaggagcc
tttaatctca 1260 ttaaagagac atgaaccagc aaaaaaaaaa aa 1292
* * * * *
References