Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
Diagnosis And Treatment Of
Tyrosine Phosphatase-Related Disorders
And Related Methods
Field Of The Invention
The present invention relates to protein tyrosine phos-
phatases. In particular, the invention concerns proteins we
have named PTP04, SAD, PTP05, PTP10, ALP, and ALK-7, nucleotide
sequences encoding these proteins, and various products and
assay methods that can be used for identifying compounds useful
for the diagnosis and treatment of various diseases and condi-
tions related to these proteins, for example cell proliferative
disorders.
Background Of The Invention
The following description is provided to aid in under-
standing the invention but is not admitted to be prior art to
the invention.
Cellular signal transduction is a fundamental mechanism
whereby external stimuli that regulate diverse cellular
processes are relayed to the interior of cells. One of the key
biochemical mechanisms of signal transduction involves the
reversible phosphorylation of proteins, which enables regula-
tion of the activity of mature proteins by altering their
structure and function. The best characterized protein kinases
in eukaryotes phosphorylate proteins on the alcohol moiety of
serine, threonine and tyrosine residues. These kinases largely
fall into two groups, those specific for phosphorylating
serines and threonines, and those specific for phosphorylating
tyrosines.
The phosphoryiation state of a given substrate is also
regulated by a class of proteins responsible for removal of the
II 41
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phosphate group added to a given substrate by a protein kinase.
The protein phosphatases can also be classified as being
specific for either serine/threonine or tyrosine. The known
enzymes can be divided into two groups - receptor and non-
receptor type proteins. Most receptor-type protein tyrosine
phosphatases (RPTPs) contain two conserved catalytic tyrosine
phosphatase domains each of which encompasses a segment of 240
amino acid residues (Saito et al, Cell Growth and Diff. 2:59-
65, 1991). The RPTPs can be subclassified further based upon
the amino acid sequence diversity of their extracellular
domains (Saito, et al, supra; Krueger, et al, Proc. Natl. Acad.
Sci. USA 89:7417-7421, 1992). Alignment of primary peptide
sequences of both types of known PTPases shows some sequence
consensus in catalytic domains and has made it possible to
identify cDNAs encoding proteins with tyrosine phosphate
activity via the polymerase chain reaction (PCR).
Many kinases and phosphatases are involved in regulatory
cascades wherein their substrates may include other kinases and
phosphatases whose activities are regulated by their
phosphorylation state. Ultimately the activity of some
downstream effector is modulated by phosphorylation resulting
from activation of such a pathway.
It is well established that the abnormal or inappropriate
activity of tyrosine kinases and/or tyrosine phosphatases plays
a role in a variety of human disorders including cell
proliferative disorders such as cancer, fibrotic disorders,
disorders of the immune system and metabolic disorders such as
diabetes. A need, therefore, exists to identify new tyrosine
kinases and phosphatases as a first step in understanding a
disease process and the subsequent identification of
therapeutic treatments for the disorder.
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Summary Of The Invention
The present invention concerns PTP04, SAD, PTP05, PTP10,
Alp, and ALK-7 polypeptides, nucleic acids encoding such
polypeptides, cells, tissues and animals containing such
nucleic acids, antibodies to the polypeptides, assays utilizing
the polypeptides, and methods relating to all of the foregoing.
A first aspect of the invention features an isolated,
enriched, or purified nucleic acid molecule encoding a PTP04, a
SAD, a PTP05, a PTP10, an ALP, or an ALK-7 polypeptide.
By "isolated" in reference to nucleic acid is meant a
polymer of 14, 17, 21 or more nucleotides conjugated to each
other, including DNA or RNA that is isolated from a natural
source or that is synthesized. The isolated nucleic acid of
the present invention is unique in the sense that it is not
found in a pure or separated state in nature. Use of the term
"isolated" indicates that a naturally occurring sequence has
been removed from its normal cellular (i.e., chromosomal)
environment. Thus, the sequence may be in a cell-free solution
or placed in a different cellular environment. The term does
not imply that the sequence is the only nucleotide sequence
present, but that it is essentially free (about 90 - 95~ pure
at least) of non-nucleotide material naturally associated with
it and thus is meant to be distinguished from isolated
chromosomes.
By the use of the term "enriched" in reference to nucleic
acid is meant that the specific DNA or RNA sequence constitutes
a significantly higher fraction (2 . 5 fold) of the total DNA
or RNA present in the cells or solution of interest than in
normal or diseased cells or in the cells from which the
sequence was taken. This could be caused by a person by
preferential reduction in the amount of other DNA or RNA
present, or by a preferential increase in the amount of the
specific DNA or RNA sequence, or by a combination of the two.
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However, it should be noted that "enriched" does not imply that
there are no other DNA or RNA sequences present, just that the
relative amount of the sequence of interest has been
significantly increased. The term "significant" here is used to
indicate that the level of increase is useful to the person
making such an increase, and generally means an increase
relative to other nucleic acids of about at least 2 fold, more
preferably at least 5 to 10 fold or even more. The term also
does not imply that there is no DNA or RNA from other sources.
The other source DNA may, for example, comprise DNA from a
yeast or bacterial genome, or a cloning vector such as pUCl9.
This term distinguishes the sequence from naturally occurring
enrichment events, such as viral infection, or tumor type
growths, in which the level of one mRNA may be naturally
increased relative to other species of mRNA. That is, the term
is meant to cover only those situations in which a person has
intervened to elevate the proportion of the desired nucleic
acid.
It is also advantageous for some purposes that a
nucleotide sequence be in purified form. The term "purified"
in reference to nucleic acid does not require absolute purity
(such as a homogeneous preparation); instead, it represents an
indication that the sequence is relatively purer than in the
natural environment (compared to the natural level this level
should be at least 2-5 fold greater, e. g. , in terms of mg/mL) .
Individual clones isolated from a cDNA library may be purified
to electrophoretic homogeneity. The claimed DNA molecules
obtained from these clones can be obtained directly from total
DNA or from total RNA. The cDNA clones are not naturally
occurring, but rather are preferably obtained via manipulation
of a partially purified naturally occurring substance
(messenger RNA).. The construction of a cDNA library from mRNA
involves the creation of a synthetic substance (cDNA) and pure
_T.
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individual cDNA clones can be isolated from the synthetic
library by clonal selection of the cells carrying the cDNA
library. Thus, the process which includes the construction of
a cDNA library from mRNA and isolation of distinct cDNA clones
5 yields an approximately 106-fold purification of the native
message. Thus, purification of at least one order of
magnitude, preferably two or three orders, and more preferably
four or five orders of magnitude is expressly contemplated.
The term is also chosen to distinguish clones already in
existence which may encode PTP04, SAD, PTP05, PTP10, ALP, or
Alk-7 but which have not been isolated from other clones in a
library of clones. Thus, the term covers clones encoding
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 which are isolated from
other non-PTP04, non-SAD, non-PTP05, non-PTP10, non-ALP, or
non-ALK-7 clones.
A PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
polypeptide can be encoded by a full-length nucleic acid
sequence or any portion of the full-length nucleic acid
sequence. In preferred embodiments the isolated nucleic acid
comprises, consists essentially of, or consists of a nucleic
acid sequence set forth in SEQ ID N0:1, SEQ ID NO:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID NO:6, SEQ ID N0:7, or
SEQ ID N0:8, a nucleic acid sequence that hybridizes to the
nucleic acid sequence set forth in SEQ ID NO: l, SEQ ID N0:2,
SEQ ID N0:3, SEQ ID NO:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
N0:7, or SEQ ID N0:8 or a functional derivative (as defined
below) of either. The nucleic acid may be isolated from a
natural source by cDNA cloning or subtractive hybridization
the natural source may be mammalian (human) blood, semen, or
tissue and the nucleic acid may be synthesized by the triester
or other method or by using an automated DNA synthesizer.
The term "hybridize" refers to a method of interacting a
nucleic acid sequence with a DNA or RNA molecule in solution or
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on a solid support, such as cellulose or nitrocellulose. If a
nucleic acid sequence binds to the DNA or RNA molecule with
high affinity, it is said to "hybridize" to the DNA or RNA
molecule. The strength of the interaction between the probing
sequence and its target can be assessed by varying the
stringency of the hybridization conditions. Various low or
high stringency hybridization conditions may be used depending
upon the specificity and selectivity desired. Stringency is
controlled by varying salt or denaturant concentrations.
As a general guideline, high stringency conditions
(hybridization at 50-65 °C, 5X SSPC, 50~ formamide, wash at 50-
65 °C, 0.5X SSPC) can be used to obtain hybridization between
nucleic acid sequences having regions which are greater than
about 90o complementary. Low stringency conditions
(hybridization at 35-37 °C, 5X SSPC, 40-45~ formamide, wash at
42 °C SSPC) can be used so that sequences having regions which
are greater than 35-45o complementarity will hybridize to the
probe. These conditions only represent examples of stringency
conditions and those skilled in the art recognize that these
conditions may be changed depending on the particular mode of
practice. Further examples of hybridization conditions are
shown in the examples below. Those skilled in the art will
recognize how such conditions can be varied to vary specificity
and selectivity. Under highly stringent hybridization
conditions only highly complementary nucleic acid sequences
hybridize. Preferably, such conditions prevent hybridization
of nucleic acids having one or two mismatches out of 20
contiguous nucleotides.
In yet other preferred embodiments the nucleic acid is an
isolated conserved or unique region, for example those useful
for the design of hybridization probes to facilitate identi
fication and cloning of additional polypeptides, or for the
.___ _ T___
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design of PCR probes to facilitate cloning of additional
polypeptides.
By "conserved nucleic acid regions", it is meant regions
present on two or more nucleic acids encoding a PTP04, a SAD, a
PTP05, a PTP10, an ALP, or an ALK-7 polypeptide, to which a
particular nucleic acid sequence can hybridize under lower
stringency conditions. Examples of lower stringency conditions
suitable for screening for nucleic acids encoding PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 polypeptides are provided in Abe,
et al. ,T. Biol. Chem. 19:13361 (1992). Preferably, conserved
regions differ by no more than 5 out of 20 continguous
nucleotides.
By ."unique nucleic acid region" it is meant a sequence
present in a full length nucleic acid coding for a PTP04, a
SAD, a PTP05, a PTP10, an ALP, or an ALK-7 polypeptide that is
not present in a sequence coding for any other known naturally
occurring polypeptide. Such regions preferably comprise 14,
17, 21 or more contiguous nucleotides present in the full
length nucleic acid encoding a PTP04, a SAD, a PTP05, a PTP10,
an ALP, or an ALK-7 polypeptide. In particular, a unique
nucleic acid region is preferably of human origin.
The invention also features a nucleic acid probe for the
detection of a nucleic acid encoding a PTP04, a SAD, a.PTP05, a
PTP10, an ALP, or an ALK-7 polypeptide in a sample. The
nucleic acid probe contains nucleic acid that will hybridize
specifically to a sequence of at least 14, preferably 17, 20 or
22, continguous nucleotides set forth in SEQ ID N0:1 or a
functional derivative thereof. The probe is preferably at
least 14, 17 or more bases in length and selected to hybridize
specifically to a unique region of a PTP04, a SAD, a PTP05, a
PTP10, an ALP, or an ALK-7 endocing nucleic acid.
In preferred embodiments the nucleic acid probe hybridizes
to nucleic acid encoding at least 14 contiguous amino acids of
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the full-length sequence set forth in SEQ ID NO:1, SEQ ID N0:2,
SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
N0:7, or SEQ ID N0:8 or a functional derivative thereof.
Various low or high stringency hybridization conditions may be
used depending upon the specificity and selectivity desired.
Under highly stringent hybridization conditions only highly
complementary nucleic acid sequences hybridize. Preferably,
such conditions prevent hybridization of nucleic acids having 1
or 2 mismatches out of 20 contiguous nucleotides.
Methods for using the probes include detecting the
presence or amount of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
RNA in a sample by contacting the sample with a nucleic acid
probe under conditions such that hybridization occurs and
detecting the presence or amount of the probe bound to PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 RNA. The nucleic acid duplex
formed between the probe and a nucleic acid sequence coding for
a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
polypeptide may be used in the identification of the sequence
of the nucleic acid detected (for example see, Nelson et al.,
in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San
Diego (Kricka, ed., 1992)). Kits for performing such methods
may be constructed to include a container means having disposed
therein a nucleic acid probe.
The invention also features recombinant nucleic acid,
preferably in a cell or an organism. The recombinant nucleic
acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ
ID N0:7, or SEQ ID N0:8 or a functional derivative thereof and
a vector or a promoter effective to initiate transcription in a
host cell. The recombinant nucleic acid can alternatively
contain a transcriptional initiation region functional in a
cell, a sequence complimentary to an RNA sequence encoding a
_ .._..---~-,~_... _ ..._..__ ~-.._.... _..-- _..T _
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PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 polypeptide
and a transcriptional termination region functional in a cell.
Another aspect of the invention features an isolated,
enriched, or purified PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide.
By "PTP04 polypeptide" it is meant an amino acid sequence
substantially similar to the sequence shown in SEQ ID N0:9, or
fragments thereof. By "SAD polypeptide" it is meant an amino
acid sequence substantially similar to the sequence shown in
SEQ ID N0:10, or fragments thereof. By "PTP05 polypeptide" or
"PTP10 polypeptide" it is meant an amino acid sequence
substantially similar to the sequence shown in SEQ ID N0:11,
SEQ ID N0:12, SEQ ID N0:13, or SEQ ID N0:19, or fragments
thereof. By "ALP polypeptide" it is meant an amino acid
sequence substantially similar to the sequence shown in SEQ ID
N0:15, or fragments thereof. By "ALK-7 polypeptide" it is
meant an amino acid sequence substantially similar to the
sequence shown in SEQ ID N0:16, or fragments thereof. Two
substantially similar sequences will preferably have at least
90g identity (more preferably at least 95o and most preferably
99-1000 to each other.
By "identity" is meant a property of sequences that
measures their similarity or relationship. Identity is
measured by dividing the number of identical residues in the
two sequences by the total number of residues and multiplying
the product by 100. Thus, two copies of exactly the same
sequence have 100 identity, but sequences that are less highly
conserved and have deletions, additions, or replacements have a
lower degree of identity. Those skilled in the art will
recognize that several computer programs are available for
determining sequence identity.
By "isolated" in reference to a polypeptide is meant a
polymer of 6, 12, 18 or more amino acids conjugated to each
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other, including polypeptides that are isolated from a natural
source or that are synthesized. The isolated polypeptides of
the present invention are unique in the sense that they are not
found in a pure or separated state in nature. Use of the term
5 "isolated" indicates that a naturally occurring sequence has
been removed from its normal cellular environment. Thus, the
sequence may be in a cell-free solution or placed in a
different cellular environment. The term does not imply that
the sequence is the only amino acid chain present, but that it
10 is essentially free (about 90 - 95% pure at least) of material
naturally associated with it.
By the use of the term "enriched" in reference to a
polypeptide it is meant that the specific amino acid sequence
constitutes a significantly higher fraction (2 - 5 fold) of the
total of amino acids present in the cells or solution of
interest than in normal or diseased cells or in the cells from
which the sequence was taken. This could be caused by a person
by preferential reduction in the amount of other amino acids
present, or by a preferential increase in the amount of the
specific amino acid sequence of interest, or by a combination
of the two. However, it should be noted that "enriched" does
not imply that there are no other amino acid sequences present,
just that the relative amount of the sequence of interest has
been significantly increased. The term significant here is
used to indicate that the level of increase is useful to the
person making such an increase, and generally means an increase
relative to other amino acids of about at least 2 fold, more
preferably at least 5 to 10 fold or even more . The term also
does not imply that there is no amino acid from other sources.
The other source amino acid may, for example, comprise amino
acid encoded by a yeast or bacterial genome, or a cloning
vector such as pUCl9. The term is meant to cover only those
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situations in which a person has intervened to elevate the
proportion of the desired nucleic acid.
It is also advantageous for some purposes that an amino
acid sequence be in purified form. The term "purified" in
reference to a polypeptide does not require absolute purity
(such as a homogeneous preparation) instead, it represents an
indication that the sequence is relatively purer than in the
natural environment (compared to the natural level this level
should be at least 2-5 fold greater, e. g. , in terms of mg/mL) .
Purification of at least one order of magnitude, preferably two
or three orders, and more preferably four or five orders of
magnitude is expressly contemplated. The substance is prefer-
ably free of contamination at a functionally significant level,
for example 90~, 95%, or 99o pure.
In another aspect the invention features an isolated,
enriched, or purified PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide fragment.
By "a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
polypeptide fragment" it is meant an amino acid sequence that
is less than the full-length PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 amino acid sequence shown in SEQ ID N0:2. Examples of
fragments include PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
domains, PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 mutants and
PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific epitopes.
By "a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
domain" it is meant a portion of the PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 polypeptide having homology to amino acid
sequences from one or more known proteins wherein the sequence
predicts some common function, interaction or activity. Well
known examples of domains are the SH2 (Src Homology 2) domain
(Sadowski, et al, Mol. Cell. Biol. 6:4396, 1986 Pawson and
Schlessinger, Curr. Biol. 3:434, 1993), the SH3 domain (Mayer,
et al, Nature 332:272, 1988; Pawson and Schlessinger, Curr.
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Biol. 3:434, 1993), and pleckstrin (PH) domain (Ponting, TIBS
21:245, 1996: Haslam, et al, Nature 363:309, 1993), all of
which are domains that mediate protein:protein interaction, and
the kinase catalytic domain (Hanks and Hunter, FASEB J 9:576-
595, 1995). Computer programs designed to detect such
homologies are well known in the art. The relative homology is
at least 20%, more preferably at least 30o and most preferably
at least 350.
By "a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
mutant" it is meant a PTP04, a SAD, a PTP05, a PTP10, an ALP, or
an ALK-7 polypeptide which differs from the native sequence in
that one or more amino acids have been changed, added or
deleted. Changes in amino acids may be conservative or non-
conservative. By "conservative" it is meant the substitution of
an amino acid for one with similar properties such as charge,
hydrophobicity, structure, etc. Examples of polypeptides
encompassed by this term include, but are not limited to, (1)
chimeric proteins which comprise a portion of a PTP04, a SAD, a
PTP05, a PTP10, an ALP, or an ALK-7 polypeptide sequence fused
to a non-PTP04, a non-SAD, a non-PTP05, a non-PTP10, a non-ALP,
or a non-ALK-7 polypeptide sequence, for example a polypeptide
sequence of hemagglutinin (HA), (2) PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 proteins lacking a specific domain, for example
the catalytic domain, and (3) PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 proteins having a point mutation. A PTP04, a SAD, a
PTP05, a PTP10, an ALP, or an ALK-7 mutant will retain some
useful function such as, for example, binding to a natural
binding partner, catalytic activity, or the ability to bind to
a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 specific
antibody (as defined below).
By "PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific
epitope" it is: meant a sequence of amino acids that is both
antigenic and unique to PTP04, SAD, PTP05, PTP10, ALP, or ALK-
_._ _ __ ____._______ _ __
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7. PTP04-, SAD-, PTP05-, PTP10-, ALP-, or ALK-7-specific
epitope can be used to produce PTP04-, SAD-, PTP05-, PTP10-,
ALP-, or ALK-7-specific antibodies, as more fully described
below. Particularly preferred epitopes are shown in Examples
below.
By "recombinant PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide" it is meant to include a polypeptide produced by
recombinant DNA techniques such that it is distinct from a
naturally occurring polypeptide either in its location (e. g.,
present in a different cell or tissue than found in nature),
purity or structure. Generally, such a recombinant polypeptide
will be present in a cell in an amount different from that
normally observed in nature.
In yet another aspect the invention features an antibody
(e. g., a monoclonal or polyclonal antibody) having specific
binding affinity to a PTP09, a SAD, a PTP05, a PTP10, an ALP,
or an ALK-7 polypeptide or polypeptide fragment. By "specific
binding affinity" is meant that the antibody binds to target
polypeptide with greater affinity than it binds to other
polypeptides under specified conditions. Antibodies or anti-
body fragments are polypeptides which contain regions that can
bind other polypeptides. The term "specific binding affinity"
describes an antibody that binds to a PTP04, a SAD, a PTP05, a
PTP10, an ALP, or an ALK-7 polypeptide with greater affinity
than it binds to other polypeptides under specified conditions.
The term "polyclonal" refers to antibodies that are
heterogenous populations of antibody molecules derived from the
sera of animals immunized with an antigen or an antigenic
functional derivative thereof. For the production of poly-
clonal antibodies, various host animals may be immunized by
injection with the antigen. Various adjuvants may be used to
increase the immunological response, depending on the host
species.
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"Monoclonal antibodies" are substantially homogenous
populations of antibodies to a particular antigen. They may be
obtained by any technique which provides for the production of
antibody molecules by continuous cell lines in culture.
Monoclonal antibodies may be obtained by methods known to those
skilled in the art. See, for example, Kohler, et al., Nature
256:495-497 (1975), and U.S. Patent No. 4,376,110.
The term "antibody fragment" refers to a portion of an
antibody, often the hypervariable region and portions of the
surrounding heavy and light chains, that displays specific
binding affinity for a particular molecule. A hypervariable
region is a portion of an antibody that physically binds to the
polypeptide target.
Antibodies or antibody fragments having specific binding
affinity to a PTP09, a SAD, a PTP05, a PTP10, an ALP, or an
ALK-7 polypeptide may be used in methods for detecting the
presence and/or amount of a PTP04, a SAD, a PTP05, a PTP10, an
ALP, or an ALK-7 polypeptide in a sample by probing the sample
with the antibody under conditions suitable for formation of an
immunocomplex between the antibody and the PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 polypeptide and detecting the presence
and/or amount of the antibody conjugated to the PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 polypeptide. Diagnostic kits for
performing such methods may be constructed to include
antibodies or antibody fragments specific for PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 as well as a conjugate of a binding
partner of the antibodies or the antibodies themselves.
An antibody or antibody fragment with specific binding
affinity to a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an
ALK-7 polypeptide can be isolated, enriched, or purified from a
prokaryotic or eukaryotic organism. Routine methods known to
those skilled in the art enable production of antibodies or
antibody fragments, in both prokaryotic and eukaryotic
_ t
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organisms. Purification, enrichment, and isolation of
antibodies, which are polypeptide molecules, are described
above.
In another aspect the invention features a hybridoma which
5 produces an antibody having specific binding affinity to a
PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK-7
polypeptide. By "hybridoma" is meant an immortalized cell line
which is capable of secreting an antibody, for example a PTP04,
a SAD, a PTP05, a PTP10, an ALP, or an ALK-7 antibody. In
10 preferred embodiments the PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 antibody comprises a sequence of amino acids that is able
to specifically bind a PTP04, a SAD, a PTP05, a PTP10, an ALP,
or an ALK-7 polypeptide.
In another embodiment, the invention encompasses a
15 recombinant cell or tissue containing a purified nucleic acid
coding for a PTP04, a SAD, a PTP05, a PTP10, an ALP, or an ALK
7 polypeptide. In such cells, the nucleic acid may be under
the control of its genomic regulatory elements, or may be under
the control of exogenous regulatory elements including an
exogenous promoter. By "exogenous" it is meant a promoter that
is not normally coupled transcriptionally to the coding
sequence for the PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide in its native state.
The invention features a method for identifying human
cells containing a PTP04, a SAD, a PTP05, a PTP10, an ALP, or
an ALK-7 polypeptide or a related sequence. The method
involves identifying the novel polypeptide in human cells using
techniques that are routine and standard in the art, such as
those described herein for identifying PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 (e. g., cloning, Southern or Northern blot
analysis, in situ hybridization, PCR amplification, etc.).
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The invention also features methods of screening cells for
natural binding partners of PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 polypeptides.
The term "natural binding partner" refers to molecules, or
portions of these molecules, that bind to the protein of
interest in cells. Natural binding partners may be
polypeptides or lipids, but do not include glutathione.
Natural binding partners can play a role in propagating a
signal in a protein signal transduction process. A change in
the interaction between a protein and a natural binding partner
can manifest itself as an increased or decreased probability
that the interaction forms, or an increased or decreased
concentration of the protein/natural binding partner complex.
A protein's natural binding partner can bind to a protein's
intracellular region with high affinity. High affinity
represents an equilibrium binding constant on the order of 10-6
M or less. In addition, a natural binding partner can also
transiently interact with a protein's intracellular region and
chemically modify it. Natural binding partners of protein are
chosen from a group that includes, but is not limited to, SRC
homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine
binding (PTB) domains, guanine nucleotide exchange factors,
protein phosphatases, and other protein kinases or protein
phosphatases. Methods of determining changes in interactions
between proteins and their natural binding partners are readily
available in the art.
In another aspect, the invention provides an assay to
identify substances capable of modulating the activity of
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7. Such assays may be
performed in vitro or in vivo can be obtained by modifying
existing assays, such as the assays described in WO 96/90276,
published December 19, 1996 and WO 96/14433, published May 17,
1996. Other possibilities include testing for phosphatase
_..._ .T_ _ _ __._. _
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17
activity on standard substrates such as Src kinase or synthetic
amino acid substrates. The substances so identified may be
enhances or inhibitors of PTP09, SAD, PTP05, PTP10, ALP, or
ALK-7 activity and can be peptides, natural products (such as
those isolated from fungal strains, for example) or small
molecular weight chemical compounds. A preferred substance
will be a compound with a molecular weight of less than 5, 000,
more preferably less than 1,000, most preferably less than 500.
The assay and substances contemplated by the invention are
discussed in more detail below.
In a preferred embodiment, the invention provides a method
for treating or preventing an abnormal condition by admi-
nistering a compound which is a modulator of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 function in vitro. The abnormal condition
preferably involves abnormality in PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 signal transduction pathway, and most preferably
is cancer. Such compounds preferably show positive results in
one or more in vitro assays for an activity corresponding to
treatment of the disease or disorder in question (such as the
assays described in examples 5, 10, 15, 20, and 21 below).
Examples of substances that can be screened for favorable
activity are provided in section XIV below.
Substances identified as modulators of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 activity can be used to study the effects
of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 modulation in animal
models of cell proliferative disorders. For example,
inhibitors of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity
can be tested as treatments for cell proliferative disorders
such as leukemia or lymphoma using subcutaneous xenograph
models in mice.
In a further aspect, the invention provides a method for
identifying modulators of protein activity. The method
involves the steps of: a) forming a captured protein by
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contacting the protein with a natural binding partner; b)
contacting the captured protein with a test compound: and c)
measuring the protein activity. Preferably, the method also
includes the step of comparing the protein activity with the
activity of a control protein, which has the same amino acid
sequence as the protein in step (a) without the natural binding
partner, to determine the extent of modulation.
The term "modulator" refers to a compound which has the
ability of altering the activity of a protein. A modulator may
activate the activity of the protein, may activate or inhibit
the activity of the protein depending on the concentration of
the compound exposed to the protein, or may inhibit the
activity of the protein.
The term "modulator" also refers to a compound that alters
the function of a protein by increasing or decreasing the
probability that a complex forms between a protein and a
natural binding partner. A modulator preferably increases the
probability that such a complex forms between the protein and
the natural binding partner, more preferably increases or
decreases the probability that a complex forms between the
protein and the natural binding partner depending on the
concentration of the compound exposed to the protein, and most
preferably decreases the probability that a complex forms
between the protein and the natural binding partner.
The term "activity of a protein", in the context of the
invention, defines the natural function of a protein in a cell.
Examples of protein function include, but are not limited to,
catalytic activity and binding a natural binding partner.
The term "activates" refers to increasing the natural
function of a protein. The protein function is preferably the
interaction with a natural binding partner and most preferably
catalytic activity.
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The term "inhibit" refers to decreasing the cellular
function of a protein. The protein function is preferably the
interaction with a natural binding partner and most preferably
catalytic activity.
The term "catalytic activity", in the context of the
invention, defines the rate at which a protein reacts with a
- substrate. Catalytic activity can be measured, for example, by
determining the amount of a substrate converted to a product as
a function of time. When the protein is a protein kinase or a
protein phosphatase, then the reaction with a substrate is the
phosphorylation or dephosphorylation of the substrate, respec-
tively. Phosphorylation or dephosphorylation of a substrate
occurs at the active-site of a protein kinase or a protein
phosphatase. The active-site is normally a cavity in which the
substrate binds to the protein kinase or protein phosphatase
and is phosphorylated.
The term "substrate" as used herein refers to a molecule
which is acted upon by an enzyme. If the enzyme is a protein
kinase then the substrate is phosphorylated by the protein
kinase. If the enzyme is a protein phosphatase then the
substrate is dephosphorylated by the protein phosphatase.
The term "compound" refers to a molecule which has at least
two types of atoms in its composition. The molecule may be a
small organic molecule. The term "organic molecule" refers to a
molecule which has at least one carbon atom in its structure.
The term "complex" refers to an assembly of at least two
molecules bound to one another. Signal transduction complexes
often contain at least two protein molecules bound to one
another. For instance, a protein tyrosine receptor protein
kinase, GRB2, SOS, RAF, and RAS assemble to form a signal
transduction complex in response to a mitogenic ligand.
The term "contacting" as used herein refers to any touching
between a compound and a protein, preferably the mixing of a
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solution comprising a compound with a liquid medium bathing the
protein of the methods. The touching may .involve interaction
between the compound and the protein. The solution comprising
the compound may be added to the medium bathing the protein by
5 utilizing a delivery apparatus, such as a pipet-based device or
syringe-based device.
The term "protein" as used herein refers to a naturally
occurring or chemically modified polypeptide chain that has
distinct secondary and tertiary structures. The chemical
10 modification may be point mutations. The term "protein" as used
herein does not include a polypeptide chain which is covalently
fused or otherwise joined through human intervention with
another distinct polypeptide chain. For example, a GST-fusion
protein is not included under the term "protein" as used herein.
15 The term "captured protein" as used herein refers to a
protein that has come to contact with one of its natural
binding partners and has formed a complex with the natural
binding partner. The natural binding partner may be free in
the solution, bound to a solid support, or free in the solution
20 with the ability to bind to a solid support.
The term "test compound" refers to a compound under study
for its potential effect on the catalytic activity of a
protein.
The term "control protein" refers to a protein which has
the same amino acid sequence of the captured protein but is not
being modulated by a test compound, nor has it come in contact
with a test compound, nor is it bound to a natural binding
partner. The activity of a control protein can be measured
using the techniques of the invention, and such activity may be
compared with the activity of a modulated protein. A
difference between the levels of the two measured activities
determines the: extent of modulation by the modulators.
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The invention provides a method for identifying modulators
of protein activity, where the method is. preferably a non-
radioactive method. The protein is preferably not a fusion
protein. Most preferably, the protein is not a GST-fusion
protein. The protein is preferably an enzyme, a receptor
enzyme, or a non-receptor enzyme, more preferably a protein
kinase, and most preferably a protein tyrosine kinase. The
protein tyrosine kinase is preferably Zap70 or Syk. In other
preferred embodiments, the protein is a protein tyrosine
phosphatase, and more preferably the protein is PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7.
The term "fusion protein" refers to a heterologous protein
formed by the covalent linkage of two distinct polypeptides.
The term "GST-fusion protein" refers to a heterologous protein
formed by the covalent linkage of a polypeptide and glutathione
S-transferase (GST).
The term °enzyme" refers to a protein that can act as a
catalyst for biological reactions. Examples of catalyzed
biological reactions include, but are not limited to, formation
of new bonds, addition of water, addition of a phosphoryl
group, and isomerization of an organic molecule.
The term "catalyst" refers to a compound or a dissolved
metal ion that increases the rate of a chemical reaction
without being consumed in the reaction.
The term "receptor enzyme" refers to an enzyme that has a
portion of its amino acid sequence within the cell membrane.
The term "non-receptor enzyme" refers to an enzyme that has
none of its amino acid sequence within the cell membrane. The
non-receptor enzyme may be associated with the membrane via
interactions, such as covalent linkage with fatty acids of the
membrane.
The term "protein kinase" refers to an enzyme that
transfers the high energy phosphate of adenosine triphosphate
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to an amino acid residue, either tyrosine, serine, or
threonine, located on a protein target.
The term "protein tyrosine kinase," or PTK, refers to an
enzyme that transfers the high energy phosphate of adenosine
triphosphate to a tyrosine residue located on a protein target.
"Zap70" and "Syk" are protein tyrosine kinases of the Syk
family which is characterized by the presence of two tandemly
arranged Src-homology 2 (SH2) domains and no membrane
localization motifs. These proteins are probably
phosphorylated by the Src family of protein tyrosine kinases at
the two tyrosine residues within the ITAM motif.
The term "ITAM motif" stands for "immunoreceptor tyrosine-
based activation motif" and refers to a 16 amino acid motif
(YXXLX6_BYXXL) that is conserved in all of the signal
transducing subunits of the T-cell antigen receptor (TCR) (c. f.
Chan, et al. (1995) The EMBO Journal, 14:11, 2499-2508).
The term "protein tyrosine phosphatase" refers to an enzyme
that removes a phosphate group from a phosphotyrosine in a
protein target.
In a preferred embodiment, the natural binding partner of
one of the above proteins is capable of binding to a solid
support. The natural binding partner is preferably a peptide,
more preferably a phosphopeptide, and most preferably the
phosphopeptide comprises an ITAM motif. In other preferred
embodiments, the natural binding partner comprises a lipid.
The term "solid support" as used herein refers to an
insoluble surface to which a molecule can be bound. Examples
of solid supports include, but are not limited to, well plates
(i.e. 96-well plates), glass beads, or resins (i.e. cellulose,
agarose, polypropylene, polystyrene, etc.). Natural binding
partners can be attached, through either covalent or non-
covalent interactions, to the solid support prior to or after
binding a protein. Examples of non-covalent interactions
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include, but are not limited to, hydrogen bonds, electrostatic
interactions, and hydrophobic interactions.
The term "peptide" refers to an arrangement of two or more
amino acids, linked together through an amide bond between the
carboxyl end of one amino acid and the amino end of another.
The term "phosphopeptide" refers to a peptide that has a
phosphate group chemically attached to one of its amino acid
residues.
The term "lipid" refers to a water-insoluble substance that
can be extracted from cells by organic solvents of low
polarity. Examples of lipids include, but are not limited to,
glycerides, steroids, and terpenes.
The modulators of protein activity being identified by the
methods of the invention preferably modulate the autocatalytic
activity, catalytic activity, or binding of a second natural
binding partner.
The activity of an enzyme is "autocatalytic activity" when
the enzyme and its substrate are identical. Some receptor
protein tyrosine kinases are capable of exhibiting
autocatalytic activity.
In preferred embodiments, the invention provides a method
for identifying modulators of protein activity, comprising the
step of contacting the captured protein with one or more
components of the group consisting of a substrate, a second
natural binding partner, and an antibody. The method
preferably further involves the step of lysing cells before
forming the captured protein. Most preferably, the method
involves the step of washing the solid support after capturing
the protein and binding the protein: natural binding partner
complex to the solid support and prior to measuring the protein
activity.
In another aspect, the invention provides a kit for the
identification of modulators of non-receptor enzyme activity
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comprising: a) a natural binding partner; b) a solid support;
and c) one or more components selected from the group
consisting of a substrate, a second natural binding partner,
and an antibody.
The natural binding partner in the above kit is preferably
a peptide, more preferably a phosphopeptide. Even more
preferably the phosphopeptide comprises an ITAM motif. In
other preferred embodiment, the natural binding partner
comprises a lipid.
The summary of the invention described above is non-
limiting and other features and advantages of the invention
will be apparent from the following detailed description, and
from the claims.
Brief Description of the Fiaures
Figure 1 shows a comparison between the amino acid
sequence of human PTP04 and the amino acid sequence of the
protein to which it is most closely related, murine 2PEP. The
relative homology between the two (approximately 70%) suggests
that the two proteins are members of the same PTP family but
are not species orthologs.
Detailed Descri tion of the Invention
The present invention relates to the isolation and
characterization of new proteins which we have called PTP04,
SAD, PTP05, PTP10, ALP, and ALK-7, nucleotide sequences
encoding PTP04, SAD, PTP05, PTP10, ALP, or ALK-7, various
products and assay methods that can be used to identify
compounds useful for the diagnosis and treatment of various
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 related diseases and
conditions, for example cancer. Polypeptides derived from
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 and nucleic acids
encoding such polypeptides may be produced using well known and
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standard synthesis techniques when given the sequences
presented herein.
I. The Polypeptides of the Invention
5
A. PTP04
PTP04 is a tyrosine phosphatase with an apparent molecular
weight of approximately 100 kDa. Primary sequence analysis
shows that PTP04 is comprised of three domains: an N-terminal
10 domain, a catalytic domain, and a C-terminal domain. The lack
of a hydrophobic stretch of amino acids generally characterized
as a transmembrane region indicates that PTP04 is a non-
receptor tyrosine phosphatase.
The full-length PTP04 was originally isolated from a human
15 leukemia cell line. Subsequent expression analysis of both
normal tissues and cancer cell lines, shown in detail below,
revealed that PTP04 is expressed in human thymus and has very
low expression in other normal cells but is significantly
overexpressed in a number of tumors, particularly in leukemias
20 and lymphomas. This suggests that PTP04 plays an important
role in the growth and persistence of these cancers.
B . SAD
SAD is a tyrosine kinase with an apparent molecular weight
25 of approximately 55 kDa. Primary sequence analysis shows that
SAD is comprised of four domains: a domain at the N-terminus
that shows no homology to any known sequence (the unique
domain), an SH3 domain, an SH2 domain and a catalytic domain.
The lack of a hydrophobic stretch of amino acids generally
characterized as a transmembrane region indicates that SAD is a
non-receptor tyrosine kinase. A comparison of the amino acid
sequences suggests that SAD is a member of the Frk family.
Like some other members of this family, SAD lacks an N-terminal
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myristylation site and a C-terminal regulatory tyrosine
characteristic of Src family members. It is most closely
related to the murine NR-TK Srm (Kohmura, et al, Mol. Cell.
Bio. 14(10):6915, 1994) with approximately 85~ sequence
homology in the catalytic domain. (Discussed in detail in the
examples below.)
SAD was originally isolated from a human breast cancer
cell line. Subsequent expression analysis of both normal
tissues and cancer cell lines, shown in detail below, revealed
that SAD has very limited expression in normal cells but is
significantly overexpressed in a number of tumors. This
suggests that SAD plays an important role in the growth and
persistence of these cancers.
C. PTP05 and PTP10
PTP05 is a tyrosine phosphatase with an apparent molecular
weight of approximately 49 kDa. Two additional isoforms have
been identified, one larger (approximately 54 kDa) and one
smaller (approximately 47 kDa). Primary sequence analysis
shows that PTP05 is comprised of three domains: an N-terminal
domain, a catalytic domain, and a C-terminal domain. The lack
of a hydrophobic stretch of amino acids generally characterized
as a transmembrane region indicates that PTP05 is a non-
receptor tyrosine phosphatase. PTP10 is also a tyrosine
phosphatase with significant homology to PTP05. Together they
define a new family of PTPs.
D. ALP
ALP is a tyrosine phosphatase with an apparent molecular
weight of approximately 160 - 200 kDa. Primary sequence
analysis shows that ALP is comprised of three domains: a
domain at the N-terminus that is rich in proline residues
(30.60 and contains several tyrosines that may be
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phosphorylated, a catalytic domain, and a C-terminal domain
containing region rich in prolines and serines (45.60 that
resenbling a PEST motif (Rogers, et al, Science 234:364, 1986).
These proline rich regions may be protein: protein interaction
sites as SH3 domains have been shown to bind to proline rich
regions (Morton and Campbell, Curr. Biol. 4:514, 1994; Ren, et
al, Science 259:1157, 1993). The lack of a hydrophobic stretch
of amino acids generally characterized as a transmembrane
region indicates that ALP is a non-receptor tyrosine
phosphatase.
The full-length ALP was originally isolated from a human
brain cancer cell line. Subsequent expression analysis of both
normal tissues and cancer cell lines, shown in detail below,
revealed that ALP has low expression in normal cells but is
significantly overexpressed in a number of tumors. This
suggests that ALP plays an important role in the growth and
persistence of these cancers.
E. ALK-7
ALK-7 is a type I receptor serine/threonine kinase (STK
receptor). Proteins with some homology have been described in
the rat (Ryden, et al. J. Biol. Chem. 271:30603, 1996;
Tsuchida, et al. Molec. Cell. Neurosci. 7:467, 1996), however,
unlike the rat proteins, the human ALK-7 is expressed in more
restricted regions of the brain, notably hippocampous,
hypothalamic nuclei, sustantia nigra, an pituitary. This
extremely restricted expression pattern strongly suggests a
role for human ALK-7 in the growth and/or survival of neurons
and its relevance in treatment of such diseases as Parkinson's,
Huntington's disease and Alzheimer's.
The polypeptide and nucleotide sequences of the invention
can be used, therefore, to identify modulators of cell growth
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and survival which are useful in developing therapeutics for
various cell proliferative disorders and conditions, and in
particular cancers related to inappropriate PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 activity. Assays to identify compounds
that act intracellularly to enhance or inhibit PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 activity can be developed by
creating genetically engineered cell lines that express PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 nucleotide sequences, as is
more fully discussed below.
II. Nucleic Acids Encoding the Poly eptides of the Invention.
A first aspect of the invention features nucleic acid
sequences encoding a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide. Included within the scope of this invention are
the functional equivalents of the herein-described isolated
nucleic acid molecules. Functional equivalents or derivatives
can be obtained in several ways. The degeneracy of the genetic
code permits substitution of certain codons by other codons
which specify the same amino acid and hence would give rise to
the same protein. The nucleic acid sequence can vary
substantially since, with the exception of methionine and
tryptophan, the known amino acids can be coded for by more than
one codon. Thus, portions or all of the PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 gene could be synthesized to give a
nucleic acid sequence significantly different from that shown
in SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
N0:5, SEQ ID N0:6, SEQ ID N0:7, or_SEQ ID N0:8. The encoded
amino acid sequence thereof would, however, be preserved.
In addition, the nucleic acid sequence may comprise a
nucleotide sequence which results from the addition, deletion
or substitution of at least one nucleotide to the 5'-end and/or
the 3'-end of ahe nucleic acid formula shown in SEQ ID N0:1,
SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID
_T__ ....... _..._....._. ._........... _..
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N0:6, SEQ ID N0:7, or SEQ ID N0:8, or a derivative thereof.
Any nucleotide or polynucleotide may be used in this regard,
provided that its addition, deletion or substitution does not
alter the amino acid sequence of SEQ ID N0:9, SEQ ID N0:10, SEQ
ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID
N0:15, or SEQ ID N0:16 which is encoded by the nucleotide
sequence. For example, the present invention is intended to
include any nucleic acid sequence resulting from the addition
of ATG as an initiation codon at the 5'-end of the PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 nucleic acid sequence or its
functional derivative, or from the addition of TTA, TAG or TGA
as a termination codon at the 3'-end of the inventive
nucleotide sequence or its derivative. Moreover, the nucleic
acid molecule of the present invention may, as necessary, have
restriction endonuclease recognition sites added to its 5'-end
and/or 3'-end.
Such functional alterations of a given nucleic acid
sequence afford an opportunity to promote secretion and/or
processing of heterologous proteins encoded by foreign nucleic
acid sequences fused thereto. All variations of the nucleotide
sequence of the PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 genes
and fragments thereof permitted by the genetic code are,
therefore, included in this invention.
Further, it is possible to delete codons or to substitute
one or more codons by codons other than degenerate codons to
produce a structurally modified polypeptide, but one which has
substantially the same utility or activity of the polypeptide
produced by the unmodified nucleic acid molecule. As recogn
ized in the art, the two polypeptides are functionally
equivalent, as are the two nucleic acid molecules which give
rise to their production, even though the differences between
the nucleic acid molecules are not related to degeneracy of the
genetic code.
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Functional equivalents or derivatives of PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 can also be obtained using nucleic
acid molecules encoding one or more functional domains of the
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide.
5 The catalytic domain of PTP04 functions as an enzymatic
remover of phosphate molecules bound onto tyrosine amino acids
and a nucleic acid sequence encoding the catalytic domain alone
or linked to other heterologous nucleic acid sequences can be
considered a functional derivative of PTP04. Other functional
10 domains of PTP04 include, but are not limited to, the proline-
rich region within the N-terminal domain, and the C-terminal
domain. Nucleic acid sequences encoding these domains are
shown in SEQ ID N0:1 as follows: N-terminal domain 53-196;
catalytic domain 197-934, C-terminal domain 935-2473.
15 The SH2 domain of SAD functions as a phosphorylated
tyrosine binding domain and a nucleic acid sequence encoding
the SH2 domain alone or linked to other heterologous nucleic
acid sequences can be considered a functional derivative of
SAD. Other functional domains of SAD include, but are not
20 limited to, the unique domain, the SH3 domain, and the
catalytic domain. Nucleic acid sequences encoding these
domains are shown in SEQ ID N0:2 as follows: N-terminal unique
domain approximately 49-213; SH3 domain approximately 214-375;
SH2 domain approximately 406-684; catalytic domain
25 approximately 736-1488.
The catalytic domain of PTP05 functions to remove
phosphate molecules bound onto tyrosine residues and a nucleic
acid sequence encoding the catalytic domain alone or linked to
other heterologous nucleic acid sequences can be considered a
30 functional derivative of PTP05. Other functional domains of
these proteins include, but are not limited to, the proline-
rich region within the N-terminal domain, and the C-terminal
domain. Nucleic acid sequences encoding these domains are
_ t -_ t
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shown in SEQ ID N0:3 as follows: N-terminal domain
approximately 199-759 ; catalytic domain approximately 760-
1458, C-terminal domain approximately 1459-1476.
The N-terminal proline-rich domain of ALP functions as a
SH3 binding domain and a nucleic acid sequence encoding the N
terminal proline-rich domain alone or linked to other
heterologous nucleic acid sequences can be considered a
functional derivative of ALP. Other functional domains of ALP
include, but are not limited to, the proline-rich region within
the N-terminal proline-rich domain, the C-terminal
proline/serine-rich domain, the proline/serine-rich region
within the C-terminal proline/serin-rich domain, and the
catalytic domain. Nucleic acid sequences encoding these
domains are shown in SEQ ID N0:7 as follows: N-terminal domain
313-2883; proline-rich region 1369-2643 : catalytic domain
approximately 2884-3600, C-terminal proline/serine-rich domain
3601-4134, proline/serine-rich region 3613-4456.
The extracellular domain of ALK-7 functions as a ligand or
co-receptor binding domain and a nucleic acid sequence encoding
the extracellular domain alone or linked to other heterologous
nuclic acid sequences can be considered a functional derivative
of ALK-7. Other functional domains of ALK-7 include, but are
not limited to, the signal sequence, the transmembrane domain,
the intracellular domain, and the catalytic domain. Nucleic
acid sequences encoding these domains are shown in SEQ ID N0:8
as follows: signal sequence 155-229; extracellular domain 155-
993; transmembrane domain 494-568; intracellular domain 569-
1633: catalytic domain approximately 731-1609. It should be
noted that the signal sequence is cleaved from the
extracellular domain in the mature protein.
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III. A Nucleic Acid Probe for the Detection of the Proteins of
the Invention.
A nucleic acid probe of the present invention may be used
to probe an appropriate chromosomal or cDNA library by usual
hybridization methods to obtain another nucleic acid molecule
of the present invention. A chromosomal DNA or cDNA library
may be prepared from appropriate cells according to recognized
methods in the art (e. g. "Molecular Cloning: A Laboratory
Manual", second edition, edited by Sambrook, Fritsch, &
Maniatis, Cold Spring Harbor Laboratory, 1989).
In the alternative, chemical synthesis is carried out in
order to obtain nucleic acid probes having nucleotide sequences
which correspond to N-terminal and C-terminal portions of the
amino acid sequence of the polypeptide of interest. Thus, the
synthesized nucleic acid probes may be used as primers in a
polymerase chain reaction (PCR) carried out in accordance with
recognized PCR techniques, essentially according to PCR
Protocols, "A Guide to Methods and Applications", edited by
Michael et al., Academic Press, 1990, utilizing the appropriate
chromosomal or cDNA library to obtain the fragment of the
present invention.
One skilled in the art can readily design such probes
based on the sequence disclosed herein using methods of
computer alignment and sequence analysis known in the art
(e. g.. "Molecular Cloning: A Laboratory Manual", second
edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring
Harbor Laboratory, 1989). The hybridization probes of the
present invention can be labeled by standard labeling
techniques such as with a radiolabel, enzyme label, fluorescent
label, biotin-avidin label, chemiluminescence, and the like.
After hybridization, the probes may be visualized using known
methods.
_ __~_. _r - _._______. _~_.__ T _ _ _.
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The nucleic acid probes of the present invention include
RNA as well as DNA probes and nucleic acids modified in the
sugar, phosphate or even the base portion as long as the probe
still retains the ability to specifically hybridize under
conditions as disclosed herein. Such probes are generated
using techniques known in the art. The nucleic acid probe may
be immobilized on a solid support. Examples of such solid
supports include, but are not limited to, plastics such as
polycarbonate, complex carbohydrates such as agarose and
sepharose, acrylic resins, such as polyacrylamide and latex
beads, and nitrocellulose. Techniques for coupling nucleic
acid probes to such solid supports are well known in the art.
The test samples suitable for nucleic acid probing methods
of the present invention include, for example, cells or nucleic
acid extracts of cells, or biological fluids. The sample used
in the above-described methods will vary based on the assay
format, the detection method and the nature of the tissues,
cells or extracts to be assayed. Methods for preparing nucleic
acid extracts of cells are well known in the art and can be
readily adapted in order to obtain a sample which is compatible
with the method utilized.
IV. A Probe Based Method And Kit For Detecting the Proteins of
the Invention.
One method of detecting the presence of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 in a sample comprises (a) contacting the
sample with the above-described nucleic acid probe, under
conditions such that hybridization occurs, and (b) detecting
the presence of the probe bound to the nucleic acid molecule.
One skilled in the art would select the nucleic acid probe
according to techniques known in the art as described above.
Samples to be tested include but should not be limited to RNA
samples of human tissue.
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A kit for detecting the presence of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 in a sample comprises at least one
container having disposed therein the above-described nucleic
acid probe. The kit may further comprise other containers
comprising one or more of the following: wash reagents and
reagents capable of detecting the presence of bound nucleic
acid probe. Examples of detection reagents include, but are
not limited to radiolabelled probes, enzymaticly labeled probes
(horseradish peroxidase, Alkaline phosphatase), and affinity
labeled probes (biotin, avidin, or steptavidin).
In detail, a compartmentalized kit includes any kit in
which reagents are contained in separate containers. Such
containers include small glass containers, plastic containers
or strips of plastic or paper. Such containers allow the
efficient transfer of reagents from one compartment to another
compartment such that the samples and reagents are not cross-
contaminated and the agents or solutions of each container can
be added in a quantitative fashion from one compartment to
another. Such containers will include a container which will
accept the test sample, a container which contains the probe or
primers used in the assay, containers which contain wash
reagents (such as phosphate buffered saline, Tris-buffers, and
the like), and containers which contain the reagents used to
detect the hybridized probe, bound antibody, amplified product,
or the like. One skilled in the art will readily recognize
that the nucleic acid probes described in the present invention
can readily be incorporated into one of the established kit
formats which are well known in the art with or without a set
of instructions concerning the use of such reagents in an
assay.
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V. DNA Constructs Comprising a PTP04, a SAD, a PTP05, a
PTP10, an ALP, or an ALK-7 Nucleic Acid Molecule and Cells
Containing These Constructs.
The present invention also relates to a recombinant DNA
5 molecule comprising, 5' to 3', a promoter effective to initiate
transcription in a host cell and the above-described nucleic
acid molecules. In addition, the present invention relates to
a recombinant DNA molecule comprising a vector and a nucleic
acid molecule described herein. The present invention also
10 relates to a nucleic acid molecule comprising a transcriptional
region functional in a cell, a sequence complimentary to an RNA
sequence encoding an amino acid sequence corresponding to a
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide or
functional derivative, and a transcriptional termination region
15 functional in said cell. The above-described molecules may be
isolated and/or purified DNA molecules.
The present invention also relates to a cell or organism
that contains a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 nucleic
acid molecule as described herein and thereby is capable of
20 expressing a peptide. The polypeptide may be purified from
cells which have been altered to express the polypeptide. A
cell is said to be "altered to express a desired polypeptide"
when the cell, through genetic manipulation, is made to produce
a protein which it normally does not produce or which the cell
25 normally produces at lower levels. One skilled in the art can
readily adapt procedures for introducing and expressing either
genomic, cDNA, or synthetic sequences into either eukaryotic or
prokaryotic cells.
A nucleic acid molecule, such as DNA, is said to be
30 "capable of expressing" a polypeptide if it contains nucleotide
sequences which contain transcriptional and translational
regulatory information and such sequences are "operably linked"
to nucleotide sequences which encode the polypeptide. An
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operable linkage is a linkage in which the regulatory DNA
sequences and the DNA sequence sought to be expressed are
connected in such a way as to permit gene sequence expression.
The precise nature of the regulatory regions needed for gene
sequence expression may vary from organism to organism, but
will in general include a promoter region which, in
prokaryotes, contains both the promoter (which directs the
initiation of RNA transcription) as well as the DNA sequences
which, when transcribed into RNA, will signal synthesis
initiation. Such regions will normally include those 5'-non-
coding sequences involved with initiation of transcription and
translation, such as the TATA box, capping sequence, CAAT
sequence, and the like.
If desired, the non-coding region 3' to the sequence
encoding a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene may be
obtained by the above-described cloning methods. This region
may be retained for its transcriptional termination regulatory
sequences, such as termination and polyadenylation. Thus, by
retaining the 3'-region naturally contiguous to the DNA
sequence encoding a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
gene, the transcriptional termination signals may be provided.
Where the transcriptional termination signals are not
satisfactorily functional in the expression host cell, then a
3' region functional in the host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and
a PTP04, SAD, PTPOS, PTP10, ALP, or ALK-7 sequence) are said to
be operably linked if the nature of the linkage between the two
DNA sequences does not (1) result in the introduction of a
frame-shift mutation, (2) interfere with the~ability of the
promoter region sequence to direct the transcription of a
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene sequence, or (3)
interfere with. the ability of the a PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 gene sequence to be transcribed by the promoter
_ _ __. ._...__T ...........__ _.... ____-_-__.._T...
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region sequence. Thus, a promoter region would be operably
linked to a DNA sequence if the promoter were capable of
effecting transcription of that DNA sequence. Thus, to express
a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene, transcriptional
and translational signals recognized by an appropriate host are
necessary.
The present invention encompasses the expression of a
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 gene (or a functional
derivative thereof) in either prokaryotic or eukaryotic cells.
Prokaryotic hosts are, generally, very efficient and convenient
for the production of recombinant proteins and are, therefore,
one type of preferred expression system for a PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 gene. Prokaryotes most frequently
are represented by various strains of E. coli. However, other
microbial strains may also be used, including other bacterial
strains.
In prokaryotic systems, plasmid vectors that contain
replication sites and control sequences derived from a species
compatible with the host may be used. Examples of suitable
plasmid vectors may include pBR322, pUC118, pUC119 and the
like: suitable phage or bacteriophage vectors may include
1gt10, lgtll and the like; and suitable virus vectors may
include pMAM-neo, pKRC and the like. Preferably, the selected
vector of the present invention has the capacity to replicate
in the selected host cell.
Recognized prokaryotic hosts include bacteria such as E.
coli and those from genera such as Bacillus, Streptomyces,
Pseudomonas, Salmonella, Serratia, and the like. However,
under such conditions, the polypeptide will not be
glycosylated. The prokaryotic host must be compatible with the
replicon and control sequences in the expression plasmid.
To express PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 {or a
functional derivative thereof) in a prokaryotic cell, it is
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necessary to operably link a PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 sequence to a functional prokaryotic promoter. Such
promoters may be either constitutive or, more preferably,
regulatable (i.e., inducible or derepressible). Examples of
constitutive promoters include the int promoter of
bacteriophage 1, the bla promoter of the b-lactamase gene
sequence of pBR322, and the CAT promoter of the chloramphenicol
acetyl transferase gene sequence of pPR325, and the like.
Examples of inducible prokaryotic promoters include the major
right and left promoters of bacteriophage 1 (PL and Pn), the
trp, recA, lacZ, lacI, and gal promoters of E. coli, the a-
amylase (Ulmanen et at., J. Bacteriol. 162:176-182, 1985) and
the sigma-28-specific promoters of B. subtilis (Gilman et al.,
Gene sequence 32:11-20(2984)), the promoters of the
bacteriophages of Bacillus (Gryczan, In: The Molecular Biology
of the Bacilli, Academic Press, Inc., NY (1982)), and
Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468-
478, 1986). Prokaryotic promoters are reviewed by Glick (J.
Ind. Microbiot. 1:277-282, 1987); Cenatiempo (Biochimie 68:505-
516, 1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
Proper expression in a prokaryotic cell also requires the
presence of a ribosome binding site upstream of the gene
sequence-encoding sequence. Such ribosome binding sites are
disclosed, for example, by Gold et at. (Ann. Rev. Microbiol.
35:365-404, 1981). The selection of control sequences,
expression vectors, transformation methods, and the like, are
dependent on the type of host cell used to express the gene.
As used herein, "cell", "cell line", and "cell culture"
may be used interchangeably and all such designations include
progeny. Thus, the words "transformants" or "transformed
cells" include the primary subject cell and cultures derived
therefrom, without regard to the number of transfers. It is
also understood that all progeny may not be precisely identical
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in DNA content, due to deliberate or inadvertent mutations.
However, as defined, mutant progeny have the same functionality
as that of the originally transformed cell.
Host cells which may be used in the expression systems of
the present invention are not strictly limited, provided that
they are suitable for use in the expression of the PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 peptide of interest. Suitable
hosts may often include eukaryotic cells. Preferred eukaryotic
hosts include, for example, yeast, fungi, insect cells,
mammalian cells either in vivo, or in tissue culture.
Mammalian cells which may be useful as hosts include HeLa
cells, cells of fibroblast origin such as VERO, 3T3 or CHO-K1,
or cells of lymphoid origin (such as 32D cells) and their
derivatives. Preferred mammalian host cells include SP2/0 and
J558L, as well as neuroblastoma cell lines such as IMR 332 and
PC12 which may provide better capacities for correct post-
translational processing.
In addition, plant cells are also available as hosts, and
control sequences compatible with plant cells are available,
such as the cauliflower mosaic virus 35S and 195, and nopaline
synthase promoter and polyadenylation signal sequences.
Another preferred host is an insect cell, for example the
Drosophila larvae. Using insect cells as hosts, the Drosophila
alcohol dehydrogenase promoter can be used. Rubin, Science
240:1953-1459, 1988). Alternatively, baculovirus vectors can
be engineered to express large amounts of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 in insects cells (Jasny, Science 238:1653,
1987); Miller et al., In: Genetic Engineering (1986), Setlow,
J.K., et al., eds., Plenum, Vol. 8, pp. 277-297).
Any of a series of yeast gene sequence expression systems
can be utilized which incorporate promoter and termination
elements from the actively expressed gene sequences coding for
glycolytic enzymes are produced in large quantities when yeast
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are grown in mediums rich in glucose. Known glycolytic gene
sequences can also provide very efficient transcriptional
control signals. Yeast provides substantial advantages in that
it can also carry out post-translational peptide modifications.
5 A number of recombinant DNA strategies exist which utilize
strong promoter sequences and high copy number of plasmids
which can be utilized for production of the desired proteins in
yeast. Yeast recognizes leader sequences on cloned mammalian
gene sequence products and secretes peptides bearing leader
10 sequences (i.e., pre-peptides). For a mammalian host, several
possible vector systems are available for the expression of
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7.
A particularly preferred yeast expression system is that
utilizing Schizosaccharmocyces pombe. This system is useful
15 for studying the activity of members of the Src family
(Superti-Furga, et al, EMBO J. 12:2625, 1993) and other NR-TKs.
A wide variety of transcriptional and translational
regulatory sequences may be employed, depending upon the nature
of the host. The transcriptional and translational regulatory
20 signals may be derived from viral sources, such as adenovirus,
bovine papilloma virus, cytomegalovirus, simian virus, or the
like, where the regulatory signals are associated with a
particular gene sequence which has a high level of expression.
Alternatively, promoters from mammalian expression products,
25 such as actin, collagen, myosin, and the like, may be employed.
Transcriptional initiation regulatory signals may be selected
which allow for repression or activation, so that expression of
the gene sequences can be modulated. Of interest are
regulatory signals which are temperature-sensitive so that by
30 varying the temperature, expression can be repressed or
initiated, or are subject to chemical (such as metabolite)
regulation.
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Expression of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 in
eukaryotic hosts requires the use of eukaryotic regulatory
regions. Such regions will, in general, include a promoter
region sufficient to direct the initiation of RNA synthesis.
Preferred eukaryotic promoters include, for example, the
promoter of the mouse metallothionein I gene sequence (Hamer et
al., J. Mol. Appl. Gen. 1:273-288, 1982) the TK promoter of
Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early
promoter (Benoist et al., Nature (London) 290:304-310, 1981);
the yeast gal4 gene sequence promoter (Johnston et al., Proc.
Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al.,
Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
Translation of eukaryotic mRNA is initiated at the codon
which encodes the first methionine. For this reason, it is
preferable to ensure that the linkage between a eukaryotic
promoter and a DNA sequence which encodes PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 (or a functional derivative thereof) does
not contain any intervening codons which are capable of
encoding a methionine (i.e., AUG). The presence of such codons
results either in a formation of a fusion protein (if the AUG
codon is in the same reading frame as a PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 coding sequence) or a frame-shift mutation
(if the AUG codon is not in the same reading frame as a PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 coding sequence).
A PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 nucleic acid
molecule and an operably linked promoter may be introduced into
a recipient prokaryotic or eukaxyotic cell either as a
nonreplicating DNA (or RNA) molecule, which may either be a
linear molecule or, more preferably, a closed covalent circular
molecule (a plasmid). Since such molecules are incapable of
autonomous replication, the expression of the gene may occur
through the transient expression of the introduced sequence.
Alternatively, permanent or stable expression may occur through
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the integration of the introduced DNA sequence into the host
chromosome.
A vector may be employed which is capable of integrating
the desired gene sequences into the host cell chromosome.
Cells which have stably integrated the introduced DNA into
their chromosomes can be selected by also introducing one or
more markers which allow for selection of host cells which
contain the expression vector. The marker may provide for
prototrophy to an auxotrophic host, biocide resistance, e.g.,
antibiotics, or heavy metals, such as copper, or the like. The
selectable marker gene sequence can either be directly linked
to the DNA gene sequences to be expressed, or introduced into
the same cell by co-transfection. Additional elements may also
be needed for optimal synthesis of single chain binding protein
mRNA. These elements may include splice signals, as well as
transcription promoters, enhancers, and termination signals.
cDNA expression vectors incorporating such elements include
those described by Okayama, Mol. Cell. Bio. 3:280, 1983.
The introduced nucleic acid molecule can be incorporated
into a plasmid or viral vector capable of autonomous
replication in the recipient host. Any of a wide variety of
vectors may be employed for this purpose. Factors of
importance in selecting a particular plasmid or viral vector
include: the ease with which recipient cells that contain the
vector may be recognized and selected from those recipient
cells which do not contain the vectors the number of copies of
the vector which are desired in a particular host; and whether
it is desirable to be able to "shuttle" the vector between host
cells of different species.
Preferred prokaryotic vectors include plasmids such as
those capable of replication in E. coil (such as, for example,
pBR322, ColEl, pSC101, pACYC 184, pVX. Such plasmids are, for
example, disclosed by Sambrook (cf. "Molecular Cloning: A
_ _T ____ ___ T
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Laboratory Manual", second edition, edited by Sambrook,
Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)).
Bacillus plasmids include pC194, pC221, pT127, and the like.
Such plasmids are disclosed by Gryczan (In: The Molecular
Biology of the Bacilli, Academic Press, NY (1982), pp. 307-
329). Suitable Streptomyces plasmids include p1J101 (Kendall
et al., J. Bacteriol. 169:4177-4183,1987), and streptomyces
bacteriophages such as fC31 (Chater et al., In: Sixth
International Symposium on Actinomycetales Biology, Akademiai
Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas
plasmids are reviewed by John et al. (Rev.-Infect. Dis. 8:693-
709, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).
Preferred eukaryotic plasmids include, for example, BPV,
vaccinia, SV40, 2-micron circle, and the like, or their
derivatives. Such plasmids are well known in the art (Botstein
et al., Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The
Molecular Biology of the Yeast Saccharomyces: Life Cycle and
Inheritance", Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204, 1982);
Bollon et at., J. Clin. Hematol. Oncol. 10:39-48, 1980);
Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,
Gene Sequence Expression, Academic Press, NY, pp. 563-608
(1980) .
Once the vector or nucleic acid molecule containing the
constructs) has been prepared for expression, the DNA
constructs) may be introduced into an appropriate host cell by
any of a variety of suitable means, i.e., transformation,
transfection, conjugation, protoplast fusion, electroporation,
particle gun technology, calcium phosphate-precipitation,
direct microinjection, and the like. After the introduction of
the vector, recipient cells are grown in a selective medium,
which selects for the growth of vector-containing cells.
Expression of the cloned gene molecules) results in the
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production of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 or
fragments or functional derivatives thereof. This can take
place in the transformed cells as such, or following the
induction of these cells to differentiate (for example, by
administration of bromodeoxyuracil to neuroblastoma cells or
the like). A variety of incubation conditions can be used to
form the peptide of the present invention. The most preferred
conditions are those which mimic physiological conditions.
VI. The Polypeptides of the Invention.
Also a feature of the invention are PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 polypeptides. A variety of methodologies
known in the art can be utilized to obtain the polypeptides of
the present invention. They may be purified from tissues or
cells which naturally produce them. Alternatively, the above-
described isolated nucleic acid sequences can be used to
express a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 protein
recombinantly.
Any eukaryotic organism can be used as a source for the
polypeptide of the invention, as long as the source organism
naturally contains such a polypeptide. As used herein, "source
organism" refers to the original organism from which the amino
acid sequence is derived, regardless of the organism the
protein is expressed in and ultimately isolated from.
One skilled in the art can readily follow known methods
for isolating proteins in order to obtain the peptide free of
natural contaminants. These include, but are not limited to:
size-exclusion chromatography, HPLC, ion-exchange
chromatography, and immuno-affinity chromatography.
A PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 protein, like
all proteins, is comprised of distinct functional units or
domains. In eukaryotes, proteins sorted through the so-called
vesicular pathway (bulk flow) usually have a signal sequence
_.___ _.T ._. __ _ _....
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(also called a leader peptide) in the N- terminus, which is
cleaved off after the translocation through the ER (endoplasmic
reticulum) membrane. Some N-terminal signal sequences are not
cleaved off, remaining as transmembrane segments, but it does
5 not mean these proteins are retained in the ER; they can be
further sorted and included in vesicles.
SAD protein lacks a hydrophobic signal sequence and is
classified as a non-receptor protein. Other motifs involved in
targeting proteins to specific cellular locations include those
10 selective for the mitochondrial matrix (Gavel and von Heijne,
Prot Eng 4:33, 1990), the nucleus (Robbins, et al, Cell 64:615,
1991), peroxisomes, endoplasmic reticulum (Jackson, et al, EMBO
J 9:3253, 1990), vesicular pathways (Bendiak, Biophys Res Comm
170:879, 1990), glycosyl-phosphatidylinositol (GPI) lipid
15 anchors, and lysosomal organelles, and motifs that target
proteins to lipid membranes such as myristylation (Towler, et
al, Annu Rev Biochem 57:69, 1988) and farnesylation sites. The
N-terminal 15 amino acids of the SAD protein conforms to the
features which define a mitochondrial membrane protein with a
20 bipartite structure of an N-terminal stretch of high arginine
content involved in membrane targeting followed by the apolar
sequence which signals translocation to the mitochondrial
intermembrane space.
Non-receptor proteins generally function to transmit
25 signals within the cell, either by providing sites for
protein: protein interactions or by having some catalytic
activity (contained within a catalytic domain), often both.
Methods of predicting the existence of these various domains
are well known in the art. Protein: protein interaction domains
30 can be identified by comparison to other proteins. The SH2
domain, for example is a protein domain of about 100 amino
acids first identified as a conserved sequence region between
the proteins Src and Fps (Sadowski, et al, Mol. Cell. Bio.
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6:4396, 1986). Similar sequences were later found in many
other intracellular signal-transducing proteins. SH2 domains
function as regulatory modules of intracellular signaling
cascades by interacting with high affinity to phosphotyrosine-
containing proteins in a sequence specific and strictly
phosphorylation-dependent manner (Mayer and Baltimore, Trends
Cell. Biol. 3:8, 1993). Kinase or phosphatase catalytic
domains can be identified by comparison to other known
catalytic domains with kinase or phosphatase activity. See,
for example Hanks and Hunter, FASEB J. 9:576-595, 1995.
Receptor proteins also have, and are somewhat defined by,
a hydrophobic transmembrane segments) which are thought to be
Alpha-helices in membranes. Membrane proteins also integrate
into the cell membrane in a specific manner with respect to the
two sides (cytoplasmic/intracellular or exo-cytoplasmic/
extracellular), which is referred to as membrane topology.
Extracellular portions of integral membrane proteins often
function as ligand binding domains whereas intracellula
portions generally function to transmit signals within the
cell, either by providing sites for protein: protein
interactions or by having some catalytic activity (contained
within a catalytic domain), often both. Methods of predicting
the existence of these various domains are well known in the
art. See, for example, D. J. McGeoch, Virus Research 3:271,
1985, or G. von Heijne, Nucl. Acids Res. 14:4683, 1986, for
signal sequences, P. Klein, et al., Biochim. Biophys. Acta
815:968, 1985, for transmembrane domains, and S. J. Singer,
Ann. Rev. Cell Biol. 6:247, 1990, or E. Hartmann, et al., Proc.
Natl. Acad. Sci. USA, 86:5786, 1989, for prediction of membrane
topology. Kinase catalytic domains can be identified by
comparison to other known catalytic domains with kinase
activity. See, for example, Hanks and Hunter, FASEB J. 9:576-
595, 1995.
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Primary sequence analysis of the PTP04 amino acid sequence
(shown in SEQ ID N0:9) reveals that it does not contain a
signal sequence or transmembrane domain and is, therefore, an
intracellular protein. Comparison to known protein sequences
revels that PTP04 is comprised of several unique domains.
These include a 48 amino acid N-terminal domain (shown from
amino acid number 1-48 of SEQ ID N0:9), a 245 amino acid
catalytic domain (shown from amino acid number 49-294 of SEQ ID
N0:9), and a 512 amino acid C-terminal domain (shown from amino
acid number 295-807 of SEQ ID N0:9).
Primary sequence analysis of the SAD amino acid sequence
(shown in SEQ ID N0:10) reveals that it contains four distinct
domains. These include an approximately 55 amino acid N-
terminal unique domain (shown from amino acid number 1-55 of
SEQ ID NO:10), an approximately 54 amino acid SH3 domain (shown
from amino acid number 56-109 of SEQ ID NO:10), an
approximately 93 amino acid SH2 domain (shown from amino acid
number 120-212 of SEQ ID NO:10), an approximately 251 amino
acid catalytic domain (amino acid number 230-480 of SEQ ID
No:lO), and a C-terminal tail of 8 amino acids (shown from
amino acid 481-488 of SEQ ID N0:10).
Primary sequence analysis of the PTP05 amino acid sequence
(shown in SEQ ID N0:11 with isoforms shown in SEQ ID N0:12 and
SEQ ID N0:13) reveals that it and its isoforms do not contain a
signal sequence or transmembrane domain, and it is, therefore,
an intracellular protein. Comparison to known protein
sequences revels that PTP05 is comprised of several unique
domains. These include a 187 amino acid N-terminal domain
(shown from amino acid number 1-187 of SEQ ID N0:11), a 242
amino acid catalytic domain (shown from amino acid number 188-
420 of SEQ ID N0:11), and a 5 amino acid C-terminal domain
(shown from amino acid number 421-426 of SEQ ID N0:11).
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Two additional isoforms of PTP05 were also identified, a
"long" form (SEQ ID N0:12) and a "C-trunc" form (SEQ ID N0:13) .
The "long" form has a 37 amino acid insertion in the N-terminal
domain (aminoacids 44-80 of SEQ ID N0:12) which extends this
domain to 224 amino acids. The catalytic domain extends from
amino acid 225-457 of SEQ ID N0:12 and the C-terminal domain
extents from amino acids 458-463 of SEQ ID N0:12. The "C-trunc"
form results from a deletion of nucleotides 1415-1507 of SEQ ID
N0:3, most likely due to alternative exon splicing. This
deletion results in a replacement of the C-terminal 21 amino
acids with a unique 7 amino acid sequence. This change
eliminates a conserved C-terminal portion of the catalytic
domain, which may affect enzymatic activity. The N-terminal
domain of the "C-trunc" form extends from amino acid 1-87 of SEQ
ID N0:13, the catalytic domain from amino acids 188-405 of SEQ
ID N0:13 and the unique C-terminal domain from 406-412 of SEQ
ID N0:13.
Primary sequence analysis of the ALP amino acid sequence
(shown in SEQ ID N0:15) reveals that it does not contain a
signal sequence or transmembrane domain and is, therefore, an
intracellular protein. Comparison to known protein sequences
revels that ALP is comprised of several unique domains. These
include a 857 amino acid N-terminal proline-rich domain (shown
from amino acid number 1-857 of SEQ ID N0:15) within which is a
proline-rich region (amino acid number 353-777 of SEQ ID
N0:15), a 238 amino acid catalytic domain (shown from amino
acid number 858-1096 of SEQ ID N0:15), and a 177 amino acid C-
terminal proline/serine-rich domain (shown from amino acid
number 1097-1274 of SEQ ID N0:15) within which is a
proline/serine-rich region (amino acid number 1101-1214 of SEQ
ID N0:15).
Primary sequence analysis for an ALK-7 amino acid sequence
(shown in SEQ ID N0:16) reveals that it contains all the motifs
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characteristic of a type I STK receptor. These include a 25
amino acid signal peptide (shown from amino acid number 1-25 of
SEQ ID N0:16), an 88 amino acid cysteine-rich extracellular
region (shown from amino acid number 2&-113 of SEQ ID N0:16), a
single 25 amino acid transmembrane domain (shown from amino
acid number 114-136 of SEQ ID N0:16), and a 355 amino acid
cytoplasmic domain (shown from amino acid number 137-493 of SEQ
ID N0:16), which includes a GS domain and a catalytic domain
(amino acid number 193-485 of SEQ ID N0:16).
The extracellular domain conserves the 10 cysteines
present in all type I STK receptors (ten Dijke, et al.,
Oncogene 8:2879, 1993; Bassinge, et al., Science 263:87, 1994;
Massague, Trends Cell Biol. 4:172, 1994) and also contains 3
potential N-=linked glycosylation sites. The divergent
extracellular domain sequence of ALK-7 {28-30o identity to ALK-
4 and ALK-5) suggests it may have a unique ligand/type II STK
receptor specificity. A rat ALK-7-like protein ahs been found
to bind TGFbeta and activin in a complex with the type II TGF
beta receptor and ACTRII. However, these ligands are not
expressed in the same cell types as human ALK-7 suggesting
alternative ligands. Candidate ALK-7-specific ligands include
other TGFbetas such as TGFbeta 2, GDF-1, and homologues of
GDNF, such as neuturin, which have been found to be expressed
in neurons in a pattern similar to that of ALK-7.
The intracellular domain is somewhat more homologous to
other ALK proteins, particularly in the catalytic domain which
shows 83~ identity to other type I STK receptors. The 40 amino
acids immediately N-terminal of the transmembrane domain (the
juxtamembrane domain) are, however, quite unique in comparison
with other ALKs.
These PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 domains have
a variety of uses. An example of such a use is to make a
polypeptide consisting of the PTP04, SAD, PTP05, PTP10, ALP, or
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ALK-7 catalytic domain and a heterologous protein such as
glutathione S-transferase (GST). Such a polypeptide can be
used in a biochemical assay for PTP04, SAD, PTP05, PTP10, ALP,
or ALK-7 catalytic activity useful for studying PTP04, SAD,
5 PTP05, PTP10, ALP, or ALK-7 substrate specificity or for
identifying substances that can modulate PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 catalytic activity. Alternatively, one
skilled in the art could create a PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 polypeptide lacking at least one of the three
10 major domains. Such a polypeptide, when expressed in a cell,
is able to form complexes with the natural binding partners)
of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 but unable to
transmit any signal further downstream into the cell, i.e.,. it
would be signaling incompetent and thus would be useful for
15 studying the biological relevance of PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 activity. (See, for example, Gishizky, et al,
PNAS :10889, 1995).
VII. An Antibody Having Binding Affinity To the Poly eptides of
20 the Invention And A Hybridoma Containing the Antibody.
The present invention also relates to an antibody having
specific binding affinity to an PTP04, SAD, PTP05, PTP10, ALP,
or ALK-7 polypeptide. The polypeptide may have the amino acid
sequence set forth in SEQ ID N0:2, or a be fragment thereof, or
25 at least 6 contiguous amino acids thereof. Such an antibody
may be identified by comparing its binding affinity to a PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide with its binding
affinity to another polypeptide. Those which bind selectively
to PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 would be chosen for
30 use in methods requiring a distinction between PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 and other polypeptides. Such
methods could include, but should not be limited to, the
analysis of altered PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
T. __ __._. _
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expression in tissue containing other polypeptides and assay
systems using whole cells.
A PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 peptide of the
present invention can be used to produce antibodies or
hybridomas. One skilled in the art will recognize that if an
antibody is desired, such a peptide would be generated as
described herein and used as an immunogen. Preferred PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 peptides for this purpose as
shown in Example 4 below. The antibodies of the present
invention include monoclonal and polyclonal antibodies, as well
fragments of these antibodies, and humanized forms. Humanized
forms of the antibodies of the present invention may be
generated using one of the procedures known in the art such as
chimerization or CDR grafting. The present invention also
relates to a hybridoma which produces the above-described
monoclonal antibody, or binding fragment thereof. A hybridoma
is an immortalized cell line which is capable of secreting a
specific monoclonal antibody.
In general, techniques for preparing monoclonal antibodies
and hybridomas are well known in the art (Campbell, "Monoclonal
Antibody Technology: Laboratory Techniques in Biochemistry and
Molecular Biology," Elsevier Science Publishers, Amsterdam, The
Netherlands, 1989: St. Groth et al., J. Immunol. Methods 35:1
21, 1980). Any animal (mouse, rabbit, and the like) which is
known to produce antibodies can be immunized with the selected
polypeptide. Methods for immunization are well known in the
art. Such methods include subcutaneous or intraperitoneal
injection of the polypeptide. One skilled in the art will
recognize that the amount of polypeptide used for immunization
will vary based on the animal which is immunized, the
antigenicity of the polypeptide and the site of injection.
The polypeptide may be modified or administered in an
adjuvant in order to increase the peptide antigenicity.
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Methods of increasing the antigenicity of a polypeptide are
well known in the art. Such procedures include coupling the
antigen with a heterologous protein (such as globulin or b-
galactosidase) or through the inclusion of an adjuvant during
immunization.
For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-
Agl4 myeloma cells, and allowed to become monoclonal antibody
producing hybridoma cells. Any one of a number of methods well
known in the art can be used to identify the hybridoma cell
which produces an antibody with the desired characteristics.
These include screening the hybridomas with an ELISA assay,
western blot analysis, or radioimmunoassay (Lutz, et al., Exp.
Cell Res. 175:109-124, 1988). Hybridomas secreting the desired
antibodies are cloned and the class and subclass is determined
using procedures known in the art (Campbell, "Monoclonal
Antibody Technology: Laboratory Techniques in Biochemistry and
Molecular Biology", supra, 1984).
For polyclonal antibodies, antibody containing antisera is
isolated from the immunized animal and is screened for the
presence of antibodies with the desired specificity using one
of the above-described procedures. The above-described anti
bodies may be detectably labeled. Antibodies can be detectably
labeled through the use of radioisotopes, affinity labels (such
as biotin, avidin, and the like), enzymatic labels (such as
horse radish peroxidase, Alkaline phosphatase, and the like)
fluorescent labels (such as FITC ox rhodamine, and the like),
paramagnetic atoms, and the like. Procedures for accomplishing
such labeling are well-known in the art, for example, see
(Stemberger, et al., J. Histochem. Cytochem. 18:315, 1970;
Bayer, et at., Meth. Enzym. 62:308, 1979; Engval, et al.,
Immunot. 109::129, 1972; Goding, J. Immunol. Meth. 13:215,
1976) . The labeled antibodies of the present invention can be
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used for in vitro, in vivo, and in in situ assays to identify
cells or tissues which express a specific peptide.
The above-described antibodies may also be immobilized on
a solid support. Examples of such solid supports include
plastics such as polycarbonate, complex carbohydrates such as
agarose and sepharose, acrylic resins and such as
polyacrylamide and latex beads. Techniques for coupling
antibodies to such solid supports are well known in the art
(Weir et al., "Handbook of Experimental Immunology" 4th Ed.,
Blackwell Scientific Publications, Oxford, England, Chapter 10,
1986; Jacoby et al., Meth. Enzym. 34, Academic Press, N.Y.,
1974). The immobilized antibodies of the present invention can
be used for in vitro, in vivo, and in situ assays as well as in
immunochromotography.
Furthermore, one skilled in the art can readily adapt
currently available procedures, as well as the techniques,
methods and kits disclosed above with regard to antibodies, to
generate peptides capable of binding to a specific peptide
sequence in order to generate rationally designed antipeptide
peptides, for example see Hurby et al., "Application of
Synthetic Peptides: Antisense Peptides", In Synthetic Peptides,
A User's Guide, W.H. Freeman, NY, pp. 289-307(1992), and
Kaspczak et al., Biochemistry 28:9230-8(1989).
vIII.An Antibody Based Method And Kit For Detecting the
Polypeptides of the Invention.
The present invention encompasses a method of detecting a
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide in a
sample, comprising: (a) contacting the sample with an above-
described antibody, under conditions such that immunocomplexes
form, and (b) detecting the presence of said antibody bound to
the polypeptide. In detail, the methods comprise incubating a
test sample with one or more of the antibodies of the present
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invention and assaying whether the antibody binds to the test
sample. Altered levels, either an increase or decrease, of
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 in a sample as compared
to normal levels may indicate disease.
Conditions for incubating an antibody with a test sample
vary. Incubation conditions depend on the format employed in
the assay, the detection methods employed, and the type and
nature of the antibody used in the assay. One skilled in the
art will recognize that any one of the commonly available
immunological assay formats (such as radioimmunoassays, enzyme-
linked immunosorbent assays, diffusion based Ouchterlony, or
rocket immunofluorescent assays) can readily be adapted to
employ the antibodies of the present invention. Examples of
such assays can be found in Chard, "An Introduction to
Radioimmunoassay and Related Techniques" Elsevier Science
Publishers, Amsterdam, The Netherlands (1986); Bullock et al.,
"Techniques in Immunocytochemistry," Academic Press, Orlando,
FL Vol. 1(1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen,
"Practice and Theory of Enzyme Immunoassays: Laboratory Techni-
ques in Biochemistry and Molecular Biology," Elsevier Science
Publishers, Amsterdam, The Netherlands (1985).
The immunological assay test samples of the present
invention include cells, protein or membrane extracts of cells,
or biological fluids such as blood, serum, plasma, or urine.
The test sample used in the above-described method will vary
based on the assay format, nature of the detection method and
the tissues, cells or extracts used as the sample to be
assayed. Methods for preparing protein extracts or membrane
extracts of cells are well known in the art and can be readily
be adapted in order to obtain a sample which is capable with
the system utilized.
A kit contains all the necessary reagents to carry out the
previously described methods of detection. The kit may
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comprise: (i) a first container containing an above-described
antibody, and (ii) second container containing a conjugate
comprising a binding partner of the antibody and a label. In
another preferred embodiment, the kit further comprises one or
5 more other containers comprising one or more of the following:
wash reagents and reagents capable of detecting the presence of
bound antibodies.
Examples of detection reagents include, but are not
limited to, labeled secondary antibodies, or in the
10 alternative, if the primary antibody is labeled, the
chromophoric, enzymatic, or antibody binding reagents which are
capable of reacting with the labeled antibody. The
compartmentalized kit may be as described above for nucleic
acid probe kits. One skilled in the art will readily recognize
15 that the antibodies described in the present invention can
readily be incorporated into one of the established kit formats
which are well known in the art.
IX. Isolation of Natural Binding Partners of the Poly eptides
20 of the Invention.
The present invention also relates to methods of detecting
natural binding partners capable of binding to a PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 polypeptide. A natural binding
partner of PTP09, SAD, PTP05, PTP10, ALP, or ALK-7 may be, for
25 example, a substrate protein which is dephosphorylated as part
of a signaling cascade. The binding partners) may be present
within a complex mixture, for example, serum, body fluids, or
cell extracts.
In general methods for identifying natural binding
30 partners comprise incubating a substance with PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 and detecting the presence of a
substance bound to PTP04, SAD, PTP05, PTP10, ALP, or ALK-7.
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Preferred methods include the two-hybrid system of Fields and
Song (supra) and co-immunoprecipitation.
X. Identification of and Uses for Substances Capable of
Modulating the Activity of the Polypeptides of the
Invention.
The present invention also relates to a method of
detecting a substance capable of modulating PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 activity. Such substances can either
enhance activity (agonists) or inhibit activity (antagonists).
Agonists and antagonists can be peptides, antibodies, products
from natural sources such as fungal or plant extracts or small
molecular weight organic compounds. In general, small
molecular weight organic compounds are preferred. Examples of
classes of compounds that can be tested for PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 modulating activity are, for example but
not limited to, thiazoles (see for example co-pending US
applications 60/033,522, 08/660,900), and naphthopyrones (US
patent number 5,602,171).
In general the method comprises incubating cells that
produce PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 in the presence
of a test substance and detecting changes in the level of
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity or PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 binding partner activity. A change
in activity may be manifested by increased or decreased
phosphorylation of a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide, increased or decreased phosphorylation of a PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 substrate, or increased or
decreased biological response in cells. A method for detecting
modulation of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 activity
using the phosphorylation of an artificial substrate is shown
in the examples below. Biological responses can include, for
example, proliferation, differentiation, survival, or motility.
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The substance thus identified would produce a change in
activity indicative of the agonist or antagonist nature of the
substance. Once the substance is identified it can be isolated
using techniques well known in the art, if not already
available in a purified form.
The present invention also encompasses a method of
agonizing (stimulating) or antagonizing PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 associated activity in a mammal comprising
administering to said mammal an agonist or antagonist to PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 in an amount sufficient to
effect said agonism or antagonism. Also encompassed in the
present application is a method of treating diseases in a
mammal with an agonist or antagonist of PTP04-, SAD-, PTP05-,
PTP10-, ALP-, or ALK-7-related activity comprising
administering the agonist or antagonist to a mammal in an
amount sufficient to agonize or antagonize PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 associated function(s). The particular
compound can be administered to a patient either by itself or
in a pharmaceutical composition where it is mixed with suitable
carriers or excipient(s). In treating a patient a
therapeutically effective dose of the compound is administered.
A therapeutically effective dose refers to that amount of the
compound that results in amelioration of symptoms or a
prolongation of survival in a patient.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell
cultures or experimental animals. For example, for determining
the LDSO (the dose lethal to 50% of the population) and the EDSo
(the dose therapeutically effective in 50~ of the population).
The dose ratio between toxic and therapeutic effects is the
therapeutic index and it can be expressed as the ratio LDSO/ED5o.
Compounds which exhibit large therapeutic indices are
preferred. The data obtained from these cell culture assays
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and animal studies can be used in formulating a range of dosage
for use in human. The dosage of such compounds lies preferably
within a range of circulating concentrations that include the
EDSO with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the
route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from
cell culture assays. For example, a dose can be formulated in
animal models to achieve a circulating plasma concentration
range. that includes the ICSO as determined in cell culture
(i.e., the concentration of the test compound which achieves a
half-maximal disruption of the protein complex, or a half-
maximal inhibition of the cellular level and/or activity of a
complex component). Such information can be used to more
accurately determine useful doses in humans. Levels in plasma
may be measured, for example, by HPLC.
The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the
patient's condition. (See e.g. Fingl et al., 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 p1).
It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administra-
tion due to toxicity, or to organ dysfunctions. Conversely,
the attending physician would also know to adjust treatment to
higher levels if the clinical response were not adequate
(precluding toxicity). The magnitude of an administrated dose
in the management of the oncogenic disorder of interest will
vary with the severity of the condition to be treated and to
the route of administration. The severity of the condition
may, for example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose fre-
quency, will also vary according to the age, body weight, and
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response of the individual patient. A program comparable to
that discussed above may be used in veterinary medicine.
Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or
locally. Techniques for formulation and administration may be
found in "Remington's Pharmaceutical Sciences," 1990, 18th ed.,
Mack Publishing Co., Easton, PA. Suitable routes may include
oral, rectal, transdermal, vaginal, transmucosal, or intestinal
administration: parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intra-
thecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections, just to name a few.
For injection, the agents of the invention may be
formulated in aqueous solutions, preferably in physiologically
~15 compatible buffers such as Hanks's solution, Ringer's solution,
or physiological saline buffer. For such transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
Use of pharmaceutically acceptable carriers to formulate
the compounds herein disclosed for the practice of the
invention into dosages suitable for systemic administration is
within the scope of the invention. With proper choice of
carrier and suitable manufacturing practice, the compositions
of the present invention, in particular, those formulated as
solutions, may be administered parenterally, such as by
intravenous injection. The compounds can be formulated readily
using pharmaceutically acceptable carriers well known in the
art into dosages suitable for oral administration. Such
carriers enable the compounds of the invention to be formulated
as tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated.
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Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary
skill in the art. For example, such agents may be encapsulated
into liposomes, then administered as described above.
5 Liposomes are spherical lipid bilayers with aqueous interiors.
All molecules present in an aqueous solution at the time of
liposome formation are incorporated into the aqueous interior.
The liposomal contents are both protected from the external
microenvironment and, because liposomes fuse with cell
10 membranes, are efficiently delivered into the cell cytoplasm.
Additionally, due to their hydrophobicity, small organic mole-
cules may be directly administered intracellularly.
Pharmaceutical compositions suitable for use in the
present invention include compositions wherein the active
15 ingredients are contained in an effective amount to achieve its
intended purpose. Determination of the effective amounts is
well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein.
In addition to the active ingredients, these
20 pharmaceutical compositions may contain suitable pharma
ceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The pre
parations formulated for oral administration may be in the form
25 of tablets, dragees, capsules, or solutions.
The pharmaceutical compositions of the present invention
may be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating, dragee
making, levigating, emulsifying, encapsulating, entrapping or
30 lyophilizing processes.
Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-
soluble form. Additionally, suspensions of the active com-
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pounds may be prepared as appropriate oily injection suspen-
sions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such
as ethyl oleate or triglycerides, or liposomes. Aqueous injec-
tion suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension
may also contain suitable stabilizers or agents which increase
the solubility of the compounds to allow for the preparation of
highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained
by combining the active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipi-
ents are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as
sodium alginate.
Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which
may optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide,
lacquer solutions, and suitable organic solvents or solvent
mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different
combinations of active compound doses.
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Pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft,
sealed capsules made of gelatin and a plasticizer, such as
glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with filler such as lactose,
binders such as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or
liquid polyethylene glycols. In addition, stabilizers may be
added.
XI. Transgenic Animals.
Also contemplated by the invention are transgenic animals
useful for the study of PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
activity in complex in vivo systems. A variety of methods are
available for the production of transgenic animals associated
with this invention. DNA sequences encoding PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 can be injected into the pronucleus of a
fertilized egg before fusion of the male and female pronuclei,
or injected into the nucleus of an embryonic cell (e.g.., the
nucleus of a two-cell embryo) following the initiation of cell
division (Brinster, et al., Proc. Nat. Acad. Sci. USA 82: 4438,
1985). Embryos can be infected with viruses, especially retro-
viruses, modified to carry inorganic-ion receptor nucleotide
sequences of the invention.
Pluripotent stem cells derived from the inner cell mass of
the embryo and stabilized in culture can be manipulated in
culture to incorporate nucleotide sequences of the invention.
A transgenic animal can be produced from such cells through
implantation into a blastocyst that is implanted into a foster
mother and allowed to come to term. Animals suitable for
transgenic experiments can be obtained from standard commercial
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sources such as Charles River (Wilmington, MA), Taconic
(Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN),
etc.
The procedures for manipulation of the rodent embryo and
for microinjection of DNA into the pronucleus of the zygote are
well known to those of ordinary skill in the art (Hogan, et
al., supra). Microinjection procedures for fish, amphibian
eggs and birds are detailed in Houdebine and Chourrout,
Experientia 47: 897-905, 1991). Other procedures for intro-
duction of DNA into tissues of animals are described in U.S.
Patent No., 4,995,050 (Sandford et al., July 30, 1990).
By way of example only, to prepare a transgenic mouse,
female mice are induced to superovulate. After being allowed
to mate, the females are sacrificed by COZ asphyxiation or
cervical dislocation and embryos are recovered from excised
oviducts. Surrounding cumulus cells are removed. Pronuclear
embryos are then washed and stored until the time of injection.
Randomly cycling adult female mice are paired with vasectomized
males. Recipient females are mated at the same time as donor
females. Embryos then are transferred surgically. The
procedure for generating transgenic rats is similar to that of
mice. See Hammer, et al., Cell 63:1099-1112, 1990).
Methods for the culturing of embryonic stem (ES) cells and
the subsequent production of transgenic animals by the
introduction of DNA into ES cells using methods such as
electroporation, calcium phosphate/DNA precipitation and direct
injection also are well known to those of ordinary skill in the
art. See, for example, Teratocarcinomas and Embryonic Stem
Cells, A Practical Approach, E.J. Robertson, ed., IRL Press,
1987).
In cases involving random gene integration, a clone
containing the sequences) of the invention is co-transfected
with a gene encoding resistance. Alternatively, the gene
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64
encoding neomycin resistance is physically linked to the
sequences) of the invention. Transfection and isolation of
desired clones are carried out by any one of several methods
well known to those of ordinary skill in the art (E. J.
Robertson, supra).
DNA molecules introduced into ES cells can also be
integrated into the chromosome through the process of
homologous recombination. Capecchi, Science 244: 1288-1292
(1989). Methods for positive selection of the recombination
event (i.e., neo resistance) and dual positive-negative
selection (i.e., neo resistance and gancyclovir resistance) and
the subsequent identification of the desired clones by PCR have
been described by Capecchi, supra and Joyner et al., Nature
338: 153-156, 1989), the teachings of which are incorporated
herein. The final phase of the procedure is to inject targeted
ES cells into blastocysts and to transfer the blastocysts into
pseudopregnant females. The resulting chimeric animals are
bred and the offspring are analyzed by Southern blotting to
identify individuals that carry the transgene. Procedures for
the production of non-rodent mammals and other animals have
been discussed by others. See Houdebine and Chourrout, supra;
Pursel, et al., Science 244:1281-1288, 1989); and Simms, et
al., Bio/Technology 6:179-183, 1988).
Thus, the invention provides transgenic, nonhuman mammals
containing a transgene encoding a PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 polypeptide or a gene effecting the expression of
a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide. Such
transgenic nonhuman mammals are particularly useful as an in
vivo test system for studying the effects of introducing a
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide, regulating
the expression of a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7
polypeptide (.z.e., through the introduction of additional
genes, antisense nucleic acids, or ribozymes).
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A "transgenic animal" is an animal having cells that
contain DNA which has been artificially inserted into a cell,
which DNA becomes part of the genome of the animal which
develops from that cell. Preferred transgenic animals are
5 primates, mice, rats, cows, pigs, horses, goats, sheep, dogs
and cats. The transgenic DNA may encode for a human PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 polypeptide. Native expres
sion in an animal may be reduced by providing an amount of
anti-sense RNA or DNA effective to reduce expression of the
10 receptor.
XII. Gene Therapy.
PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 or its genetic
sequences, both mutated and non-mutated, will also be useful in
15 gene therapy (reviewed in Miller, Nature 357:455-460, (1992).
Miller states that advances have resulted in practical
approaches to human gene therapy that have demonstrated
positive initial results. The basic science of gene therapy is
described in Mulligan, Science 260:926-931, (1993).
20 In one preferred embodiment, an expression vector
containing a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 coding
sequence or a PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 mutant
coding sequence as described above is inserted into cells, the
cells are grown in vitro and then infused in large numbers into
25 patients. In another preferred embodiment, a DNA segment
containing a promoter of choice (for example a strong promoter)
is transferred into cells containing an endogenous PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 in such a manner that the promoter
segment enhances expression of the endogenous PTP04, SAD,
30 PTP05, PTP10, ALP, or ALK-7 gene (for example, the promoter
segment is transferred to the cell such that it becomes
directly linked to the endogenous PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 gene).
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The gene therapy may involve the use of an adenovirus
containing PTP04, SAD, PTP05, PTP10, ALP, or ALK-7 cDNA
targeted to an appropriate cell type, systemic PTP04, SAD,
PTP05, PTP10, ALP, or ALK-7 increase by implantation of
engineered cells, injection with PTP04, SAD, PTP05, PTP10, ALP,
or ALK-7 virus, or injection of naked PTP04, SAD, PTP05, PTP10,
ALP, or ALK-7 DNA into appropriate cells or tissues, for
example neurons.
Expression vectors derived from viruses such as
retroviruses, vaccinia virus, adenovirus, adeno-associated
virus, herpes viruses, several RNA viruses, or bovine papilloma
virus, may be used for delivery of nucleotide sequences (e. g.,
cDNA) encoding recombinant PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 protein into the targeted cell population (e.g.., tumor
cells or neurons). Methods which are well known to those
skilled in the art can be used to construct recombinant viral
vectors containing coding sequences. See, for example, the
techniques described in Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989),
and in Ausubel et al., Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience, N.Y.
(1989). Alternatively, recombinant nucleic acid molecules
encoding protein sequences can be used as naked DNA or in
reconstituted system e.g., liposomes or other lipid systems for
delivery to target cells (See e.g., Felgner et al., Nature
337:387-8, 1989). Several other methods for the direct
transfer of plasmid DNA into cells exist for use in human gene
therapy and involve targeting the DNA to receptors on cells by
complexing the plasmid DNA to proteins. See, Miller, supra.
In its simplest form, gene transfer can be performed by
simply injecting minute amounts of DNA into the nucleus of a
cell, through a process of microinjection. (Capecchi MR, Cell
22:479-88, 1980). Once recombinant genes are introduced into a
_ .~__ __._.___
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67
cell, they can be recognized by the cells normal mechanisms for
transcription and translation, and a gene product will be
expressed. Other methods have also been attempted for
introducing DNA into larger numbers of cells. These methods
include: transfection, wherein DNA is precipitated with CaPO,
and taken into cells by pinocytosis (Chen C. and Okayama H,
Mol. Cell Biol. 7:2745-52, 1987): electroporation, wherein
cells are exposed to large voltage pulses to introduce holes
into the membrane (Chu G., et al., Nucleic Acids Res., 15:1311-
26, 1987); lipofection/liposome fusion, wherein DNA is packaged
into lipophilic vesicles which fuse with a target cell (Felgner
PL., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7, 1987));
and particle bombardment using DNA bound to small projectiles
(Yang NS. et al., Proc. Natl. Acad. Sci. 87:9568-72, 1990).
Another method for introducing DNA into cells is to couple the
DNA to chemically modified proteins.
It has also been shown that adenovirus proteins are
capable of destabilizing endosomes and enhancing the uptake of
DNA into cells. The admixture of adenovirus to solutions
containing DNA complexes, or the binding of DNA to polylysine
covalently attached to adenovirus using protein crosslinking
agents substantially improves the uptake and expression of the
recombinant gene. Curiel DT et al., Am. J. Respir. Cell. Mol.
Biol., 6:247-52, 1992).
As used herein "gene transfer" means the process of
introducing a foreign nucleic acid molecule into a cell. Gene
transfer is commonly performed to enable the expression of a
particular product encoded by the gene. The product may
include a protein, polypeptide, anti-sense DNA or RNA, or
enzymatically active RNA. Gene transfer can be performed in
cultured cells or by direct administration into animals.
Generally gene transfer involves the process of nucleic acid
contact with a target cell by non-specific or receptor mediated
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68
interactions, uptake of nucleic acid into the cell through the
membrane or by endocytosis, and release of nucleic acid into
the cytoplasm from the plasma membrane or endosome. Expression
may require, in addition, movement of the nucleic acid into the
nucleus of the cell and binding to appropriate nuclear factors
for transcription.
As used herein "gene therapy" is a form of gene transfer
and is included within the definition of gene transfer as used
herein and specifically refers to gene transfer to express a
therapeutic product from a cell in vivo or in vitro. Gene
transfer can be performed ex vivo on cells which are then
transplanted into a patient, or can be performed by direct
administration of the nucleic acid or nucleic acid-protein
complex into the patient.
In another preferred embodiment, a vector having nucleic
acid sequences encoding a PTP04, SAD, PTP05, PTP10, ALP, or
ALK-7 is provided in which the nucleic acid sequence is
expressed only in specific tissue. Methods of achieving
tissue-specific gene expression as set forth in International
Publication No. WO 93/09236, filed November 3, 1992 and
published May 13, 1993.
In all of the preceding vectors set forth above, a further
aspect of the invention is that the nucleic acid sequence
contained in the vector may include additions, deletions or
modifications to some or all of the sequence of the nucleic
acid, as defined above.
In another preferred embodiment, a method of gene
replacement is set forth. "Gene replacement" as used herein
means supplying a nucleic acid sequence which is capable of
being expressed in vivo in an animal and thereby providing or
augmenting the function of an endogenous gene which is missing
or defective in the animal.
_. _ 1 _____-__
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XIII.Compounds that Modulate the Function of PTP04, SAD, PTP05,
PTP10, ALP, or ALK-7 Proteins.
In an effort to discover novel treatments for diseases,
biomedical researchers and chemists have designed, synthesized,
and tested molecules that inhibit the function of protein
kinases. Some small organic molecules form a class of com-
pounds that modulate the function of protein kinases. Examples
of molecules that have been reported to inhibit the function of
protein kinases include, but are not limited to, bis
monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO
92/20642, published November 26, 1992 by Maguire et al.),
vinylene-azaindole derivatives (PCT WO 94/14808, published July
7, 1994 by Ballinari et aI.), 1-cyclopropyl-4-pyridyl-
quinolones (U. S. Patent No. 5,330,992), styryl compounds (U. S.
Patent No. 5,217,999), styryl-substituted pyridyl compounds
(U. S. Patent No. 5,302,606), certain quinazoline derivatives
(EP Application No. 0 566 266 A1), selenoindoles and selenides
(PCT WO 94/03427, published February 17, 1994 by Denny et al.),
tricyclic polyhydroxylic compounds (PCT WO 92/21660, published
December 10, 1992 by Dow), and benzylphosphonic acid compounds
(PCT WO 91/15495, published October 17, 1991 by Dow et al).
The compounds that can traverse cell membranes and are
resistant to acid hydrolysis are potentially advantageous
therapeutics as they can become highly bioavailable after being
administered orally to patients. However, many of these
protein kinase inhibitors only weakly inhibit the function of
protein kinases. In addition, many inhibit a variety of
protein kinases and will therefore cause multiple side-effects
as therapeutics for diseases.
Some indolinone compounds, however, form classes of acid
resistant and membrane permeable organic molecules. PCT WO
96/22976, published August 1, 1996 by Ballinari et al.
describes hydrosoluble indolinone compounds that harbor
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tetralin, naphthalene, quinoline, and indole substituents fused
to the oxindole ring. These bicyclic substituents are in turn
substituted with polar moieties including hydroxylated alkyl,
phosphate, and ether moieties. International Patent Publica-
5 tion WO 96/22976, published August l, 1996 by Ballinari et al.
describe indolinone chemical libraries of indolinone compounds
harboring other bicyclic moieties as well as monocyclic
moieties fused to the oxindole ring. WO 96/22976, published
August 1, 1996 by Ballinari et al. teach methods of indolinone
10 synthesis, methods of testing the biological activity of
indolinone compounds in cells, and inhibition patterns of
indolinone derivatives.
Other examples of substances capable of modulating PTP04,
SAD, PTP05, PTP10, ALP, or ALK-7 activity include, but are not
15 limited to, tyrphostins, quinazolines, quinoxolines, and
quinolines.
The quinazolines, tyrphostins, quinolines, and
quinoxolines referred to above include well known compounds
such as those described in the literature. For example,
20 representative publications describing quinazoline include
Barker et al., EPO Publication No. 0 520 722 A1; Jones et al.,
U.S. Patent No. 4,447,608; Kabbe et al., U.S. Patent No.
4,757,072; Kaul and Vougioukas, U.S. Patent No. 5, 316,553;
Kreighbaum and Comer, U.S. Patent No. 4,343,940 Pegg and
25 Wardleworth, EPO Publication No. 0 562 734 A1~ Barker et al.,
Proc. of Am. Assoc. for Cancer Research 32:327 (1991); Bertino,
J.R., Cancer Research 3:293-304 (1979); Bertino, J.R., Cancer
Research 9(2 part 1):293-304 (1979); Curtin et al., Br.
J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research
30 43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178;
Fry et al., Science 265:1093-1095 (1994); Jackman et al.,
Cancer Research 51:5579-5586 (1981); Jones et al. J. Med. Chem.
29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362
_ _.
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71
(1987) Lemus et al., J. Org. Chem. 54:3511-3518 (1989); Ley
and Seng, Synthesis 1975:415-522 (1975); Maxwell et al.,
Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al.,
Cancer Research 45:325-330 (1985); Phillips and Castle, J.
Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al.,
Cancer Research 47(11):2996-2999 (1977); Sculier et al., Cancer
Immunol. and Immunother. 23:A65 (1986); Sikora et al., Cancer
Letters 23:289-295 (1984); Sikora et al., Analytical Biochem.
172:344-355 (1988).
Quinoxaline is described in Kaul and Vougioukas, U.S.
Patent No. 5,316,553.
Quinolines are described in Dolle et al., J. Med. Chem.
37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131
(1994); Burke et al., J. Med. Chem. 36:425-432 (1993); and
Burke et al. BioOrganic Med. Chem. Letters 2:1771-1774 (1992).
Tyrphostins are described in Allen et al., Clin. Exp.
Immunol. 91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529
(1993); Baker et al., J. Cell Sci. 102:593-555 (1992); Bilder
et al., Amer. Physiol. Soc. pp. 6363-6143:C721-C730 (1991);
Brunton et al., Proceedings of Amer. Assoc. Cancer Rsch. 33:558
(1992); Bryckaert et al., Experimental Cell Research 199:255-
261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993);
Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,
J. Med. Chem. 32:2344-2352 (1989); Gazit et al., " J. Med.
Chem. 36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs
5:213-222 (1994); Kaur et al., King et al., Biochem. J.
275:413-418 (1991); Kuo et al., Cancer Letters 74:197-202
(1993): Levitzki, A., The FASEB J. 6:3275-3282 (1992); Lyall
et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson et al.,
The Prostate 22:335-395 (1993); Pillemer et al., Int. J. Cancer
50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-
683 (1993); Rendu et al., Biol. Pharmacology 44(5):881-888
(1992); Sauro and Thomas, Life Sciences 53:371-376 (1993);
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72
Sauro and Thomas, J. Pharm. and Experimental Therapeutics
267(3):119-1125 (1993); Wolbring et al., J. Biol. Chem.
269(36):22470-22472 (1994); and Yoneda et al., Cancer Research
51:4430-4435 (1991).
Other compounds that could be used as modulators include
oxindolinones.
Examples
The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention. The examples below show the isolation and
characterization of the novel proteins, protein expression in
normal and tumor cells, generation of protein specific
antibodies, and recombinant expression in mammalian and yeast
systems. Also shown are assays useful for identifying
compounds that modulate protein activity.
Example 1: Isolation Of cDNA Clones Encoding PTP04
The example below describes the isolation and identi
fication of a new PTP sequence from primary cancer tissues and
the subsequent cloning of a full-length human PTP04. Also
described are probes useful for the detection of PTP04 in cells
or tissues.
Materials and Methods:
Poly A+ RNA was isolated from 30uM cryostat sections of
frozen samples from primary human lung and colon carcinomas
(Micro-FastTrack, InVitrogen, San Diego, CA). This RNA was
used to generate single-stranded cDNA using the Superscript
Preamplification System (GIBCO BRL, Gaithersburg, MD.; Gerard,
GF et al. (1989), FOCUS 11, 66) under conditions recommended by
the manufacturer. A typical reaction used 10 /.cg total RNA or 2
~cg poly (A) RNA with 1. 5 ,ug oligo (dT) 12_ie in a reaction volume of
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60 ~.L. The product was treated with RNaseH and diluted to 100
/,cL with H20. For subsequent PCR amplification, 1-4 /.cL of this
sscDNA was used in each reaction.
Degenerate oligonucleotides were synthesized on an Applied
Biosystems 394 DNA synthesizer using established phosphora
midite chemistry, precipitated with ethanol and used unpurified
for PCR. The sequence of the degenerate oligonucleotide primers
follows:
PTPDFW - 5'-GAYTTYTGGVRNATGRTNTGGGA- (sense) (SEQ ID
N0:17) and
PTPHCSA - 5'-CGGCCSAYNCCNGCNSWRCARTG -3' (antisense) (SEQ
ID N0:18).
These primers were derived from the peptide sequences
DFWXMXW(E/D) (SEQ ID N0:19) (sense strand from PTP catalytic
domain) and HCXAGXG (antisense strand from PTP catalytic
domain) (SEQ ID N0:20), respectively. Degenerate nucleotide
residue designations are: N = A, C, G, or T: R = A or G; and Y
- C or T.
PCR reactions were performed using degenerate primers
applied to the single-stranded cDNA listed above. The primers
were added at a final concentration of 5 ~M each to a mixture
containing 10 mM Tris'HC1 (pH8.3) , 50 mM KC1, 1. 5 mM MgCl2, 200
~M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U
AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ~cL cDNA.
Following 3 min denaturation at 95 °C, the cycling conditions
were 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min
45 s
for 35 cycles. PCR fragments migrating between 350-400 by were
isolated from 2o agarose gels using the GeneClean Kit (Bio101),
and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.)
according to the manufacturer's protocol.
Colonies were selected for mini plasmid DNA-preparations
using Qiagen columns and the plasmid DNA was sequenced using
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74
cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction
products were run on an ABI Prism 377 DNA Sequencer, and
analyzed using the BLAST alignment algorithm (Altschul, S.F. et
al., J. Mol. Bio1.215:403-10). One novel clone novel clone
(G77-4a-117), designated PTP09, was isolated from human HLT370
primary lung carcinoma sample.
To obtain full-length cDNA encoding the novel phosphatase,
RACE (rapid amplification of cDNA ends) was performed with
sense or anti-sense oligonucleoides derived from the original
PCR fragments. Marathon-Ready cDNA (Clontech, Palo Alto, CA)
made from human Molt-4 leukemia cells was used in the RACE
reactions with the following primers:
RACE primers:
5'-CACCGTTCGAGTATTTCAGATTGTGAAGAAG-TCC-3' (6595) (SEQ ID
N0:21) ,
5'-GGACTTCTTCACAATCTGAAATACTCGAACGGTG-3' (6596) (SEQ ID
N0:22),
5'-CCGTTATGTGAGGAAGAGCCACATTACAGGACC-3' (6599) (SEQ ID
N0:23},
5'-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3' (6600) (SEQ ID
N0:24),
AP-1, and AP-2 (Clontech).
RT-PCR primers for PTP04:
5'-GGCATGCATGGAGTATGAAATGG-3' (6618) (SEQ ID N0:25},
5'-CGTACATCCCAGATGAGCTCAAGAATAGGG-3' (6632) (SEQ ID N0:26).
Isolated cDNA fragments encoding PTP04 were confirmed by
DNA sequencing and subsequently used as probes for the
screening of a human leukocyte cDNA library.
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A human leukocyte cDNA library (lTriplEx, Clontech) and a
Molt-4 leukemia cell library (1gt11, Clontech) were then
screened to isolate full-length transcripts encoding PTP04.
The 5' or 3'-RACE fragments were 32P-labeled by random priming
5 and used as hybridization probes at 2x106 cpm/mL following
standard techniques for library screening. Pre-hybridization
(3 h) and hybridization (overnight) were conducted at 42 °C in
5X SSC, 5 X Denhart's solution, 2.5~ dextran sulfate, 50 mM
Na2P09/NaHPOq [pH 7.0], 50~ formamide with 100 mg/mL denatured
10 salmon sperm DNA. Stringent washes were performed at 65 °C in
O.1X SSC and O.lo SDS. Several overlapping clones were
isolated and found to span the sequence of the PCR fragment
(G77-4a-117). The final sequence was verified by sequencing of
both strains using a cycle sequencing dye-terminator kit with
15 AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing
reaction products were run on an ABI Prism 377 DNA Sequencer.
Results:
The 3,580 by human PTP04 nucleotide sequence encodes a
20 polypeptide of 807 amino acids. The PTP04 coding sequence is
flanked by a 52 nucleotide 5'-untranslated region and a 1086
nucleotide 3'-untranslated region ending with a poly(A) tail.
While there are no upstream in frame stop codons, the first ATG
beginning at nucleotide position 53 conforms to the Kozak
25 consensus for an initiating methionine. This predicted first 6
amino acids are identical to those of murine ZPEP (SwissProt:
P29352, GeneBank: M90388), further supporting this is the true
translational start site. One cDNA clone had an insert after
nucleotide 30 in the 5'UTR, but otherwise had no sequence
30 differences.
The 807 amino acid sequence shows no signal sequence or, a
transmembrane domain and PTP04 is, therefore, an intracellular
protein. PTP04 has an N-terminal region from amino acids 1-48,
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76
a catalytic domain from amino acids 49-294, and a C-terminal
tail from amino acids 295-807. PTP04 is most related to murine
ZPEP with an overall homology of 70~. ZPEP is a member of a
subfamily of PTPs that includes PTP-PEST, HSC, BDP1 and PTP20,
all of which are cytoplasmic PTPs with a single catalytic
domain and a region rich in Pro, Glu, Ser and Thr residues
(PEST domain). PTP04 also has a C-terminal PEST domain, from
amino acids 495-807, where there are 57 serine residues (180)
and 35 proline residues(11~). A comparison of the amino acid
sequences of PTP04 and ZPEP is shown in Figure 1.
The homology between PTP04 and ZPEP is concentrated in the
N terminal and C-terminal ends of the proteins with significant
divergence in the middle. The N-terminal region of PTP04, from
amino acids 1-48, is 81o homologous to murine ZPEP. The
catalytic domain of PTP04, from amino acids 49-294, is 89°a
homologous to murine ZPEP. The region of PTP04 from 294-600 is
approximately 50% homologous to murine ZPEP. The C-terminal
region of PTP04, from 680-817, is 80o homologous to murine
ZPEP. The human SuPTP04 sequence defines a novel member of the
PTP-PEST subfamily of PTPs.
Example 2: Expression Of PTP04
The example below shows the evaluation of PTP04 expression
in normal human tissues and in cancer cell lines.
Materials and Methods:
Northern blots were prepared by running 20 /,cg total RNA
per lane isolated from 22 human adult normal tissues (thymus,
lung, duodenum, colon, testis, brain, cerebellum, salivary
gland, heart, liver, pancreas, kidney, spleen, stomach, uterus,
prostate, skeletal muscle, placenta, mammary gland, bladder,
lymph node, adipose tissue), 2 human fetal normal tissues
(fetal liver, fetal brain), and 24 human tumor cell lines
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77
HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, A549,
HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3,
SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, SR,
DU-145, PC-3) (obtained from Nick Scuidero, National Cancer
Institute, Developmental Therapeutics Program, Rockville, MD).
The total RNA samples were run on a denaturing formaldehyde 1%
agarose gel and transferred onto a nitrocellulose membrane
(BioRad, CA). An additional human normal tissue Northern blot
containing 2 /,cg polyA+ mRNA per lane from 8 different human
cancer cell lines (NCI-H522, K-562, MOLT-9, HL-60, S3, Raji,
SW480, 6361) on a charge-modified nylon membrane (human cancer
cell line blot #7757-1, Clontech, Palo Alto, CA) were also
hybridized.
For the total RNA samples, nitrocellulose membranes were
hybridized with randomly primed [a-32P]dCTP-labeled probes
synthesized from a 579 by StuI-BstXI fragment of
pCR2.l.mini298. Hybridization was performed overnight at 42°C
in 4X SSPE, 2.SX Denhardt's solution, 50% formamide, 0.2 mg/mL
denatured salmon sperm DNA, 0.1 mg~/mL yeast tRNA (Boehringer
Mannheim,IN), 0.2% SDS, with 5 x 106 cpm/mL of [a-3zP]dCTP
labeled DNA probes on a Techne hybridizer HB-1. The blots were
washed with 2X SSC, 0.1% SDS, at 65 °C for 20 min twice followed
by in 0.5 X SSC, 0.1% SDS at 65 °C for 20 min. The blots were
exposed to a phospho-imaging screen for 24 hours and scanned on
a Molecular Dynamics Phosphoimager SF.
A 351 by EcoRI-HindIII fragment of G77-4a-117 was used to
generate a probe for 2 ~g poly A+ mRNA samples on a Clontech
nylon membrane. Hybridization was performed at 42 °C overnight
in 5X SSC, 2% SDS, lOX Denhardt's solution, 50% formamide, 100
/.cg/mL denatured salmon sperm DNA with 1-2 x 106 cpm/mL of [a-
s2P]dCTP -labeled DNA probes. The membrane was washed at room
temperature in 2X SSC/0.05% SDS for 30 min and followed by at
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50 °C in 0.2X SSC/O.lo SDS for 30 min, twice, and exposed for 48
hours on Kodak XAR-2 film.
RT-PCR Detection of novel PTPs -
Total RNA was isolated from various cell lines or fresh
frozen tissues by centrifugation thrugh a cesium chloride
cushion. Twenty ,ug of total RNA was reverse transcribed with
random hexamers and Moloney murine leukemia virus reverse
transcriptase (Super-ScriptII, GIBCO BRL, Gaithersburg, MD).
PCR was then used to amplify cDNA encoding SuPTP04. RT-PCR
reactions lacking only the reverse transcriptase were performed
as controls. PCR products were electrophoresed on 3~ agarose
gels, visualized by ethidium bromide staining and photographed
on a UV light box. The intensity for a 270-by fragment
specific to PTP04 were compared among different RNA samples.
Results:
A single SuPTP04 mRNA transcript of approximately 4.5 kb
was identified by Northern analysis, and found to be
exclusively in the Thymus. The rest of 23 human normal tissues
(fetal brain, fetal liver, lung, duodenum, colon, testis,
brain, cerebellum, salivary gland, heart, liver, pancreas,
kidney, spleen, stomach, uterus, prostate, skeletal muscle,
placenta, mammary gland, bladder, lymph node, adipose tissue)
were all negative. Six of the human tumor cell lines (CCRF-
CEM, K-562, MOLT-4, HL-60, SR, Raji) were positive. The rest
of 26 human tumor cell lines were negative. RT-PCR with gene
specific primer-pairs showed that expression of the transcripts
encoding SuPTP04 confirmed the results from Northern analysis
and also detected low levels in adipose, kidney, small
intestine, hematopoietic tissues and various cell types
(spleen, thymus, lymph node, bone marrow, peripheral leukocytes
and lymphocytes.
_ __ ____T ___
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The selective expression of PTP04 in cells of hematopoetic
origin including normal human thymus and several leukemia cell
lines suggests a potential involvement in immune regulation
including T and B cell survival, differentiation or co-
y stimulation, and/or inflammatory, immunosuppressive or
autoimmune disorders. Additionally, expression in adipose
tissue suggests a possible role in metabolic disorders such as
diabetes.
Example 3: Recombinant Expression Of PTP04
The following example illustrates the construction of
vectors for expression of recombinant PTP04 and the creation of
recombinant cell lines expressing PTP04.
Construction of Expression Vectors -
Expression constructs were generated by PCR-assisted
mutagenesis in which the entire coding domains of PTP04 was
tagged on its carboxy-terminal end with the hemophilus
influenza hemaglutinin (HA) epitope YPYDVPDYAS (SEQ ID N0:55)
(Pati, 1992). The construct was introduced into two mammalian
expression vectors: pLXSN (Miller, A.D. & Rosman, G.J.,
Biotechniques 7, 980-988, 1989) for the generation of virus
producing lines; and pRKS for transient expression in
mamma 1 i an .
Dominant negative (signaling incompetent) PTP04 constructs
were also made in both pLXSN and pRKS by mutation of the
invariant Cys in the conserved HCSAG (SEQ ID N0:56) motif to
an Ala by PCR mutagenesis.
The entire PTP04 open reading frames (no HA-tag) excluding
the initiating methionines were generated by PCR and ligated
into pGEX vector (Pharmacia Biotech, Uppsala, Sweden) for
bacterial production of GST-fusion proteins for immunization of
rabbits for antibody production. The entire PTP04 open reading
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frame excluding the initiating methionines was generated by PCR
and ligated into pGEX vector for bacterial production of GST-
fusion proteins for immunization of rabbits for antibody
production. This vector contains the glutathione-S-transferase
5 coding sequence followed by a polylinker for generating
recombinant fusion proteins. The GST moiety comprises the N-
terminal portion of the fusion protein.
Transient Expression in Mammalian Cells -
10 The pRK5 expression plasmids (10 ~,g DNA/100 mm plate)
containing the HA-tagged PTP04 gene can be introduced into COS
and 293 cells with lipofectamine (Gibco BRL). After 72 hours,
the cells were harvested in 0.5 mL solubilization buffer (20 mM
HEPES pH 7.35, 150 mM NaCl, 10% glycerol, to Triton X-100, 1.5
15 mM MgCl2, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ,ug/mL
aprotinin). Sample aliquots were resolved by SDS
polyacrylamide gel electrophoresis (PAGE) on 15%acrylamide/0.5~
bis-acrylamide gels and electrophoretically transferred to
nitrocellulose. Non-specific binding was blocked by
20 preincubating blots in Blotto (phosphate buffered saline
containing 5~ w/v non-fat dried milk and 0.2°s v/v nonidet P-40
(Sigma)), and recombinant protein was detected using a murine
Mab to the HA decapeptide tag. Alternatively, recombinant
protein can be detected using various PTP04-specific antisera.
Generation of Virus Producin Cell Lines
pLXSN recombinant constructs containing the PTP04 gene
were transfected into an amphotropic helper cell line PA317
using CaCl2 mediated transfection. After selection on 6418,
the cells were plated on normal media without 6418 (500 ~.g/mL).
Supernatants from resistant cells were used to infect the
ecotropic helper cell line GP+E86, and cells again selected on
r
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6418. Resistant cells were again taken off 6418, and the
supernatants harvested every 8-12 hours and pooled as virus
stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498.
Viral stock titers were typically ~106/mL.
Stable Expression in Mammalian Cells
NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates
with DMEM (Gibco) containing loo fetal calf serum (FCS). The
cells were superinfected with the PTP04 retrovirus by adding
approximately 3 mL viral supernatant to 15 mL culture media for
approximately 24 hours. Cells expressing the retroviral
constructs were then selected by growth in DMEM/l0o FCS
supplemented with 500 /.cg/mL 6418.
Example 4: Generation of Anti-PTP04 Antibodies
PTP04-specific immunoreagents were raised in rabbits
against a mixture of three KLH-conjugated synthetic peptides
corresponding to unique sequences present in human PTP04. The
peptides (see below) were conjugated at the C-terminal residue
with KLH.
peptide 428A: SWPPSGTSSKMSLDDLPEKQDGTVFPSSLLP (SEQ ID
N0:27)
peptide 429A: YSLPYDSKHQIRNASNVKHHDSSALGVYSY (SEQ ID
N0:28)
peptide 430A: HTLQADSYSPNLPKSTTKAAKMMNQQRTKC (SEQ ID
N0:29)
Additional immunoreagents were generated by immunizing
rabbits with the bacterially expressed entire coding region of
PTP04 expressed as a GST-fusion protein. GST fusion proteins
were produced in DH5-alpha E. coli bacteria as descaribed in
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Smith, et al Gene 67:31, 1988. Bacterial protein lysates were
purified on glutathione-sepharose matrix as described in Smith,
et al, supra.
Example 5: Assay for PTP04 Activity
MatAri al c anrl mathnric~
Recombinant wild-type and dominant negative (signaling
incompetent) PTP04 (see Example 3, supra) were purified from
bacteria as GST-fusion proteins. Lysates were bound to a
glutathione-sepharaose matrix and washed twice with 1X HNTG,
followed by one wash with a buffer containing 100 mM 2-(N-
morpholino)ethansulfonic acid (MES), pH 6.8, 150 mM NaCl, and 1
mM EDTA.
The assay for phosphatase activity was essentially done as
described by Pei et al.(1993) using p-nitrophenolphosphate
(PNPP) as a generic PTP substrate. Briefly, after the last
washing step, reactions were started by adding 50 mL Assay
Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA,
and 50 mM PNPP) to the matrix bound proteins. Samples were
incubated for 20 min. at 23 °C. The reactions were terminated
by mixing 40 E.cL of each sample with 960 ~L 1 N NaOH, and the
absorbance of p-nitrophenol was determined at 450 nm. To
control for the presence of PTP04 in the precipitates, the
precipitates were boiled in SDS sample buffer and analyzed by
SDS-PAGE. The presence of PTP04 was then detected by
immunoblot analysis with anti-PTP04 antibodies.
Example 6: Isolation and Characterization of SAD
This example describes the isolation and characterization
of the non-receptor tyrosine kinase SAD. Initially we set out
to identify novel members of the Src family, a group of nine
related cytoplasmic tyrosine kinases which play key roles in
several signal transduction pathways. Based on comparison of
~ ___ __..
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ail known tyrosine kinases, we designed a pair of degenerate
oligonucleotide primers that specifically recognize Src family
members plus three more distantly related proteins Srm, Brk,
and MKK3 or Frk (the Srm/Brk/Frk group). The sequence FGE/DVW
(SEQ ID N0:30) is located near the amino terminus of the kinase
domain and is unique to Src family members and the Srm/Brk/Frk
group. The sequence WTAPE (SEQ ID N0: 31) is located just
amino terminal to the highly conserved DVWS motif of tyrosine
kinases and is contained in the Src family and the Srm/Brk/Frk
group as well as the Eph, Csk, Abl, and Fes families.
When we used the FGE/DVW and WTAPE primers in PCR
amplification reactions with HME (human mammary epithelial)
cell sscDNA as a template, we isolated multiple copies of known
Src relatives as well as a novel DNA fragment (HME 1264) of 483
by with homology to other kinases. The novel sequence was most
similar to mouse Srm (GeneBank Accession #D26186) and the clone
was designated human SAD.
A SAD probe was used to screen a cDNA library constructed
from human breast cancer cell line mRNA to isolate two
overlapping, independent clones spanning the kinase domain, but
containing apparent introns and presumably arising from
incompletely processed transcripts. The 5' end of the coding
region was subsequently isolated by sequential RACE reactions
from MCF7 RNA, and the entire coding region was re-isolated by
PCR from HME cDNA.
Materials And Methods
Total RNA was isolated using the Guanidine Salts/Phenol
extraction protocol of Chomczynski and Sacchi (P. Chomczynski
and N. Sacchi, Anal. Biochem. 162, 156 (1987) from HME (human
mammary epithelial) cells. This RNA was used as a template to
generate single-stranded cDNAs using the Superscript Pre-
amplification System for First Strand Synthesis kit purchased
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from GibcoBRL (Life Technologies, U.S.A.; Gerard, GF et al,
FOCUS 11:66, 1989) under conditions recommended by
manufacturer. A typical reaction used 10 ,ug total RNA or 2 ~g
poly (A) + RNA with 1. 5 ,ug oligo (dT) 12_i8 in a reaction volume of
60 ~cL. The product was treated with RNaseH and diluted to 100
uL with H20. For subsequent PCR amplification, 1-4 ,uL of these
sscDNAs were used in each reaction.
Oligonucleotides were synthesized on an Applied Biosystems
394 DNA synthesizer using established phosphoramidite chemistry
and were used unpurified after precipitation with ethanol. The
degenerate oligonucleotide primers are:
FGE/DVW - 5'-GGNCARTTYGGNGANGTNTGG-3' (SEQ ID N0:30) (sense)
and
WTAPE = 5'-CAGNGCNGCYTCNGGNGCNGTCCA-3' (SEQ ID N0:31}
(antisense).
These primers were derived from the peptide sequences
GQFG(E/D)VW (SEQ ID N0:32) (sense strand) and WTAPEALL (SEQ ID
N0:33) (antisense strand}, respectively. Degenerate nucleotide
residue designations are: N = A, C, G, or T; R = A or G: and Y
- C or T. Using Src as a template, these primers produce a
product of 480 bp.
A PCR reaction was performed using primers FGE/DVW and
WTAPE applied to HME cell cDNA. The primers were added at a
final concentration of 0.5 uM each to a mixture containing 10
mM Tris.HCl (pH8.3), 50 mM KCl, 1.5 mM MgCl2, 200 uM each
deoxynucleoside triphosphate, 0.0010 gelatin, and 1.5 U
AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ~L cDNA.
Following 3 min denaturation at 94 °C, the cycling conditions
were 94 °C for 30 sec, 37 °C for 1 min, a 2 min ramp to 72
°C,
and 72°C for lmin for the first 3 cycles, followed by 94 °C for
30 sec, 60°C for 1 min, and 72 °C for 1 min for 35 cycles . PCR
fragments migrating at between 450-550 by were isolated from 2%
_ ~ _ . ______.~_ ____ _ _
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agarose gels, phosphorylated and repaired by treatment with T4
polynucleotide kinase and Klenow fragment, and blunt-end cloned
into the EcoRV site of the vector pBluescriptSK+ (Stratagene
U.S.A.).
5 Plasmid DNAs were isolated from single colonies by DNA
minipreparations using QIAGEN columns and were sequenced using
cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction
products were run on an ABI Prism 377 DNA Sequencer, and
10 analyzed using the BLAST alignment algorithm (Altschul, S.F. et
al., J. Mol. Bio1.215:403-10, 1990). A novel clone (HME1264)
was isolated by PCR with primers FGE/DVW and WTAPE on single-
stranded cDNA from HME cells as a template. This clone was
subsequently designated as a fragment of human SAD.
15 A lambda ZapII (Stratagene Cloning Systems, La Jolla, CA)
cDNA library was constructed using mRNA from a pool of breast
carcinoma cell lines as a template for first strand cDNA
synthesis with both oligo-(dT) and random priming (library
created by Clonetech custom library synthesis department, Palo
20 Alto, CA}. The cell lines used for the pool were MCF7, HBL100,
MDA-MB231, MDA-MB175IIV, MDA-MB435, MDA-MB453, MDA-MB468, BT20,
T47D and SKBR3, all of which are available from the ATCC.
Phage were screened on nitrocellulose filters with the random
primed 32P-labeled insert from HME1264 at 2x106 cpm/mL in
25 hybridization buffer containing 6xSSPE, 50~ formamide, 2x
Denhardt's reagent, 0.1% SDS, with 0.05 mg/mL denatured,
fragmented salmon sperm DNA. After overnight hybridization at
42 °C, filters were washed in lxSSC, 0.1~ SDS at 65 °C. Two
overlapping partial clones were isolated and sequenced through
30 the coding region using manual sequencing with T7 polymerase
and oligonucleotide primers (Tabor and Richardson, Proc. Natl.
Acad. Sci. U.S.A. 84: 4767-71, 1987). These isolates encompass
the kinase domain of SAD and extend from within an apparent
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intron 5' to the kinase domain and extend 3' to an in-frame
termination codon, but are interrupted by four more apparent
introns.
Two sequential 5' RACE (rapid amplification of cDNA ends)
reactions (Frohman et al., Proc. Natl. Acad. Sci. U.S.A. 85:
8998, 1988) were subsequently used to isolate the 5' end of the
coding region. Single strand cDNA was prepared as described
above using the Superscript Pre-amplification System (GibcoBRL)
using 6 ~g total RNA from MCF7 cells as a template and gene
specific primers,5556 (5'-AGTGAGCTTCATGTTGGCT-3' (SEQ ID N0:39}
for RACE 1 or 5848 (5'-GGTAGAGGCTGCCATCAG-3' (SEQ ID N0:35))
for RACE 2 to prime reverse transcription. Following treatment
with RNase H, sscDNA was recovered using two sequential ethanol
precipitations with ammonium acetate and carrier glycogen
homopolymer tail of dA was added by treatment with deoxy-
terminal transferase (GibcoBRL) and two reaction mixtures
diluted to 50 /.cL with TE. Second strand cDNA synthesis by
AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus) was primed with
0.2 uM PENN(dT)1~ (5'- GACGATCGGAATTCGCGA(dT)17-3' (SEQ ID N0:36)
using 1-5 ~L of tailed cDNA as a template and buffer conditions
given above. Following 5 min denaturation at 94 °C, 1 min
annealing at 50 °C, and 90 min extension at 72 °C, primers PENN
(5'-GACGATCGGAATTCGCGA-3' (SEQ ID N0:37) and 5555 (5'-
CCCAGCCACAGGCCTTC-3' (SEQ ID N0:38} were added at 1 ~tM and PCR
done with cycling conditions of 94 °C for 30 s, 49 °C for 1
min,
and 72 °C for 1 min, 45 sec for 40 cycles. A second, nested PCR
was done using 0.2 ~,L of the initial PCR reaction as a template
and primers PENN (see SEQ ID N0:37) and 5554 (5'-
CCACACCTCCCCAAAGTA-3' (SEQ ID N0:39) at 1 ACM with an initial 3
min denaturation at 94 °C, followed by cycling conditions of 94
°C for 30 s, 49 °C for 1 min, and 72 °C for 1 min, 45 sec
for 35
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cycles. PCR products were separated on 1~ agarose gels and
visualized by ethidium bromide staining and Southern
hybridization using oligonucleotide 5557 (5'-
TGGGAGCGGCCACACTCCGAATTCGCCCTT-3' (SEQ ID N0:40) end-labeled
with 32P. Reaction products of 500-700 by were digested with
EcoRI and cloned into the EcoRI site of pBluescriptSK+
(Stratagene U.S.A.), and positive clones were identified by
colony hybridization with oligonucleotide 5557 as a probe.
Clone 16A1 (which encompasses nucleotides 195 to 783 of SEQ ID
NO:10) was isolated and sequenced by a combination of ABI and
manual sequencing.
A second set of 5' RACE reactions was done based on the
sequence of clone 16A1 using the procedure given above except
as noted. Gene specific primers were 5848 (SEQ ID N0:35) for
the first strand synthesis, 6118 (5'-GCCTGCGTGCGAAGATG-3' (SEQ
ID N0:41) for the first PCR, and 6119 (5'-CTTCGAGGGCACAGAGCC-3'
(SEQ ID N0:42) for the second PCR, and the probe for Southern
and colony hybridization was random primed 32P-labeled insert
from 16A1. PCR fragments migrating at between 250-450 by were
isolated from 2~ agarose gels, phosphorylated and repaired by
treatment with T4 polynucleotide kinase and Klenow fragment,
and blunt-end cloned into the EcoRV site of the vector
pBluescriptSK+ (Stratagene U.S.A.). Clone 20E2 (which
encompasses nucleotides 1 to 267 of SEQ ID N0:10) was isolated
and sequenced by a combination of ABI and manual sequencing.
The coding region of SAD was subsequently isolated from
HME cDNA as two overlapping PCR fragments. Single stranded
cDNA was prepared from poly(A)+ RNA from HME cells using the
Superscript Preamplification System (GibcoBRL) as described
above. PCR with AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus)
used 1-2 ~cL of cDNA as a template, an initial 3 min
denaturation at 94°C, followed by cycling conditions of 99oC for
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30 s, 55 °C for 1 min, and 72 °C for 1 min, 45 sec for 30 cycles
and the buffer conditions given above. Primers 6642 (5'-
ATGGAGCCGTTCCTCAGGAGG-3' (SEQ ID N0:43) and 6644 (5'-
TCACCCAGCTTCCTCCCAAGG-3' (SEQ ID N0:44) were used to amplify an
approximately 710 by 5' fragment of SAD, and primers 6643 (5'-
AGGCCAACTGGAAGCTGATCC-3' (SEQ ID N0:45) and 6645 (5'-
GCTGGAGCCCAGAGCGTTGG-3' (SEQ ID N0:46) were used to amplify an
approximately 860 by 3' fragment of SAD. PCR fragments were
isolated from 1% agarose gels, phosphorylated and repaired by
treatment with T4 polynucleotide kinase and Klenow fragment,
and blunt-end cloned into the EcoRV site of the vector
pBluescriptSK+ (Stratagene U.S.A.). Positive clones were
identified by colony hybridization with the random primed 32P-
labeled insert from 16A1 (for the 5' fragment) and the random
primed 32P-labeled insert from HME1264 or 32P-labeled
oligonucleotide 5557 (for the 3' fragment) as probes. The
overlapping 5' and 3' PCR fragments were ligated together via
the unique EcoRI site to give the full length SAD coding
region. The complete sequence of the coding region of huma SAD
was determined from overlapping 5' and 3' PCR clones amplified
from cDNA prepared from HME cells. 5' noncoding sequence was
determined from the overlapping RACE fragment 16A1. Sequence
was determined manually on both strands using cycle sequencey
dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI,
Foster City, CA).
Results
The 1,548 by human SAD (SAD h) nucleotide sequence shown
in SEQ ID N0:10 contains a single open reading frame encoding a
polypeptide of 488 amino acids. The SAD h coding region is
preceded by a 48 nucleotide 5'-untranslated region including an
in-frame termi~iation codon four codons before the initiating
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methionine and a 33 nucleotide 3'-untranslated region that
includes two in-frame stop codons.
The sequences of SAD cDNAs were determined from
overlapping PCR-amplified fragments from normal HME cell cDNA
(nucleotides 49-1548), clones from a breast carcinoma cell
lambda cDNA library (nucleotides 694-1548), and overlapping 5'
RACE products from MCF7 cDNA (nucleotides 1-783) with the
following sequence differences including some likely
polymorphic sites. Ambiguities include a change of nucleotide
636 (see SEQ ID N0:10) from a C in the HME PCR clone to a T in
the MCF& RACE product, nucleotide 1477 from a T in the HME PCR
clone to a C in the breast carcinoma libray, a deletion of
nucleotides GT at positions 919 - 920 in the breast carcinoma
library and apparent introns inserted at positions (relataive
to SEQ ID N0:10) 694, 995, 1117, and 1334 in the breast
carcinoma library.
The domain structure of SAD consists of an N-terminal
unique domain followed by an SH3 domain, an SH2 domain and a
kinase domain. This overall topology is shared by members of
the Src, Srm/Brk/Mkk3, and Csk families. SAD is most similar
to mouse Srm (GeneBank Accession #D26186) (Kohmura et al., Mol.
Cell. Biol. 14: 6915-6925, 1994), a distant SRC relative of
unknown function. SAD and Srm share sequence identities in the
individual domains of 55~ (unique region), 72~ (SH3 domain),
78~ (SH2 domain), and 85~ (kinase domain). Unlike true Src
family members, SAD and Srm lack both an N-terminal membrane
attachment sequence and a potential C-terminal negative
regulatory tyrosine. In addition, the characteristic HRDLRXAN
(SEQ ID N0:47) sequence in the Src family kinase domain is
HRDLAXRN (SEQ ID N0:48) in SAD and other Srm/Brk/Mkk3 group
members. Like most other tyrosine kinases, SAD and Srm both
contain a potential autophosphorylation site (380Y of SAD).
The N-terminal sequences of SAD and Srm are similar with twenty
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identical residues out of the first twenty-two amino acids, but
the extreme C-termini are quite distinct.
Available evidence suggests that SAD h and Srm m are
distinct genes rather than species orthologues. Overall, the
5 levels of homology between SAD h and Srm m listed above are
comparable to those of close Src family members (for example
Src h and Yes h), but lower than those of species counterparts
(for example Src h and Src m). SAD h and Srm m also exhibit
distinct expression patterns with SAD h expression detected by
10 PCR in the duodenum and perhaps testes, but not in other
tissues tested, while the Srm m expression was detected by
Northern with highest levels in lung, liver, spleen, kidney,
and testes (Kohmura et al., Mol. Cell. Biol. 14: 6915-6925,
1994) (See Example 2 below.). Lastly, disruption of the Srm
15 gene in mice has no detectable phenotype (Kohmura et al. , Mol .
Cell. Biol. 14: 6915), suggesting that other related proteins
might compensate for its function.
Example 7: SAD Expression Analysis
Materials And Methods
RNA was isolated from a variety of human cell lines and
fresh frozen normal tissues. (Tumor cell lines were obtained
from Nick Scuidero, National Cancer Institute, Developmental
Therapeutics Program, Rockville, MD)Single stranded cDNA was
synthesized from 10 ~g of each RNA as described above using the
Superscript Preamplification System. (GibcoBRL). These single
strand templates were then used in a 35 cycle PCR reaction
using an annealing temperature of 65 °C with two SAD-specific
oligonucleotides (5284: 5'-TCGCCAAGGAGATCCAGACAC-3' (SEQ ID
N0:49), and 5285: 5'-GAAGTCAGCCACCTTGCAGGC-3' (SEQ ID N0:50).
Reaction products were electrophoresed on 2% agarose gels,
_~ __
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stained with ethidium bromide and photographed on a UV light
box. The relative intensity of the approximately 320-by SAD-
specific band was estimated for each sample. The results are
shown with a numerical rating with 4 being the highest relative
expression and 0 being the lowest.
Results
The SAD expression profile in normal human tissue and
multiple cell lines of diverse neoplastic origin was determined
by the semi-quantitative PCR assay using primers from sequences
in the kinase domain. The results are included in Tables 1 and
2. In normal tissue samples (Table 1), modest SAD expression
was detected in the duodenum and possible low levels in testes
with all other samples negative. Much higher expression was
found in a subset of cancer cell lines (Table 2) with the
highest levels in some human colon tumor cell lines (HCT-15,
SW480, and HT-29), an ovarian carcinoma (IGROV1), and an
intestinal carcinoma (SNU-C2B). Lesser expression of SAD was
also seen in some other tumor cell lines derived from colon,
breast, lung, ovary, and kidney as shown in Table 2.
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Table 1
cell type Origin exp. level
duodenum Normal Tissue 2
testes Normal Tissue 1?
brain Normal Tissue 0
heart Normal Tissue 0
kidney Normal Tissue 0
lung Normal Tissue 0
pancreas Normal Tissue 0
placenta Normal Tissue 0
salivary gland Normal Tissue 0
skeletal muscle Normal Tissue 0
spleen Normal Tissue 0
stomach Normal Tissue 0
thymus Normal Tissue 0
cerebellum Normal Tissue 0
liver Normal Tissue 0
uterus Normal Tissue 0
(prostate Normal Tissue 0
_T. _ _~
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Table 2
Cell Line Origin exp. Cell Line Origin ~xp.
HCT-15 colon 4 LOX IMVI melanoma 1?
IGROV1 ovary 4 KATO III gastric 0
carcinoma
SW480 colon 3 R-48 meta Bast. 0
adenoca adenocarcin
rcin oma
oma
SNU-C2B cecum 3 HFL1 lung, 0
primary diploid
carcino
ma
HT-29 colon 3 HOP62 lung 0
Colo 205 colon 2 OVCAR-4 ovary 0
carcino
ma
SW948 colon 2 SKOV3 ovary 0
adenoca
rcinoma
HCT116 colon 2 NCIH23 lung 0
EKVX lung 2 NCI-H460 lung 0
NCI-H23 lung 2 COL0205 colon 0
HCC-2998 colon 2 NCI-H460 lung 0
HCT116 colon 2 A549/ATCC LUNG 0
MCF7 breast 2 HOP-62 lun 0
T-47D breast 2 COLD 205 colon 0
OVCAR-3 ovar 2 KM-12 colon 0
OVCAR-5 ovary 2 MDA-MB- breast 0
231
OVCAR-8 ovary 2 MDA-MB- breast 0
435
SN12C renal 2 MDA-N breast 0
ACHN renal 2 BT-549 breast 0
786-0 renal 2 SNB-19 CNS 0
TK-10 renal 2 SNB-75 CNS 0
HT29 colon 1 U251 CNS 0
adenoca
rcinoma
RF-1 gastric 1 SF-268 CNS 0
carcino
ma
AGS gastric 1 SF-295 CNS 0
carinom
a
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EKVX lung 1 CCRF-CEM leukemia 0
HOP-92 lung 1 MOLT-4 leukemia 0
NCI-H226 lung 1 HL-60(TB) leukemia 0
NCI-H322M lung 1 RPMI8226 leukemia 0
MCF7/ADR breast 1 SR leukemia 0
OVCAR-4 ovary 1 UO-31 renal 0
SF-539 CNS 1 A498 renal 0
K-562 leukemi 1 Caki-1 renal 0
a
RXF393 renal 1 SK-MEL-2 melanoma 0
Calu-3 lung 1? SK-MEL-5 melanoma 0
adenoca
rcinoma
NCI-H522 lung 1? SK-MEL-28 melanoma 0
SW620 colon 1? UACC-62 melanoma 0
Hs578T breast 1? UACC-257 malanoma 0
Sk-OV-3 ovary 1? M14 melanoma 0
__. ~ _ . _~ _ _ __.__ i _
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Example 8: Generation of SAD-specific Immunoreagents
A SAD-specific antisera was raised in. rabbits against a
KhH-conjugated synthetic peptide derived from the C-terminal
region of SAD (amino acids 978 to 488 of SEQ ID N0:35) with a C
5 to S substitution at position 486 essentially as described in
Gentry and Lawton, Virology 152:421, 1984.
Example 9: Recombinant Expression of SAD
10 Construction Of Vectors
Expression constructs were generated by PCR-based
mutagenesis in which a BamHI site was introduced upstream of
the initiating Met giving a 5' untranslated sequence of 5'-
GGATCCCCGGACC-3' (SEQ ID N0:51). An N-terminal hexahistidine
15 tagged construct was also created by PCR with the coding
sequence for MRGSHHHHHH (SEQ ID N0:52)
(ATGAGAGGATCGCATCACCATCACCATCAC, SEQ ID NO: 53) followed by the
second nucleotide of the SAD coding sequence (a glutamate).
Proteins tagged with this sequence can be recognized by the
20 RGS~His Antibody (QIAGEN Inc.) and affinity purified with Ni
NTA resin (QIAGEN Inc.). These constructs were cloned into the
5'-BamHI and 3'-EcoRI sites of pBluescriptSK+ (Stratagene
U.S.A.) and the 5'-BamHI and 3'-Xhol sites of the mammalian
expression pcDNA3 (Invitrogen) for transient expression
25 analysis.
The SpeI-XhoI full length SAD constructs were also cloned
from pBluescriptSK+ (Stratagene U.S.A.) into the yeast
expression vector pRSP (Superti-Furga et al., EMBO J. 12, 2625-
2634). This vector contains a thiamine-repressible promoter in
30 a shuttle vector for inducible expression in Schizo-
saccharomyces pombe. This system has been useful in studies of
SRC family members for testing negative regulation by CSK,
screening for additional regulators, and purifying recombinant
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protein (Superti-Furga et al., EMBO J. 12, 2625-2634; Superti-
Furga et al., Nature Biotech. 14, 600-605).
Transient Expression of SAD in Mammalian Cells
The pcDNA3 expression plasmids (5 ~g DNA/60 mm plate)
containing the unmodified and hexahistidine-tagged SAD genes
were introduced into 293 cells with lipofectamine (Gibco BRL).
After 48 hours, the cells were harvested in 0.25 mL RIPA (20 mM
Tris-C1 pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate,
0.1% SDS, 1mM DTT, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 ~Cg/mL aprotinin, 1 ~g/mL
leupeptin, and 25 ~,g/mL trypsin inhibitor). Sample aliquots
were resolved by SDS polyacrylamide gel electrophoresis (PAGE)
on 10% acrylamide gels and electrophoretically transferred to
nitrocellulose. Non-specific binding was blocked by preincu-
bating blots in Blotto (Tris buffered saline containing 5% w/v
non-fat dried milk and 0.1% v/v Tween-20), and recombinant
protein was detected using affinity-purified SAD-specific
polyclonal antibody and peroxidase-linked secondary antibody
with the ECL kit (Amersham Life Science). Hexahistidine tagged
protein was also detected using RGS~His Antibody (QIAGEN Inc.).
Phosphotyrosine-containing proteins were detected by Western
blotting with monoclonal antibody 4610 (Upstate Biotechnology)
with 3% BSA as the blocking agent.
Affinity purified antipeptide antibody raised against the
C-terminus of SAD (see Example 8) recognized a specific ~55 kDa
protein in transfected 293 cells with greater than 90% of the
expressed protein being RIPA-insoluble. This molecular weight
is consistent with the molecular weight predicted based on SAD's
primary amino acid sequence (54,510 kD). SAD-transfected cells
contain a prominent approximately 55 kDa tyrosine
phosphorylated protein that is absent in vector controls. The
~___.._ .._ _ r ...__ '. __. 1.
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phosphorylated protein is most likely SAD itself because the
band is clearly detected in IP-Westerns using anti-SAD
crosslinked to protein A beads and 4610 as the blotting
antibody although anti-SAD only inefficiently immuno
precipitates.
Expression of Recombinant SAD in Schizosaccharomyces Pombe
S. pombe was used to express recombinant SAD as an
approach to studying its function and regulation since this
expression system has proven useful for studying Src family
members (Superti-Furga et al., EMBO J. 12, 2625-2634; Superti-
Furga et al., Nature Biotech. 14, 600-605). S. pombe strain
SP200 (h-s leu1.32 ura4 ade210) was grown as described and
transformations with pRSP expression plasmids were done by the
lithium acetate method (Moreno et al., 1991; Superb -Furga et
al., EMBO J. 12, 2625-2634). Cells were grown in the presence
of 1 uM thiamine to repress expression from the nmtl promoter
or in the absence of thiamine to induce expression.
Under derepressing conditions, SAD-expressing strains show
no growth defect compared to vector controls in either liquid
culture or solid media. This result contrasts with the toxicity
caused by expression of several other tyrosine kinases
including Src and Frk. SAD protein can be detected in these
strains as a weak band on Western blots using the polyclonal
antibody against the C-terminus. On anti-phosphotyrosine
Western blots, SAD itself is the only detectable
phosphotyrosine-containing protein, however in the presence of
pervanadate, cellular proteins are also phosphorylated. This
observation contrasts with the results seen for Src and MKK3
which phosphorylate many yeast proteins even in the absence of
phosphatase inhibitors. These findings suggest that SAD
exhibits relatively limited substrate specificity and
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autophosphorylates itself. These results are consistent with
the transient expression experiments in 293 cells.
Example 10: Assay for SAD Kinase Activity
The example below describes an in vitro assay for SAD
kinase activity. The assay is useful for the identification of
modulators of SAD activity.
Materials And Methods
S. pombe expressing hexahistidine-tagged SAD were
harvested by centrifugation and lysed by the glass bead method
(Superti-Furga et al., EMBO J. 12, 2625-2634) in NP-40 lysis
buffer (50 mM Tris-C1 pH 7.5, 150 mM NaCl, to NP-40, 5 mM 2-
mercaptoethanol , 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 ~,g/mL aprotinin, 1 ~g/mL
leupeptin, and 25 ~,g/mL trypsin inhibitor). Immunoprecipita-
tions were done by mixing yeast extract (100 ~.g total protein
in 100 ~,L NP-90 lysis buffer) with 0.6 ~g the RGS~His Antibody
(QIAGEN Inc.) and 10 ~.L Protein A/G agarose (Upstate
Biotechnology) for 3 hrs at 4 °C. IP complexes were washed four
times in 1 mL lysis buffer and once in 1 mL kinase buffer (20
mM Na-HEPES pH 7.5, 10 mM MnCl2, 2 mM 2-mercaptoethanol, and 10
~M sodium vanadate). Kinase assays were for 10 min at 30 °C in
40 ul kinase buffer containing 15 ~,M ATP, 0.5 uCi g'-32P-ATP,
and either 3 ~.g denatured enolase or 10 ~g poly-Glu-Tyr (4:1)
as the substrate. Extracts were assayed using 2-10 ~g total
protein per reaction and IP complexes were assayed using 5 ul
Protein A/G beads per assay. Reactions were terminated by the
addition of SDS sample buffer and the samples were resolved on
an loo SDS polyacrylamide gel and visualized by auto-
radiography.
_.. __. ___
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Results
SAD was able to phosphorylate both denatured enolase and
poly-Glu-Tyr in vitro. Phosphorylation of both substrates was
detected in crude yeast lysates expressing SAD but not in
lysates from vector control strains. In addition, anti-His IP
complexes from SAD-expressing strains but not control strains
phosphorylated both denatured enolase and poly-Glu-Tyr.
Example 11: Isolation Of cDNA Clones Encoding PTP05 and
PTP10
The example below describes the isolation and identifica-
tion of new PTP sequences from primary murine fat and rat basal
forebrain and the subsequent cloning of a full-length PTP05
sequence Also described are probes useful for the detection of
PTP05 and/or PTP10 in cells or tissues.
Materials and Methods:
Total RNAs were isolated using the Guanidine Salts/Phenol
extraction protocol of Chomczynski and Sacchi (P. Chomczynski
and N. Sacchi, Anal. Biochem. 162, 156 (1987) from ob/ob mouse
fat and, separately, rat basal forebrain. These RNAs were used
to generate single-stranded cDNA using the Superscript
Preamplification System (GIBCO BRL, Gaithersburg, MD.: Gerard,
et al, FOCUS 11:66, 1989) under conditions recommended by the
manufacturer. A typical reaction used 10 ,ug total RNA with 1.5
/.cg oligo (dT) 12_18 in a reaction volume of 60 ~L. The product was
treated with RNaseH and diluted to 100 ~cL with H20. For
subsequent PCR amplification, 1-4 ~cL of this sscDNA was used in
each reaction.
Degenerate oligonucleotides were synthesized on an Applied
Biosystems 394 DNA synthesizer using established
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phosphoramidite chemistry, precipitated with ethanol and used
unpurified for PCR. The sequence of the degenerate
oligonucleotide primers follows:
PTPDFW - 5'-GAYTTYTGGVRNATGRTNTGGGA- (sense) (SEQ ID N0:
17) and
PTPHCSA - 5'-CGGCCSAYNCCNGCNSWRCARTG -3' (antisense) (SEQ
ID N0: 18).
These primers were derived from the peptide sequences
DFWXMXW(E/D) (SEQ ID N0: 19) (sense strand from PTP catalytic
domain) and HCXAGXG (SEQ ID N0: 20) (antisense strand from PTP
catalytic domain), respectively. The standard UIPAC
designations for degenerate residue designations are: N = A, C,
G, or T; R = A or G; Y = C or T; V = A, C or G; W = C or T: S =
C or G; M = A or C; and H = A, C or T.
PCR reactions were performed using degenerate primers
applied to the single-stranded cDNA listed above. The primers
were added at a final concentration of 5 ~M each to a mixture
containing 10 mM Tris'HC1 (pH8.3), 50 mM KC1, 1.5 mM MgCl2, 200
~,M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U
AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ~.L cDNA.
Following 3 min denaturation at 95 °C, the cycling conditions
were 94 °C for 30 sec, 50 °C for 1 min, and 72 °C far 1
min 45
sec for 35 cycles. PCR fragments migrating between 350-400 by
were isolated from 2% agarose gels using the GeneClean Kit
(Bio101), and T-A cloned into the pCRII vector (Invitrogen
Corp. U.S.A.) according to the manufacturer's protocol.
Colonies were selected for mini-plasmid DNA-preparations
using Qiagen columns and the plasmid DNA was sequenced using
cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction
products were run on an ABI Prism 377 DNA Sequencer, and
analyzed using the BLAST alignment algorithm (Altschul, S.F. et
_T .._ _
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al., J. Mol. Bio1.215:403-10). Several copies of a clone
encoding a novel PTP (R90-2-22), designated SuPTP05, was
isolated from murine adipose tissue. A related clone, PTP10,
was isolated from rat basal forebrain.
To obtain full-length cDNA encoding the novel phosphatase
PTP05, RACE (rapid amplification of cDNA ends) was performed
with sense or anti-sense oligonucleoides derived from the
original PCR fragments. Marathon-Ready cDNA (Clontech, Palo
Alto, CA) made from mouse testis was used in the RACE reactions
with the following primers:
RACE primers:
5'-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3' (6595) (SEQ ID N0:21),
5'-GGACTTCTTCACAATCTGAAATACTCGAACGGTG-3' (6596) (SEQ ID N0:22),
5'-CCGTTATGTGAGGAAGAGCCACATTACAGGACC-3' (6599) (SEQ ID N0:23),
5'-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3' (6600) (SEQ ID N0:24),
AP-1, and AP-2 (Clontech).
RT-PCR primers for PTP05 sequeqncing:
5'-CACCGTTCGAGTATTTCAGATTGTGAAGAAGTCC-3' (6595) (SEQ ID N0:21),
5'-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3' (6600) (SEQ ID N0:24).
Isolated cDNA fragments encoding SuPTP05 were confirmed by
DNA sequening and subsequently used as probes for the screening
of a murine testis cDNA library.
Two murine testis cDNA libraries (lZapII, Stratagene, La
Jolla, CA and 1gt10, Clontech), were screened to isolate full-
length transcripts encoding PTP05. The 5' or 3'-RACE fragments
were 32P-labeled by random priming and used as hybridization
probes at 2x106 cpm/mL following standard techniques for library
screening. Pre-hybridization (3 hrs) and hybridization (over-
night) were conducted at 42 °C in 5X SSC, 5 X Denhart's
solution, 2.5~ dextran sulfate, 50 mM Na2P04/NaHP04 [pH 7.0],
50~ formamide with 100 mg/mL denatured salmon sperm DNA.
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Stringent washes were performed at 65 °C in O.1X SSC and 0.1~
SDS. Several overlapping clones were isolated and found to
span the collective sequences of the PCR fragment (R90-2-22)
and the RACE products. The final sequence weas verified by
sequencing of both strains using a cycle sequencing dye-
terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster
City, CA). Sequencing reaction products were run on an ABI
Prism 377 DNA Sequencer. A full-length PTP10 clone can be
obtained using the same techniques.
Results:
The primary murine PTP05 transcript is 1785 nucleotides
and encodes a predicted polypeptide of 426 amino acids with a
predicted molecular weight of 49122 daltons (SEQ ID N0:3 and
SEQ ID N0:11). The PTP05 coding sequence is flanked by a 198
nucleotide 5'-untranslated region and a 279 nucleotide 3'-
untranslated region ending with a poly(A) tail. There are
inframe stop codons in all three frames upstream of the primary
open reading frame. The ATG beginning at nucleotide position
199 conforms to the Kozak consensus for an initiating
methionine. One clone (#6.1) containes an insertion of 111 by
at nucleotide 328 resulting in an addition 37 amino acids added
inframe to the coding sequence. A second clone (#10.1) has a
deletion of 93 by beginning at nucleotide 1415, resulting in a
frame-shift and premature termination. Upstream of the 198bp
5'UTR, the numerous clones diverge into 2 groups, extending the
5'UTR an additional 98-153 bp. Furthermore, one clone (#15.3)
lacks the polyA tail at nucleotide 1758 extends the 3' UTR by
another 300 nucleotides.
The amino acid sequence shows no signal sequence or a
transmembrane domain, and PTP05 is therefore predicted to be an
intracellular protein. The N-terminal domain of murine PTP05
extends from amino acid 1 to 187 and is unique, i.e. contains
no significant homology to any protein in the non-redundant
_ _rr_ ._ ____._____ _
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protein database. The non-redundant protein database consists
of peptide sequences from GenBank Genpept, PIR, and SwissProt.
There is a single protein tyrosine phosphatase catalytic domain
extending from amino acids 188-420. The catalytic domain
shares a relatively low level of identity at the amino, acid
level (40-97~) to PTPs from 5 distinct families: ZPEP (mouse)
(46.7$), PTP-BAS (human) (45.6x), DEP (human) (40.50 , PTP-g
(human) (40.60), suggesting that it represents a new family of
PTPs. The C-terminal tail of PTP05 extends beyond the cata-
lytic domain from amino acids 421-426 and is not homologous to
other protein tyrosine phosphatases. Motifs found in the
cytoplasmic domain of other mammalian PTPs that are absent from
PTP05 include: SH2, Talin/Ezrin-like, PEST, GLGF, and
Retinaldehyde-binding protein domains. Owing to its divergent
catalytic domain and absence of well-known non-catalytic
motifs, we have designated PTP05 as a new and distinct family
of protein tyrosine phosphatases.
An alternative form of murine PTP05 contains an insertion
of 111-by in the N-terminal coding region, extending the
sequence by 37 as (SEQ ID N0:4 and SEQ ID N0:12). This 1,896
by "long" form of murine PTP05 encodes a polypeptide of 463
amino acids with a predicted molecular weight of 53716 daltons.
The insertion is located at amino acid positions 44-80 and is
not significantly homologous to other proteins in the non
redundant protein database.
A third form of PTP05 has a deletion of nucletotides 1415-
1507 resulting in a frame shift. and C-terminal truncation
leading to an alternate sequence from amino acids 406-412 (SEQ
ID N0:5 and SEQ ID N0:13). The 1,692 by "C-trunc" murine PTP05
encodes a polypeptide of 412 amino acids with a predicted
molecular weight of 47233 daltons.
The rat PTP10 clone shares 92~ identity at the DNA level
(320 nucleotides) and 85~ amino acid identity at the protein
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level ( 107 amino acids ) with murine PTP05 ( See Figure 1 ) . The
level of homology of the two catalytic domains suggests that
PTP05 and PTP10 are distinct but related genes, and thus PTP10
is considered to be a second member of this new PTP family.
Partial sequences of rat PTP10 are shown in SEQ ID N0:6
(nucleic acid) and SEQ ID N0:14 (amino acid).
Example 12: Expression of PTP05
The example below shows the evaluation of PTP05 and PTP10
expression in normal murine tissues. A similar analysis can be
done in human tissues using a human PTP05 or PTP10.
Materials and Methods:
A mouse normal tissue Northern blot containing 2 /.cg polyA+
mRNA per lane from 8 different mouse adult tissues (lung,
testis, brain, heart, liver, kidney, spleen, skeletal muscle)
on a charge-modified nylon membrane was obtained from Clontech
(#7762-l, Palo Alto, CA).
The membrane was hybridized with randomly primed
[a32P]dCTP-labeled probe synthesized from a 241 by EcoRI
fragment of R90-2-22 (see above). Hybridization was performed
at 42 °C overnight in 5X SSC, 2% SDS, 10X Denhardt's solution,
50% formamide, 100 ~,g/mL denatured salmon sperm DNA with 1-2 x
106 cpm/mL of 32P-labeled DNA probe. The membrane was washed at
room temperature in 2X SSC/0.05% SDS for 30 min and followed by
50 °C in 0.2X SSC/0.1% SDS for 30 min, and exposed overnight on
Kodak XAR-2 film.
A similar analysis was performed using the 320 by rat
PTP10 fragment as a probe of a rat normal tissue Norther blot.
_ __.__..~.r._ _ ___~ _ __. ~
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RT-PCR Detection of Novel PTPs
Total RNA was isolated from fresh frozen mouse or rat
(separately) tissues by centrifugation thrugh a cesium chloride
cushion. Twenty ~g of total RNA was reverse transcribed with
random hexamers and Moloney murine leukemia virus reverse
transcriptase (Super-ScriptII, GIBCO BRL, Gaithersburg, MD).
PCR was then used to amplify cDNA encoding SuPTP05. RT-PCR
reactions lacking only the reverse transcriptase were performed
as controls. PCR products were electrophoresed on 3o agarose
gels, visualized by ethidium bromide staining and photographed
on a UV light box. The intensity for a 161-by fragment
specific to murine PTP05 were compared among different RNA
samples. A rating of 3 represents large quantities of PTP05
transcript identified by Northern blot analysis while a rating
of 0 represents little or none of the transcript was detected.
Results:
By Northern analysis, a single murine PTP05 mRNA
transcript of approximately 3.4 kb was identified, and found to
be exclusively expressed in the testis. The lung, brain,
heart, liver, kidney, spleen, skeletal muscle samples were
negative. PTP10 hybridized to a slightly smaller band and was
also found only in the testis in this analysis. Northern
analysis identified two rat PTP10 mRNA transcripts of
approximately 3.3 kb and 1.8 kb, exclusively expressed in the
testis. The rat heart, brain, spleen, lung, liver, skeletal
muscle, and kidney samples were negative.
RT-PCR with gene specific primer-pairs showed that
expression of the transcripts encoding PTP05 confirmed the
results from Northern analysis and also detected low levels in
adipose, kidney, small intestine, and cells/tissues of
hematopoietic.or immune origin including spleen, thymus, lymph
node, bone marrow, and peripheral blood lymphocytes). RT-PCR
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with rat PTP10 gene specific primers confirmed the results from
the Northern analysis, detecting a strong signal only in rat
testis sscDNA and not in templates corresponding to rat
skeletal muscle, heart, kidney, spleen, adrenal gland, lung,
liver, intestine, uterus, spinal cord, brain, cortex and ovary.
The reletively selective expression of PTP05 in cells of
hematopoetic or immune origin suggests a potential involvement
in immune regulation including T and B cell survival,
differentiation or co-stimulation, and/or inflammatory,
immunosuppressive or autoimmune disorders. Additionally,
expression in adipose tissue (also the source from which PTP05
was originally isolated) suggests a possible role in metabolic
disorders such as diabetes.
Example 13: Recombinant Expression Of PTP05
The following example illustrates the contruction of
vectors for expression of recombinant PTP05 and the creation of
recombinant cell lines expressing PTP05. Similar vectors and
recombinant cell lines can be generated using PTP10 and the
techniques described herein.
Contruction of Ex ression Vectors
Expression constructs were generated by PCR-assisted
mutagenesis in which the entire coding domain of PTP05 was
tagged on its carboxy-terminal end with the hemophilus
influenza hemaglutinin (HA) epitope YPYDVPDYAS (SEQ ID N0:55)
(Pati, supra). This construct were introduced into two
mammalian expression vectors: pLXSN (Miller, A.D. & Rosman,
G.J., Biotechniques 7, 980-988, 1989) for the generation of
virus producing lines; and pRKS for transient expression in
mammalian cells .
Dominant negative PTP05 constructs were also made in both
pLXSN and pRK5 by mutation of the invariant Cys in the
_. __ ___ ~. _ _...__ _. 1
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conserved His-Cys-Ser-Ala-Gly motif (SEQ ID N0:56) to an Ala by
PCR mutagenesis.
The entire PTP05 open reading frame excluding the
initiating methionines was generated by PCR and ligated into
pGEX vector for bacterial production of GST-fusion proteins for
immunization of rabbits for antibody production. This vector
contains the glutathione-S-transferase coding sequence followed
by a polylinker for generating recombinant fusion proteins.
The GST moiety comprises the N-terminal portion of the fusion
protein.
Transient Expression in Mammalian Cells
The pRKS expression plasmids (10 ~,g DNA/100 mm plate)
containing the HA-tagged PTP05 gene can be introduced into COS
and 293 cells with lipofectamine (Gibco BRL). After 72 hours,
the cells were harvested in 0.5 mL solubilization buffer (20 mM
HEPES pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5
mM MgCl2, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ~,g/mL
aprotinin). Sample aliquots were resolved by SDS polyacryla-
mide gel electrophoresis (PAGE} on 15% acrylamide/0.5% bis-
acrylamide gels and electrophoretically transferred to nitro-
cellulose. Non-specific binding was blocked by preincubating
blots in Blotto (phosphate buffered saline containing 5% w/v
non-fat dried milk and 0.2% v/v nonidet P-40 (Sigma)), and
recombinant protein was detected using a murine Mab to the HA
decapeptide tag. Alternatively, recombinant protein can be
detected using various PTP05-specific antisera.
Generation of Virus Producing Cell Lines
pLXSN recombinant constructs containing the PTP05 gene
were transfected into an amphotropic helper cell line PA317
using CaCl2 _mediated transfection. After selection on 6418,
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the cells were plated on normal media without 6418 (500 ~,g/mL).
Supernatants from resistant cells were .used to infect the
ecotropic helper cell line GP+E86, and cells again selected on
6418. Resistant cells were again taken off 6418, and the
supernatants harvested every 8-12 hours and pooled as virus
stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498.
Viral stock titers were typically ~106/mL.
Stable Expression in Mammalian Cells
NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates
with DMEM (Gibco) containing 10% fetal calf serum (FCS). The
cells were superinfected with the PTP05 retrovirus by adding
approximately 3 mL viral supernatant to 15 mL culture media for
approximately 24 hours. Cells expressing the retroviral
constructs were then selected by growth in DMEM/l0o FCS
supplemented with 500 ~g/mL 6418.
Example 14: Generation Of Anti-PTP05 Antibodies
PTP05-specific immunoreagents were raised in rabbits
against a pool of three KLH-conjugated synthetic peptides
corresponding to unique sequences present in human PTP04. The
peptides (see below) were conjugated at the C-terminal residue
with KLH.
Peptides used for immunizing rabbits:
PTP05:
peptide 433A - MSSPRKVRGKTGRDNDEEEGNSGNLNLRN (SEQ ID
N0:57)
peptide 931A - SPVLSGSSRLSKDTETSVSEKELTQLAQI (SEQ ID
N0:58) and
peptide 432A - WDVSDRSLRNRWNSMDSETAGPSKTVSPV (SEQ ID
N0: 59) .
~ _ __ __ ._ i
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Additional immunoreagents were generated by immunizing
rabbits with a purified preparation of a GST-fusion protein
containing the entire coding region of PTP05. The GST-fusion
protiens were produced in DH5-alpha E. coli bacteria as
described in Smith, et al Gene 67:31, 1988. Bacterial protein
lysates were purified on glutathione-sepharose matrix as
described in Smith, et al., supra.
Example 15: Assay for PTP05 Activity
Materials and Methods:
Recombinant wild-type and dominant negative (signaling
incompetant) PTP05 (see Example 13, supra) were purified from
bacteria as GST-fusion proteins. Lysates were bound to a
glutathione-sepharaose matrix and washed twice with 1X HNTG,
followed by one wash with a buffer containing 100 mM 2-(N-
morpholino)ethansulfonic acid (MES), pH 6.8, 150 mM NaCl, and 1
mM EDTA.
The assay for phosphatase activity was essentially done as
described by Pei et al.(1993) using p-nitrophenolphosphate
(PNPP) as a generic PTP substrate. Briefly, after the last
washing step, reactions were started by adding 50 mL Assay
Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA,
and 50 mM PNPP) to the matrix bound proteins. Samples were
incubated for 20 min. at 23 °C. The reactions were terminated
by mixing 40 ~,L of each sample with 960 ~,L 1 N NaOH, and the
absorbance of p-nitrophenol was determined at 450 nm. To
control for the presence of PTP05 in the precipitates, the
precipitates were boiled in SDS sample buffer and analyzed by
SDS-PAGE. The presence of PTP05 was then detected by
- immunoblot analysis with anti-PTP05 antibodies.
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Example 16: Isolation Of cDNA Clones Encoding ALP
The example below describes the isolation and identifi-
cation of a new PTP sequence from mouse tissues and the sub-
sequent cloning of a full-length human ALP. Also described are
probes useful for the detection of ALP in cells or tissues.
Materials and Methods:
Total RNAs were isolated using a commonly known guanidine
salts/phenol extraction protocol from normal mouse fat and rat
pituitary. Chomczynski & Sacchi, 1987, Anal. Biochem. 162:
156. These RNA extracts were used to generate single-stranded
cDNA using the Superscript Pre-amplification System (GIBCO BRL,
Gaithersburg, MD.; Gerard et al., 1989, FOCUS 11: 66) under
conditions recommended by the manufacturer. a typical reaction
used 10 ~g total RNA with 1. 5 ~,g oligo (dT) lz-is in a reaction
volume of 60 ~.L. The product was treated with RNaseH and
diluted to 100 ~L with H20. For subsequent PCR amplification,
1-4 ~L of this sscDNA was used in each reaction.
Degenerate oligonucleotides were synthesized on an Applied
Biosystems 394 DNA synthesizer using established
phosphoramidite chemistry, precipitated with ethanol and used
unpurified for PCR. The sequence of the degenerate
oligonucleotide primers were as follows:
PTPDFW - 5'-GAYTTYTGGVRNATGRTNTGGGA-3' (SEQ ID N0:17)
PTPHCSA = 5'-CGGCCSAYNCCNGCNSWRCARTG-3' (SEQ ID N0:18)
PTPYINA - 5'-ATCCCCGGCTCTGAYTAYATHMAYGC-3' (SEQ ID N0:60)
These primers were derived from the peptide sequences
DFWXMXW(E/D) (SEQ ID N0:19) (sense strand from PTP catalytic
region) and HCXAGXG (SEQ ID N0:20)(antisense strand from PTP
catalytic region), and IPGSDYI(N/H)A (SEQ ID N0:61) respec-
tively. The standard UIPAC designations for degenerate residue
_.- T. ._._ ~_
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designations are: N = A, C, G, or T; R = A or G; Y = C or T; V
- A, C or G; W = C or T; S = C or G; M = A or C; and H = A, C
or T.
PCR reactions were performed using degenerate primers
applied to the single-stranded cDNA listed above. The primers
were added at a final concentration of 5 ~,M each to a mixture
containing 10 mM TrisHCl (pH8.3), 50 mM KC1, 1.5 mM MgCl2, 200
~,M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U
AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ~L cDNA.
Following 3 min denaturation at 95°C, the cycling conditions
were 94 °C for 30 s, 50 °C for 1 min, and 72°C for 1 min
45 s
for 35 cycles. PCR fragments migrating between 350-400 by were
isolated from 2% agarose gels using the GeneClean Kit (Bio101),
and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.)
according to the manufacturer's protocol.
Colonies were selected for mini plasmid DNA-preparations
using Qiagen columns and the plasmid DNA was sequenced using
cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction
products were run on an ABI Prism 377 DNA Sequencer, and
analyzed using the BLAST alignment algorithm. Altschul et al.,
J. Mol. Biol. 215: 403-410. A single clone encoding a novel
PTP (S50-I51), designated murine ALP, was isolated from murine
adipose tissue using degenerate oligonucleotides PTPDFW (SEQ ID
NO: 17) and PTPHCSA (SEQ ID N0:18), and a related rat ALP clone
was isolated from rat pituitary using degenerate
oligonucleotides PTPYINA (SEQ ID N0:60) and PTPHCSA (SEQ ID
N0:18).
To isolate a full-length human ALP a human cDNA library
was constructed in lambda ZapII (Stratagene, La Jolla, CA) from
polyA+ RNA isolated from the human neuroblastoma cell line
IMR32. The library was screened to isolate full-length
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transcripts encoding ALP. The murine ALP fragment was 32P-
labeled by random priming and used as a hybridization probe at
2x106 cpm/mL following standard techniques for library
screening. Pre-hybridization (3 h} and hybridization (over-
night) were conducted at 42 °C in 5X SSC, 5 X Denhart's
solution, 2.5% dextran sulfate, 50 mM Na2P04/NaHP04 [pH 7.0],
50% formamide with 100 mg/mL denatured salmon sperm DNA.
Stringent washes were performed at 65 °C in O.1X SSC with 0.1%
SDS. Multiple clones were isolated and one 4.5 kb clone
spanned the entire coding region of ALP. The final sequence
was verified by sequencing of both strands using a cycle
sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS
(ABI, Foster City, CA). Sequencing reaction products were run
on an ABI Prism 377 DNA Sequencer.
Results:
The 4,456 by human ALP nucleotide sequence encodes a
polypeptide of 1,279 amino acids. The amino acid sequence
shows no signal sequence or a transmembrane domain and is
therefore an intracellular protein. The N-terminal end extends
from amino acids 1-857 and contains several putative tyrosine
phosphorylation sites and a proline-rich region (30.6%
prolines) from amino acids 353-777. This proline-rich region
is distantly related to plant extensin proteins (30.2% amino
acid identity with Zea mat's extensin-like protein GB:Z34465
using Smith-Waterman alignment) and may represent a protein
interaction domain as well as the site for interaction with
proteins containg SH3 motifs. The C-terminal tail of ALP
extends from amino acid 1097-1274 and contains a proline/serine
rich region (45.6% serines plus prolines from amino acids 1101-
1214) resembling a PEST motif. This region also could serve as
a target for binding proteins via their SH3 motifs.
_ _ ___-_~ _ _ __- __
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The catalytic domain extends from amino acids 858-1096 and
shares 32-37% amino acid identity to PTPs from multiple
subfamilies: TC-PTP (P17706: 37.1%) PTP-BAS (D21209: 32.9%),
PTPa (M34668: 34.2%), PTP~3 (P23467: 34.2%), PTPa (A49109:
33.2%), PTP1B (P20417: 39.9%), suggesting that it represents a
new family of PTPs. While all other cytoplasmic PTPs have
their catalytic domain at either the N- or C-terminal portion
of the protein, ALP has a central catalytic domain flanked by
large N- and C-terminal domains. Its catalytic domain
conserves most of the invariant residues present in other PTPs,
but does has several atypical amino acids. In ALP, the amino
acid sequence HCSAG (SEQ ID N0:56), is changed to HCSSG (amino
acid positions 1029-1033) (SEQ ID N0:75). This motif is in the
catalytic site of the crystal stucture of PTP1B and PTPa, and
the Ala to Ser change may effect catalyitic activity or
specificty. ALP also has a change from WPD to WPE (amino acids
positions 993 - 995) in its predicted surface loop of the
catalytic domain. In PTP1B this Aspartate participates in a
salt bridge and falls into the catalytic site on binding to a
specific peptide substrate. This Asp to Glu alteration is also
present in three other mammalian PTPs (PTPD1, PCP2, PTPS31).
Example 17: Expression Of ALP
The example below shows the evaluation of ALP expression
in normal human tissues and in a wide variety of cancers.
Materials and Methods:
Northern blots were prepared by running 20 ~,g total RNA
per lane isolated from 60 different tumor cell lines (HOP-92,
EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A599,
HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3,
SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, K-562,
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MOLT-9, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29, HCC-2998,
HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498,
CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Malme-3M, SK-MEL-
2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, MCF-7, MCF-
7/ADR RES, Hs578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549,
T47D). (obtained from Nick Scuidero, National Cancer Institute,
Developmental Therapeutics Program, Rockville, MD). The total
RNA samples were run on a denaturing formaldehyde 1% agarose
gel and transferred onto a nitrocellulose membrane (BioRad,
CA). Additional human normal tissue Northern blots containing 2
~.cg polyA+ mRNA per lane from 16 different human normal tissues
(thymus, lung, colon, testis, brain, heart, liver, pancreas,
kidney, spleen, uterus, prostate, skeletal muscle, PBLs,
placenta, small intestine) on charge-modified nylon membranes
(multiple tissue blots #7760-1 and #7766-1, Clontech, Palo
Alto, CA) were also hybidized.
Nitrocellulose membranes for the total RNA samples were
hybridized with randomly primed [gamma-32P]dCTP-labeled probes
synthesized from a 1 kb fragment of EcoRI-NotI of ALP.
Hybridization was performed overnight at 42 °C in 4X SSPE, 2.5X
Denhardt's solution, 50% formamide, 200 ~cg/mL denatured salmon
sperm DNA, 100 ~,g/mL yeast tRNA (Boehringer Mannheim,IN), 0.20
SDS with 5 x 106 cpm/mL of [gamma-32P] dCTP-labeled DNA probe on
a Techne Hybridizer H-1. The blots were washed with 2X SSC,
0.1% SDS, at 65 °C for 20 min twice followed by 0.5 X SSC in
0.1% SDS at 65 °C for 20 min. The blots were exposed to a
phospho-imaging screen for 24 hours and scanned on a Molecular
Dynamics Phosphoimager SF.
For Clontech nylon-membrane blots, hybridization was
performed at 42 °C overnight in 5X SSC, 2% SDS, lOX Denhardt's
solution, 50~ formamide, 100 ~,g/mL denatured salmon sperm DNA
with 1-2 x 106 cpm/mL of [gamma-32P] dCTP-labeled DNA probe. The
-___~. __ __.__. _
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blots were washed at room temperature in 2X SSC/0.05~ SDS for
30 min and followed by at 50 °C in 0.2X SSC/O.lo SDS for 30 min,
and exposed for 48 hours on Kodak XAR-2 film.
For analysis of expression using reverse-transcriptase-PCR
detection, total RNA was isolated from various cell lines or
fresh frozen tissues by centrifugation through a cesium
chloride cushion. 20 ~,g of total RNA was reverse transcribed
with random hexamers and Moloney human leukemia virus reverse
transcriptase (Super-ScriptII, GIBCO BRL, Gaithersburg, MD).
PCR was then used to amplify cDNA encoding ALP. Reverse
transcriptase PCR (RT-PCR) reactions lacking only the reverse
transcriptase were performed as controls. PCR products were
electrophoresed on 3o agarose gels, visualized by ethidium
bromide staining and photographed on a UV light box.
The intensity of the fragment specific to ALP were
compared among different RNA samples. A rating of 4 represents
large quantities of ALP transcript while a rating of 0
represents little or none of the transcript was detected. It
should be noted that detection of proteins by RT-PCR indicates
a relatively higher abundance than detection by Northern blot
as the RT-PCR technique utilizes total RNA whereas Northern
blot analysis is performed using an enriched RNA source (mRNA).
Results:
A single ALP mRNA transcript of approximately 5.0 kb was
visualized by Northern analysis. This transcript was identi-
fied in most of the normal tissue samples tested. However, the
Northern analysis results shown in the Table 1 illustrate that
the relative abundance of ALP mRNA is quite divergent. In
normal tissues, ALP was identified in highest quantities in
pancreas, followed by heart, testis, and skeletal muscle.
Lower levels of the ALP transcript were identified in placenta,
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thymus, lung, brain, liver, spleen, uterus, prostate and small
intestine. None of the ALP transcript was detected in colon,
kidney and peripheral blood leucocytes (PBLs). ALP expression
was also detected in normal human adipocytes by RT-PCR methods.
In Northern blots of total RNA from human tumor cell
lines, the ALP RNA transcript was most abundant in NCI-H226
(lung tumor), SK-OV-3 (ovarian tumor), and RPMI 8226 (leukemia)
cell lines. The transcript was identified at lower amounts in
SNB-19 (CNS tumor), SF-268 (CNS tumor), SN12C (kidney tumor),
SK-MEL-2 (melanoma), UACC-62 (melanoma), and UACC-257
(melanoma) cell lines. The ALP transcript was not detected in
the remaining of 44 human tumor cell lines. A summary of
expression of ALP is shown in Table 1 below.
_ _t _-_~..__ _.
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Table 1
Cell type Origin AI,p
Thymus Normal tissue 0.5*
Lung Normal tissue 0.5*
Colon Normal tissue p*
Testis Normal tissue 2*
Brain Normal tissue 0.5*
Heart Normal tissue 2*
Liver Normal tissue 0.5*
Pancreas Normal tissue 3*
Kidney Normal tissue 0*
Spleen Normal tissue 0.5*
Uterus Normal tissue 0.5*
Prostate Normal tissue 0.5*
Skeletal Normal tissue 2*
muscle
PBLs Normal tissue 0*
Placenta Normal tissue 1*
Small Normal tissue 0.5*
intestine
NCI-H226 Lung tumor 4
SK-OV-3 Ovarian tumor 3
SNB-19 CNS tumor 2
0251 CNS tumor 1
SF-268 CNS tumor 2
RPMI 8226 Leukemia 3
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Cell type Origin ALP
HTC15 Colon tumor 1
UO-31 Colon tumor 1
SN12C Kidney tumor 2
SK-MEL-2 Melanoma 2
SK-MEL-28 Melanoma 1
UACC-62 Melanoma 2
UACC-257 Melanoma 2
T47D Breast tumor 1
* mRNA Northern blot.
__
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ALP exhihits increased expression in tumor cells compared
to their normal tissue counterparts. This differential
expression suggests a possible disregulation or involvement of
ALP in development or maintenance of the transformed phenotype.
Example 18: Recombinant Expression of ALP
The following example illustrates the contruction of
vectors for expression of recombinant ALP and the creation of
recombinant cell lines expressing ALP.
Contruction of Expression Vectors
Expression constructs were generated by PCR-assisted muta-
genesis in which the entire coding regions of ALP was
introduced into the mammalian expression vectors pcDNAIII
(Invitrogen) for transient expression analysis. Additional ALP
constructs were made by oligonucleotide based PCR mutagenesis
to convert atypical residues in the PTP-related domain back to
the amino acids more commonly present in other catalytically
active PTPs . These changes include : His to Tyr at amino acid
861 (See SEQ. ID. N0.:2); Ala to Gly at amino acid 902; Phe to
trp at amino acid 941; Glu to Asp at amino acid 995; and Ser to
Ala at amino acid 1032. Additional constructs containing
paired mutations as above were generated for amino acid
positions 941/1032 and 902/1032. These constructs were ligated
into the pcDNAIII mammalian expression vector behind the CMV
promoter.
The entire ALP open reading frame excluding the initiating
methionines was generated by PCR and ligated into pGEX vector
(Pharmacia Biotech, Upsala, Sweden) for bacterial production of
GST-fusion proteins for immunization of rabbits for antibody
production. This vector contains the glutathione-S-transferase
coding sequence followed by a polylinker for generating
recombinant fusion proteins. The GST moiety comprises the N-
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terminal portion of the fusion protein. The various ALP
mutants were also inseted into the pGEX vecotr for production
of recombinant protein reagents.
Transient Expression in Mammalian Cells
The pcDNAIII expression plasmids (10 ,ug DNA/100 mm plate)
containing the wild-type and mutant forms of the ALP gene were
introduced into 293 cells with lipofectamine (Gibco BRL).
After 72 hours, the cells were harvested in 0.5 mL
solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10~
glycerol, to Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 2 mM
phenylmethylsulfonyl fluoride, 1 &g/mL aprotinin). Sample
aliquots were resolved by SDS polyacrylamide gel electro-
phoresis (PAGE) on 15%acrylamide/0.5% bis-acrylamide gels and
electrophoretically transferred to nitrocellulose. Non-
specific binding was blocked by preincubating blots in Blotto
(phosphate buffered saline containing 5~ w/v non-fat dried milk
and 0.2o v/v nonidet P-40 (Sigma)), and recombinant protein was
detected using antisera specific to the amino-terminal 352
residues (see below). Recombinant ALP protein migrated appro-
ximately 180 kDa, consistent with the predicted molecular
weight of the 1274 amino acid protein.
Endogenous ALP was detected as a 200 kD protein in Western
blots of lysates from a variety of tumor cell lines including
human glioblastomas (U87MG, ATCC HTB 14; U118MG, ATCC HTB 15;
U138MG, ATCC HTB 16; A172, ATCC CRL 1620; Hs683, ATCC HTB 138),
rodent gliomas (C6, ATCC 107), rodent pituitary tumors (ATT20,
ATCC CCL 89; GH3, ATCC CCL 82.1), human neuroblastomas (SKNMC,
ATCC HTB 10; IMR 32, ATCC CCL 127), and rodent adrenal
pheochromocytomas (PC12, ATCC CRL 1721). ALP protein could not
be immunoprecipitated from the non-transformed cell line NIH
3T3 (ATCC CRL 1658).
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It is unclear why native ALP protein appears to be larger
(200 kDa) than recombinant ALP detected in transfected 293
cells (180 kDa). The difference could be the result of
alternative RNA splicing, or a post-translational modification
in the cell lines where it is endogenously expressed. Prelimi-
nary experiments indicate that ALP is phosphorylated on serine
and threonine residues in transfected 293 cells. In addition,
several tyrosine-phosphorylated proteins are associated with
ALP since they are detected in Western blots using an anti-
phosphotyrosine antibody following immunoprecipitation of
endogenous ALP from human tumor cell lines such as IMR32 after
treatments with the phosphatase inhibitor pervanadate.
Generation Of Virus Producing Cell Lines
pLXSN recombinant constructs containing the ALP gene are
transfected into an amphotropic helper cell line PA317 using
CaCl2 _mediated transfection. After selection on 6418, the
cells are plated on normal media without 6418 (500 ~,g/mL).
Supernatants from resistant cells are used to infect the
ecotropic helper cell line GP+E86, and cells again selected on
6418. Resistant cells are again taken off 6418, and the
supernatants harvested every 8-12 hours and pooled as virus
stock. Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498.
Viral stock titers are typically ~106/mL.
Stable Ex ression In Mammalian Cells
NIH-3T3, BALB/3T3 or other suitable cells are grown in 100
mm plates with DMEM (Gibco) containing 10~ fetal calf serum
(FCS). The cells are superinfected with the ALP retrovirus by
adding approximately 3 mL viral supernatant to 15 mL culture
media for approximately 24 hours. Cells expressing the
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retroviral constructs are then selected by growth in DMEM/l0a
FCS supplemented with 500 ~g/mL 6418.
Example 19: Generation Of Anti-Alp Antibodies
ALP-specific immunoreagents were generated by immunizing
rabbits with the bacterially expressed N-terminal 352 amino
acid portion of ALP expressed as a GST-fusion protein. Fusion
protein was affinity purified using glutathione-sepharose
colums (Pharmacia). Polyclonal anti-serum against the N-termi-
nal portion of ALP was generated by repeatedly immunizing
rabbits with the purified GST-futions protein. Affinity-
purified ALP antibody was obtained by binding serum IgG to ALP-
GST-fusion protein immobilized on glutathione-sepharose and
eluting with low pH and high salt.
Example 20: Assay For ALP Activity Assay For Modulators Of
Catalytic Activity
Materials And Methods:
Recombinant wild-type and mutant ALP proteins are purified
from bacteria as GST-fusion proteins. Lysates are bound to a
glutathione-sepharose matrix and eluted with glutathione. The
purified proteins are then washed with 2 x 1 mL HNTG, followed
by one wash with 1 mL of a buffer containing 100 mM 2-(N-
morpholino)ethansulfonic acid (MES), pH 6.8, 150 mM NaCl, and 1
mM EDTA. The assay for phosphatase activity is essentially
done as described by Pei et al.(1993) using p-
nitrophenolphosphate (PNPP) as a generic PTP substrate.
Briefly, after the last washing step, reactions are started by
adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10
mM DTT, 2 mM EDTA, and 50 mM p-nitrophenylphosphate) to the
precipitates. Samples are incubated for 20 min. at 23 °C. The
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reactions are terminated by mixing 40 ~L of each sample
(without beads) with 960 ~,L 1 N NaOH, and the absorbance of p-
nitrophenol was determined at 450 nm. To control for the
presence of ALP in the precipitates, the precipitates are
boiled in SDS sample buffer and analyzed by SDS-PAGE. The
presence of ALP is then detected by immunoblot analysis with
anti-ALP antibodies.
Example 21: A Consistent Method For Determination Of ZAP70
Kinase Activity.
The following protocol describes the reagents and
procedures used to determine Zap70 protein kinase activities
measuring phosphorylation of Band III-GST as readout. This
assay is used in search for inhibitors of Zap70.
Materials and Reagents
1. Baculovirus (Pharmingen, CA) encoding for muta
tionally activated form of Zap70, in which a tyrosine residue
at position 492 is replaced with a phenylalanine residue
(Y492F), containing a C-terminal HA tag and a N-terminal GST
tag (GST-Zap70-HA) is used. The modified protein is termed GZH
(i.e. Y492F GST-Zap70-HA = GZH).
2. Cell lysates: SF9 cells were infected with the GZH
virus at MOI of 10 for 96 hours. The cells were then washed
once with PBS and lysed in lysis buffer. Insoluble material
was removed by centrifugation (5 min. at
10 000 x g). Aliquots of lysates were frozen in dry
ice/ethanol and stored at -80 °C until use.
3. Band III-GST: Band III-GST fusion protein (amino acid
sequence: MEELQDYEDMMEEN (SEQ ID N0:62)) was expressed in XL1
Blue cells transformed with pGEX -2TK-Band III. Protein
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expression was induced by addition of 0.5 mM IPTG while shaking
the bacterial culture for 18 hours at 25 °C. Band III-GST by
was purified by Glutathione affinity chromatography, Pharmacia,
Alameda, CA
4. Biotinylated ITAM peptide 242 (ZETA-pY),
Sequence: YQQGQNQLpYNELNLGRREEpYDVLDKRRGRD (SEQ ID N0:63)
(Protein Chemistry Laboratory, SUGEN, INC., Redwood City, CA).
5. DMSO, Sigma, St. Louis, MO
6. 96 Well ELISA Plate: Corning 96 Well Easy Wash,
Modified Flat Bottom Plate. Catalog # 25805-96.
7. NUNC 96-well V-bottom polypropylene plates for
dilution of compounds. Applied Scientific Catalog No.
AS-72092
8. Streptavidin: Sigma S-8276
9. Purified Rabbit anti-GST antiserum. AMRAD catalog #
9001605
10. Goat anti-Rabbit-IgG-HRP. Amersham Catalog No.
V010301
Buffer solutions:
Lysis buffer: Kinase buffer:
10 mM Tris, pH 7.5 10 mM MgCl2
150 mM NaCl 10 mM MnCl2
1~ NP40 10 mM DTT
1 mM PMSF 20 mM HEPES/C1, pH 7.5
0.4 mM Na3V0q 20 mM (3-glycerophosphate
2 mg/ml Leupeptin 200 Na3V0q
mM
2 mg/ml Aprotinin
Blocking buffer: Wash buffer (TBST):
10 mM Tris, pH 7.5 50 mM Tris, pH 7.5
100 mM NaCl 150 mM NaCl
0.1% Tween 20 O.lo Tween 20
_ _~.._ _______ ~ _____-__
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lg BSA
Prnr~c~~irc
Preparation of Streptavidin Coated ELISA Plates:
Prepare borate buffer by titrating 0.1 M boric acid with
0.1 M sodium borate to pH 8.7. Add sodium azide to a final
concentration of 0.050 and store at 4 °C. Prepare
1 mg/ml Streptavidin in borate buffer and store at 100 ~L
aliquots at -80 °C. Coat 0.1 ~,g/well Streptavidin in
100 ~L of borate buffer at room temperature for 18 hours. Wash
wells with 200 ~,L cold TBST twice. Invert the plate and blot
the plate dry, cover with parafilm, and store at
4 °C for no more than one week. For longer storage, plates
should be stored at -80 °C.
Preparation of phosphotyrosine antibody-coated ELISA plates:
Coat 1 ~g/well 4610 (Upstate Biotechnology, NY) in 100 ~L
of PBS overnight at 4 °C and block with 200 ~.L of blocking
buffer for at least hour.
Kinase Assay Procedure
Biotinated peptide 242 was bound to the ELISA Plate by
incubating 1 ~g/well in 100 ~.L PBS overnight at 4 °C with
streptavidin coated ELISA Plate (see above). The wells were
blocked with 200 ~L blocking buffer for 30 minutes at room
temperature, after which the blocking buffer was removed by
aspiration. Insect cell lysate containing the Zap70 fusion
protein (GZH) was added (30 ~.g/well, volume adjusted to 100
~tL/well with lysis buffer) and left to incubate at 4 °C for 2
hours. The lysate was removed by aspiration and the wells
washed with TBST. Substrate and test compound (if any) were
100 mM NaCl 150 mM NaCl
0.1%
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added and allowed to stand for 15 minutes (GST-Band III, 5
~g/well in 90 ~,L final volume). The kinase reaction was
started by the addition of 10 ~.L of
0.1 mM ATP per well for a final concentration of 10 ~M. The 96
well plate was left for 30 minutes at room temperature
(shaking) after which 90 ~,L of the reaction liquid was
transferred to wells in a 96 well plate previously coated with
an anti-phosphotyrosine antibody (UB40, Upstate Biotechnology,
NY). This plate was allowed to stand for 30 minutes at room
temperature, after which the liquid was removed and the wells
washed with TBST. Rabbit anti-GST antibody was added (0.1
~,g/well in 100 ~L blocking buffer) and incubated for 30 minutes
at room temperature. The liquid was again removed and the
wells washed with TBST. Goat anti-Rabbit-IgG-HRP was added at
1:40,000 dilution in 100 ~,L of blocking buffer for 30 minutes
at room temperature, after which it was removed and the wells
washed with TBST and developed with ABTS. The plate is then
read in an ELISA plate reader at 410 nm. If the protein being
tested is a captured protein, the reading from the ELISA plate
reader can be related to the modulating activity of the test
compound when it is compared with the activity of a control
protein.
Example 22: Isolation And Characterization Of ALK-7
In order to isolate ALK-7, we designed degenerate oligo-
nucleotides encoding amino acid motifs within kinase subdomains
II and VI common to all known mammalian STK receptors. (Hanks
and Hunter, FASEB J. 9:576-595, 1995) Subdomain II is at the N-
terminus of the kinase domain and contains the invariant lysine
residue that is essential for enzyme activity and is involved
in ATP binding by interacting with the a- and b-phosphates of
all kinases whose structure has been elucidated. Subdomain VI
r _. - ___ i
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is referred to as the catalytic loop and contains the consensus
motif HRDLKXXN (SEQ ID N0: 64 ) . The Asp residue is involved in
accepting the proton from the hydroxyl group during the
phosphotransfer process key to all protein kinases. Based on
comparison of all STK receptors, we designed degenerate oligo-
nucleotide primers to these subdomains that would recognize
both type I and type II STK receptors.
When this PCR strategy was applied to a human
neuroblastoma cell line (SYSY) sscDNA as a template, multiple
copies of a novel DNA fragment (ALK-7) were isolated that
exhibited significant homology to other STK receptors. The
novel sequence was most similar to ALK-9 (Franzen, et al., Cell
75(4):681, 1993) and ALK-5 (ten Dijke, et al., Oncogene
8(10):2879, 1993) and was referred to as ALK-7.
Materials And Methods
Total RNAs were isolated using the Guanidine Salts/Phenol
extraction protocol of Chomczynski and Sacchi (P. Chomczynski
and N. Sacchi, Anal. Biochem. 162, 156 (1987) from normal human
tissues, from regional sections of human brain, from cultured
human tumor cell lines, and from primary neonatal rat
sympathetic, motor, and sensory neuronal cells, as well as
mesothalamic dopaminergic neurons.
These RNAs were used as templates to generate single
stranded cDNAs using the Superscript Preamplification System
for First Strand Synthesis kit purchased from GibcoBRL (Life
Technologies, U.S.A.; Gerard, G.F..et a1. (1989), FOCUS 11, 66)
under conditions recommended by manufacturer. A typical
reaction used 10 ~g total RNA or 2 ~g poly (A) + RNA with 1. 5 ~g
oligo (dT) i2-ie in a reaction volume of 60 ~,L. The product was
treated with RNaseH and diluted to 100 ~L with H20. For
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subsequent PCR amplification, 1-4 ~,L of these sscDNAs were used
in each reaction.
Oligonucleotides were synthesized on an Applied Biosystems
394 DNA synthesizer using established phosphoramidite chemistry
and were used unpurified after precipitation with ethanol. The
degenerate oligonucleotide primers are:
STK1 = 5'-GARRARGT6GC6GT6AARRT6TT-3' (SEQ ID N0:65)(sense)
STK3- -
5'-TTRATRTC6CKRTG6GM6AT6GM6GGYTT-3' (SEQ ID N0:66) (antisense).
These primers were derived from the peptide sequences
E(K/E)VAVK(V/I)F (SEQ ID N0:67) (sense strand from kinase
subdomain II) and
KP(A/S)I(A/S)HRDIK (SEQ ID N0:68) (antisense strand from kinase
subdomain VI), respectively. Degenerate
nucleotide residue
designations are: N = A, C, G, or T; R = A or G; Y = C or T;
M
- A or C; K - G or T; and 6 - Inosine. Using ALK1 as a
template, these primers produce product of 321 bp.
a
A PCR reaction was performed using primers STK1 and STK3-
applied .to the single-stranded sources listed above. The
primers were added at a final concentration
of 5 ~M each to a
mixture containing 10 mM Tris HC1 (pH 8.3), 50 mM KC1, 1.5 mM
MgCl2, 200 uM each deoxynucleoside triphosphate, O.OOlo gelatin,
and 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4
ul cDNA. Following 3 min denat uration at 95~C, the cycling
conditions were 94 C for 30 s, 37 C for 1 min, a 2 min ramp to
72 C, and 72 C for 1 min for the
first 3 cycles, followed by
94 C for 30 s, 50 C for 1 min, and C for 1 min 45 s for 35
cycles. PCR fragments migrating at 320 by were isolated from
2$ agarose gels using GeneClean (Bio101), and T-A cloned into
the pCRII vector (Invitrogen Corp.
U.S.A.) according to the
manufacturer's protocol.
_t _ i
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Colonies were selected from mini plasmid DNA-preparations
using Qiagen columns and the plasmid DNAs were sequenced using
cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction
products were run on an ABI Prism 377 DNA Sequencer, and
analyzed using the BLAST alignment algorithm (Altschul, S.F. et
al., J. Mol. Biol. 215:403-20). A novel clone (STKR6.22) was
isolated by PCR with primers STK1 and STK3- on single-stranded
cDNA from human SYSY cells as a template. This clone was
subsequently designated as a fragment of human ALK-7.
A lambda gtll (Clontech, Palo Alto, CA) cDNA library was
constructed using mRNA from a pool of nine whole human
pituitary glands. Phage were screened on nitrocellulose fil-
ters with the random primed 32P-labeled insert from STKR6.22
encoding human ALK-7 at 2x106 cpm/mL in hybridization buffer
containing 6xSSC, lx Denhardt's reagent, 0.1$ SDS, with 0.1
mg/mL denatured, fragmented salmon sperm DNA. After overnight
hybridization at 65 °C, filters were washed in O.IxSSC, O.la
SDS at 65 °C. Full length cDNA clones were sequenced on both
strands using manual sequencing with T7 polymerase and
oligonucleotide primers (Tabor and Richardson, 1987, Proc.
Natl. Acad. Sci., U.S.A. 84:4767-71).
Results
Two overlapping cDNA clones (P6 and P7), spanning 1794
nucleotides were isolated from a human pituitary library. This
sequence contains an ATG at position 156 that conforms to the
Kozak consensus for translational initiation and is followed by
a 1,482 nucleotide open reading frame with the capacity to
encode a polypeptide of 493 amino acids. There are no other
initiation codons 5' to the ATG located at position 156. The
coding region for human ALK-7 is flanked by 5' and 3'
untranslated regions of 155 and 157, respectively. There is no
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polyadenylated region although the 3' end of the sequence shown
in SEQ ID N0:8 is noticeably AT-rich, a feature characteristic
of sequences from 3'-untranslated regions. An additional cDNA
clone (P4) extended an additional 1 kb 3' of this sequence.
DNA sequence determination was performed with dideoxy
terminators using Sequenase 2Ø A primer walking strategy on
both strands was used to confirm the complete nucleotide
sequence. Oligonucleotide primers were made with an ABI 348
DNA synthesizer.
A Smith-Waterman search with the human ALK-7 gene sequence
of the public nonredundant nucleic acid and EST databases
revealed no identical matching sequences confirming that this
is a novel human gene. The closest match to the human ALK-7
sequence (85% nucleic acid identity) is a recent entry (GenBank
ACC:U69702) which appears to be the rat orthologue of human
ALK-7.
The 493 amino acid human ALK-7 sequence contains two
hydrophobic regions from 1-25 and 114-138. (See SEQ ID N0:16)
The first hydrophobic region meets the criteria of a signal
peptide domain, with a discriminant score of 5.76 using the
method of McGeoch (D. J. McGeoch, Virus Research, 3, 271,
1985), and with a weight matrix score of +6.75 (threshold -
3.5) using the von Heijne algorithm (G. von Heijne, Nucl. Acids
Res., 14, 4683, 1986). The second hydrophobic region generates
a likelihood score of -9.34, using the ALOM method of Klein et
a1. (P. Klein, M. Kanehisa, and C. DeLisi, Diochim. Biophys.
Acta, 815, 468, 1985) to predict transmembrane domains. This
algorithm predicts a maximal range of the transmembrane domain
to be from as 108-138.
Based on this analysis, ALK-7 is predicted to be a type Ia
integral membrane protein with a molecular weight of 52.35 kD
after cleavage of the N-terminal signal peptide.
.~ __._ _ I
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Example 23: Expression Of ALK-7
Using both Northern blots and PCR analysis with the novel
fragment originally cloned from SYSY cells as described above
as a probe, we screened RNAs using from a large number of tumor
cell lines and multiple human tissues, demonstrating an
apparent selectivity in expression of ALK-7 in neuronal cells
from the pituitary and substantiate nigra.
Materials And Methods
Northern Blot Analysis
Northern blots were obtained from Clontech (Palo Alto, CA)
containing 2 uq polvA+ RNA from i6 different adult human
tissues (spleen, thymus, prostate, testis, ovary, small
intestine, colonic mucosa, heart, brain, placenta, lung, liver,
skeletal muscle, kidney, pancreas, and peripheral blood
leukocytes), and four different human fetal tissues (brain,
lung, liver, and kidney), on a charge-modified nylon membrane.
Additional Northern blots were prepared by running 20 ~g total
RNA on formaldehyde 1.2% agarose gel and transferring to nylon
membranes.
Filters were hybridized with random prime (32P]dCTP-labeled
probes synthesized from the 320 by insert from human ALK-7
clone STKR6.22. Hybridization was performed at 60 °C overnight
in 6XSSC, 0.1% SDS, 1X Denhardt's solution, 100 mg/mL denatured
herring sperm DNA with 1-2 x 106 cpm/mL of 32P-labeled DNA
probes. The filters were washed in O.1XSSC/0.1% SDS, 65 °C,
and exposed overnight on Kodak XAR-2 film.
Semi-Quantitative RT-PCR Detection
The expression pattern of ALK-7 was also investigated
using a PCR technique, RNA was isolated from a variety of human
cell lines, fresh frozen tissues, and primary tumors as
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detailed above. Single stranded cDNA was synthesized from 10
ug of each RNA as described above using the Superscript
Preamplification System (GibcoBRL) These single strand temp-
lates were then used in a 35 cycle PCR reaction with two human
ALK-7-specific oligonucleotides:
ALK-7a: 5'-AACTTTGGCTGGTATCTGAATATC-3' (SEQ ID N0:69), and
ALK-7b: 5'-CCTTGTGTACCAACAATCTCCATA-3' (SEQ ID N0:70).
Reaction products were electrophoresed on 2% agarose gels,
stained with ethidium bromide and photographed on a UV light
box. The relative intensity of the -150-by ALK-7-specific
bands were estimated for each sample. A similar pair of
oligonucleotides was designed for detection of rat ALK-7:
4076: 5'-CTCCAGAGATGAGAGATCTTGG-3' (SEQ ID N0:71), and
4077: 5'-TTCCAGCCACGGTCACTATGTT-3') (SEQ ID N0:72),
encompassing a -210 by region of the rat gene.
Results
ALK-7 mRNA transcript was not detectable by Northern
analysis from multiple human tissue sources, suggesting its
expression is highly restricted. Using a more sensitive PCR-
based detection, ALK-7 was found to be expressed in human
substantia nigra, anterior pituitary, and Calu-6 lung carcinoma
cell line (see below). Weak expression was found in several
other locations including whole brain, cerebellum, and
prostate. Multiple other normal human tissues and tumor cell
lines showed no detectable ALK-7 expression.
_ _ ___~ __ -_ _ ~
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HUMAN ALK-7 RNA EXPRESSION ANALYSIS
Medium (++) Negative
Substantia Nigra IMR-32 (neuroblastoma)
Anterior Pituitary SYSY (neuroblastoma)
Calu-6 (Lung Ca) SK-N-SH (neuroblastoma)
SWI763 (astrocytoma)
SW1388 (astrocytoma)
Weak (+) U-138 (glioblastoma)
U87MG (glioblastoma)
Brain Menirigiomas (lo tumors)
Posterior Pituitary SKOV-3 (ovarian Ca)
Cerebellum ASPC (pancreas Ca)
Ovary CAPAN-1(pancreas Ca)
Prostate HS766T (pancreas Ca)
Fetal Intestine PANC (pancreas Ca)
Duodenum HOS (osteoSarcoma)
T48 (colon Ca) KHOS (osteoSarcoma)
HTB227 (breast Ca)
HTB131 (breast Ca)
LS123 (colon Ca)
LS147T (colon Ca)
SkC04 (colon Ca)
SW11E {colon Ca)
HTC15 (colon Ca)
SW403 (colon Ca)
HT29 (colon Ca)
SW627 (colon Ca)
SW948 (colon Ca)
HUVEC (h, endothelial)
Fibroblasts (Primary)
Pancreas
Testis
Thymus
Liver
Heart
Placenta
Lung
Skel. Muscle
Kidney
Spleen
Ovary
Colon
Leukocytes
In situ EXPESSION PROFILE of RAT ALK-7
The neuronal expression of ALK-7 was assessed by in situ
analysis in sagittal and coronal sections from neonatal and
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adult rat brains using a fragment of the extracellular domain
of rat ALK-7 as a probe. This region was selected because its
dissimilarity with the related ALK-4 and ALK-5. Other groups
have performed in situs with the catalytic domain of rat ALK-7
demonstrating specific expression in neuronal tissues
(cerebellum, hippocampus, and brainstem nuclei), kidney,
testis, lung, dorsolateral and anterior prostate, and adipose
tissue. However, the probe used in these studies contained an
ALK-7 catalytic domain which may cross-react with the related
ALK-4 and ALK-5 (77o nucleotide sequence identity with
stretches of 27/29 and 25/26 by identity to rat ALK-7) and
thereby broaden the expression profile. Using a more selective
ALK-7 probe our analysis revealed the more restricted
expression. In sagital sections, a moderate strength granular
band was visible in the CA2 and CA3 regions of the hippocampus,
dentate dyrus, olfactory tubercle, dorsal outer layer of the
cortex, and in a band crossing the frontal cortex area 2 from
the exterior to the corpus callosum. A moderate signal was
detected in the caudate putamen and thalamic nuclei. In
addition, signals of moderate strength were detected in the
region of the magnocellular nucleus of the lateral hypothalamus
and the medial tuberal nucleus. A similar signal was observed
in the region of the cuneiform nucleus on the anterior border
of the cerebellum. The cerebellum was devoid of hybridizing
ALK-7.
Coronal sections support the finding of expression in the
CA2, CA3 region of the hippocampus, dentate gyrus, caudate
putamen, and in the region underlying the exterior of the
cortex. In addition, a signal of moderate strength was
detected in the dorsomedial part of the ventromedial
hypothalamic nucleus. A dispersed nuclei signal of lesser
strength was detected in the area of the amygdalopiriform tran-
sition.
_.____~ _ _.._ _ _ _ _
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Example 24: ALK-7-Specific Antibodies
ALK-7-specific immunoreagents were raised in rabbits
against KLH-conjugated synthetic peptide YRKKKRPNVEEPL {SEQ ID
N0:76) from the juxtamembrane portion of the cytoplasmic domain
of ALK-7. This region is unique to ALK-7 compared to other
type I STK receptors, thereby allowing for the generation of
ALK-7 specific antisera. The N-terminal extracellular domain
of ALK-7 expressed as a GST-fusion was also used as an
immunogen to raise polyclonal antibodies in rabbits and to
generate monoclonal antibodies in mice using the techniques
described above. These antibodies were used to localize
expression of the endogenous and recombinant protein as
describe below.
Example 25: Recombinant Alk-7 Expression
The following example describes the construction of
vectors for transient and stable expression in mammalian cells.
Expression constructs were generated to make wild type ALK-7 as
well as a signaling incompetent ALK-7 {ALK-7DN) and a
constitutively activated ALK-7 (ALK-7TA).
Materials and Methods
Construction of Vectors
Expression constructs were generated by PCR-assisted
mutagenesis in which the entire coding domain of ALK-7 was
tagged at its carboxy-terminal ends with the hemophilus
influenza hemaglutinin (HA) epitope YPYDVPDYAS (SEQ ID N0:77)
(Pati, Gene 119:285, 1992). This constructs were introduced
into two mammalian expression vectors: pAdRSVOES-, a modified
adenovirus vector for the generation of virus producing
recombinant protein, and pRKS for transient expression
analysis.
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Recombinant adenoviruses were generated by in vivo
ligation as follows.
The transfer vector used Contains the following DNA
sequences in order: The left terminal region of adenovirus type
5 encoding the packaging sequences (adenovirus type 5
nucleotides 1-959); the Rous Sarcoma Virus long terminal repeat
promoter and the SV40 polyA region, isolated as an expression
cassette from the plasmid pREP (Invitrogen Corporation);
nucleotides 3320-5790 of the type 5 adenoviral genome: and the
on and beta-lactamase genes derived from the E. coli plasmid
pBluescript. Two additional forms of the plasmid were
generated. The first, pAdRSVIacZ, was prepared by the
insertion of a double stranded synthetic oligonucleotide into
the BamHI restriction site between the RSV promotor and the
SV40 polyA sequence with the following nucleotide sequence
(upper strand shown): 5'
CTTCGAA.AGCTTGAAATCGGTACCATCGATTCTAGAGTTAACTTCGAA. (SEQ ID NO:
73) The E. coli lacZ gene was excised from the expression
plasmid pCMVb (Clontech, Inc.) with the enzyme Not I and
inserted into the Not I site between the promoter and the polyA
sequence. This generated a plasmid that expressed the lacZ
gene, and had two BstBI restriction sites between the lacZ gene
and the polyA region. The second plasmid (pAdRSVOES-) was
generated by inserting a double stranded synthetic
oligonucleotide into the same region as above. Its 'nucleotide
sequence was the following: 5'
CTCTAGAACGCGTTAAGGCGCGCCAATATCGATGAATTCTTCGAAGC. (SEQ ID N0:74)
This plasmid allowed the introduction of exogenous cDNAs into
the plasmid for expression purposes.
The viral DNA used for generation of recombinant viruses
was derived from a virus (AdlacZBstBI) in which the left end of
the adenovirus genome has been replaced by the homologous
region of pAdRSVIacZ. To achieve this, DNA ,vas isolated from
___--.~_.._.
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the Ad5 d1327 strain of adenovirus (Jones and Shenk, Cell,
1978) (deleted in the E3 region), cleaved with ClaI enzyme, and
cotransfected into the HEK2934 cell line via calcium phosphate
coprecipitation with the pAdRSVIacZ plasmid. Recombinant
adenovirus plaques resulting from this transfection were
screened for the ability to express the lacZ gene by
histochemical staining with X-Gal. The resulting recombinant
adenovirus, AdlacZBstBI, provided the backbone for additional
adenovirus constructs, allowing a screen for recombinant
plaques based on the presence or absence of lacZ activity in
that further recombination would replace the lacZ gene with the
cotransfected cDNA. To achieve this, the transfer vector
construct is linearized by digestion with BstBI, and
cotransfected with AdlacZBstBI DNA which has also been cleaved
with BstBI. Typically, 5 mg of transfer vector plasmid DNA are
corecipitated with 2 mg of viral DNA for the transfection; in
vivo ligation of viral DNA and linearized transfer vector
produces a novel recombinant virus directing expression of the
new transgene.
A signaling incompetent ALK-7 construct was also made in
both vectors pAdRSVOES- and pRK5 by insertion of an HA-tag at
as 230 in the ALK-7 coding region just after catalytic domain
II. Truncation of other Type I STKRs in an analogous location
has functioned in a dominant negative manner. This construct
was called ALK-7DN. A constitutively active form of ALK-7 was
generated by a Thr to Asp mutation at amino acid 194 just
upstream of the catalytic domain I GXGXXG motif. In other Type
I STKRs, this residue undergoes ligand-dependent trans-
phosphorylation by the associated Type II STKR, resulting
receptor activation and initiation of a signaling cascade. A
similar mutation in other Type I STKR's results in a ligand-
independent, constitutively activated receptor. This construct
was called ALK-7TD.
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Generation Of Recombinant ALK-7 - Adenovirus
Early passage HEK293 cells (Graham, et al., J. Gen.
Virol. 36:59, 1977) were maintained in Dulbecco's modified
Eagles medium + loo calf serum. HEK293 monolayers were
transfected with the ALK-7-encoding transfer vectors and
cultured from five to seven days to allow plaques to appear.
The monolayers were then stained with 25 mg/mL 5-bromo-4-
chloro73-indolyl-b-D-galactopyranoside for several hours to
identify non-recombinant (blue-stained) plaques. Putative
recombinant plaques were screened for expression of the
transgene by infection of HEK293 cultures followed by
immunohistochemistry with the monoclonal antibody recognizing
the HA epitope. Viruses which were positive for transgene
protein expression were picked and subjected to several rounds
of claque purification prior to amplification and purification
on cesium chloride gradients. Banded viruses were diluted
five-fold with dilution buffer (Curiel et al., Proc. Natl.
Acad. Sci., USA 88:8850-8854, 1991) and stored at -80 °C.
Approximate titers of the virus preparations were determined
immunohistochemically on HEK293 cultures. The following
viruses were generated: AdRSVALK-7-HA; AdRSVALK-7-DN; and
AdRSVALK-7-TD.
Transient Expression
The pRKS expression plasmids (10 ~g DNA/100 mm plate)
containing the KA-tagged ALK-7, the ALK-7DN, and ALK-7TD
constructs were introduced into COS and 293 cells with lipo-
fectamine (Gibco BRL). After 72 hours, the cells were
harvested in 0.5 ml solubilization buffer (20 mM HEPES pH 7.35,
150 mM NaCl, 10~ glycerol, to Triton X-100, 1.5 mM MgCl2, 1 mM
EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ~,g/mL aprotinin).
_. _.__T ... . ._............_.. . ~ ... ..... _-~___ . _...
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Sample aliquots were resolved by SDS polyacrylamide gel
electrophoresis (PAGE) on 15~ acrylamide/0.5~ bis-acrylamide
gels and electroplicretically transferred to nitrocellulose.
Non-specific binding was blocked by preincubating blots in
Blotto (phosphate buffered saline containing 5$ w/v non-fat
dried milk and 0.2~ v/v nonidet P-40 (Sigma)), and recombinant
protein was detected using a murine Mab to the HA decapeptide
tag. Alternatively, recombinant protein can be detected using
various ALK-7-specific antisera.
Expression In Neuronal Cells
The recombinant ALK-7 protein described above were
expressed in PC12 cells and primary rat neuronal cultures by
adenovirus mediated infection. These cells will allow further
investigation into ALK-7 function. Recombinant protein expres-
sion was confirmed by immunostaining with an anti-HA antibody.
PC12 cultures (Greene, et al., Methods Enzymol. 147:207,
1987) were maintained in RPMI medium containing 10~ horse serum
and 5°s fetal calf serum. Four differentiation experiments the
medium was changed to RPMI containing 1X N2 supplement and 0.1~
BSA, and the cells were grown on a collagen I substrate. For
PC12 cell survival, the cells were grown in RPMI containing
0.1~ BSA. All cultures also contained 1X penicillin/
streptomycin. For adenoviral infections, PC12 cells were
incubated overnight with recombinant viruses at a multiplicity
of infection (MOI) between 1 and 10. The cells were then
washed and replated either into differentiation or survival
conditions for two days. Nerve Growth Factor (50 ng/mL) served
as a positive control. For differentiation, the cultures were
fixed with 2~ paraformaldehyde and the percentage of cells
bearing processes longer than 1 cell diameter was determined.
For survival, the cultures were incubated with 0.05 MTT for
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1.5 hours to stain living cells, and the relative number of
cells surviving in each condition was determined.
Sympathetic and sensory neurons were isolated as described
(Hawrot and Patterson, Methods Enzymol. 53:574, 1979; Fields et
al., Cell 14:43, 1978) and cultured in a defined medium (Hawrot
and Patterson, supra). Sympathetic neurons were isolated from
superior cervical ganglia dissected from E20 - E21 rat fetuses,
while dorsal root ganglion sensory neurons were obtained from
E16 - E18 rats. The ganglia were treated with 0.250 trypsin
for 10 minutes, washed, and triturated to obtain a single cell
suspension. Sensory neurons were preplated for 1 hour on
tissue culture plastic to deplete adherent cells. Dopaminergic
neurons were isolated as described (Shimoda, et al., Brain
Research 586:319-331, 1992) and cultured in Neurobasal medium,
supplemented with B27 supplements (Life Technologies). Neurons
were infected with adenoviruses for two hours on collagen I-
coated tissue culture plastic (supplemented with NGF for
sensory and sympathetic neurons), and the cells were then
washed and allowed to recover for two to four additional hours
(with NGF if appropriate). After the recovery period, the
cells were washed extensively to remove the growth factor, and
plated onto polylysine-laminin coated chamber slides. The
addition of NGF at 50 ng/mL served as a positive control for
survival of sensory and sympathetic neurons. After an
additional two days to three days, the sensory and sympathetic
cultures were stained with calcein AM (1 mg/mL) for 45 minutes,
mounted and examined by immunofluorescence. Generally, five
disperse fields representing 7% of the well were photographed
and the number of surviving neurons quantitated. To determine
dopaminergic neuron survival, the cultures were fixed and the
number of tyrosine hydroxylase positive neurons was determined.
_~
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Results
Recombinant ALK-7 protein expressed in COS cells migrated
with apparent Mr of 52kD-63kD, consistent with its predicted
molecular weight of 54kD based on its primary amino acid
sequence and the presence of multiple glycosylation sites. The
ALK-7TD constitutive active form produced proteins
indistinguishable from the wild type construct on SDS-PAGE.
The ALK-7DN construct expressed proteins of Mr 23.5 kd, 28 kD
and 32 kD consistent with the presence of varying amounts of
glycosylation on this truncated receptor. This analysis
confirms the recombinant protein can be stably produced in
mammalian cells.
One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those
inherent therein. The molecular complexes and the methods,
procedures, treatments, molecules, specific compounds described
herein are presently representative of preferred embodiments
are exemplary and are not intended as limitations on the scope
of the invention. Changes therein and other uses will occur to
those skilled in the art which are encompassed within the
spirit of the invention are defined by the scope of the claims.
it will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
All patents and publications mentioned in the
specification are indicative of the levels of those skilled in
the art to which the invention pertains.
The invention illustratively described herein suitably may
be practiced in the absence of any element or elements,
limitation or limitations which is not specifically disclosed
herein. Thus, for example, in each instance herein any of the
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terms "comprising", "consisting essentially of" and "consisting
of" may be replaced with either of the other two terms. The
terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described
or portions thereof, but it is recognized that various
modifications are possible within the scope of the invention
claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation
of the concepts herein disclosed may be resorted to by those
skilled in the art, and that such modifications and variations
are considered to be within the scope of this invention as
defined by the appended claims.
In addition, where features or aspects of the invention
are described in terms of Markush groups, those skilled in the
art will recognize that the invention is also thereby described
in terms of any individual member or subgroup of members of the
Markush group. For example, if X is described as selected from
the group consisting of bromine, chlorine, and iodine, claims
for X being bromine and claims for X being bromine and chlorine
are fully described.
In view of the degeneracy of the genetic code, other
combinations of nucleic acids also encode the claimed peptides
and proteins of the invention. For example, all four nucleic
acid sequences GCT, GCC, GCA, and GCG encode the amino acide
alanine. Therefore, if for an amino acid there exists an
average of three codons, a polypeptide of 100 amino acids in
length will, on average, be encoded by 3100, or 5 x 10Q', nucleic
acid sequences. It is understood by those skilled in the art
that, with, Thus, a nucleic acid sequence can be modified to
form a second nucleic acid sequence, encoding the same
_ ____~ _ _.
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polypeptide as endoded by the first second nucleic acid
sequences, using routine procedures and without undue
experimentation. Thus, all possible nucleic acids that encode
the claimed peptides and proteins are also fully described
herein, as if all were written out in full taking into account
the codon usage, especially that preferred in humans.
Furthermore, changes in the amino acid sequences of
polypeptides, or in the corresponding nucleic acid sequence
encoding such polypeptide, may be designed or selected to take
place in an area of the sequence where the significant activity
of the polypeptide remains unchanged. For example, an amino
acid change may take place within a (3-turn, away from the
active site of the polypeptide. Also changes such as deletions
(e.g. removal of a segment of the polypeptide, or in the
corresponding nucleic acid sequence encoding such polypeptide,
which does not affect the active site) and additions (e. g.
addition of more peptides to the polypeptide sequence without
affecting the function of the active site, such as the
formation of GST-fusion proteins, or additions in the
corresponding nucleic acid sequence encoding such polypeptide
without affecting the function of the active site) are also
within the scope of the present invention. Such changes to the
polypeptides can be performed by those with ordinary skill in
the art using routine procedures and without undue
experimentation. Thus, all possible nucleic and/or amino acid
sequences that can readily be determined not to affect a
significant activity of the peptide or protein of the invention
are also fully described herein.
Other embodiments are within the following claims.
CA 02288221 1999-10-27
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SUGEN, INC.
351 Galveston Drive
Redwood City, CA 94063
U.S.A.
1 (ii) TITLE DIAGNOSIS AND TREATMENT
O OF OF
INVENTION:
TYROSINE PHOSPHATASE-RELATED
DISORDERS AND RELATED
METHODS
Z (iii) NUMBER 76
S OF
SEQUENCES:
(iv) CORRESPONDENCE
ADDRESS:
2 (A) ADDRESSEE: Lyon & Lyon
O
(B) STREET: 633 West Fifth Street
Suite 4700
(C) CITY: Los Angeles
(D) STATE: California
2 (E) COUNTRY: U.S.A.
5
(F) ZIP: 90071-2066
(v) COMPUTER READABLE
FORM:
30
(A) MEDIUM TYPE: 3.5" Diskette, 1.44
Mb
storage
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM:IBM P.C. DOS 5.0
3 (D) SOFTWARE: FastSEQ for Windows
5 2.0
(vi) CURRENT
APPLICATION
DATA:
4 (A) APPLICATION NUMBER:To be assigned
O
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
4 (vii) PRIOR
5 APPLICATION
DATA:
(A) APPLICATION NUMBER:US 60/044,428
(B) FILING DATE: April 28, 1997
S (A) APPLICATION NUMBER:US 60/047,222
O
(B) FILING DATE: May 20, 1997
(A) APPLICATION NUMBER:US 60/049,477
(B) FILING DATE: June 12, 1997
55
(A) APPLICATION NUMBER:US 60/049,756
(B) FILING DATE: June 12, 1997
(A) APPLICATION NUMBER:US 60/049,914
6O (B) FILING DATE: June 18, 1997
(A) APPLICATION NUMBER:US 60/063,595
(B) FILING DATE: October 23, 1997
~
_ ____-~ __ __..___._ _.
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(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Warburg, Richard J.
(B) REGISTRATION NUMBER: 32,327
(C) REFERENCE/DOCKET NUMBER: 233/032-PCT
(ix) TELECOMMUNICATION INFORMATION:
_ 10
(A) TELEPHONE: (213) 989-1600
(B) TELEFAX: (213) 955-0440
(C) TELEX: 67-3510
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3580 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
CCCGGGTGCCCTCCCTCAACCTACTTATAGACTATTTTTC TTGCTCTGCAGCATGGACCA60
AAGAGAAATTCTGCAGAAGTTCCTGGATGAGGCCCAAAGC AAGAAAATTACTAAAGAGGA120
3 GTTTGCCAATGAATTTCTGAAGCTGAAAAGGCAATCTACC AAGTACAAGGCAGACAAAAC180
O
CTATCCTACAACTGTGGCTGAGAAGCCCAAGAATATCAAG AAAAACAGATATAAGGATAT240
TTTGCCCTATGATTATAGCCGGGTAGAACTATCCCTGATA ACCTCTGATGAGGATTCCAG300
CTACATCAATGCCAACTTCATTAAGGGAGTTTATGGACCC AAGGCTTATATTGCCACCCA360
GGGTCCTTTATCTACAACCCTCCTGGACTTCTGGAGGATG ATTTGGGAATATAGTGTCCT420
3 TATCATTGTTATGGCATGCATGGAGTATGAAATGGGAAAG AAAAAGTGTGAGCGCTACTG980
5
GGCTGAGCCAGGAGAGATGCAGCTGGAATTTGGCCCTTTC TCTGTATCCTGTGAAGCTGA540
AAAAAGGAAATCTGATTATATAATCAGGACTCTAAAAGTT AAGTTCAATAGTGAAACTCG600
AACTATCTACCAGTTTCATTACAAGAATTGGCCAGACCAT GATGTACCTTCATCTATAGA660
CCCTATTCTTGAGCTCATCTGGGATGTACGTTGTTACCAA GAGGATGACAGTGTTCCCAT720
ATGCATTCACTGCAGTGCTGGCTGTGGAAGGACTGGTGTT ATTTGTGCTATTGATTATAC780
ATGGATGTTGCTAAAAGATGGGATAATTCCTGAGAACTTC AGTGTTTTCAGTTTGATCCG840
GGAAATGCGGACACAGAGGCCTTCATTAGTTCAAACGCAG GAACAATATGAACTGGTCTA900
CAATGCTGTATTAGAACTATTTAAGAGACAGATGGATGTT ATCAGAGATAAACATTCTGG960
AACAGAGAGTCAAGCAAAGCATTGTATTCCTGAGAAAAAT CACACTCTCCAAGCAGACTC1020
4 TTATTCTCCTAATTTACCAAAAAGTACCACAAAAGCAGCA AAAATGATGAACCAACAAAG1080
5
GACAAAAATGGAAATCAAAGAATCTTCTTCCTTTGACTTT AGGACTTCTGAAATAAGTGC1140
AAAAGAAGAGCTAGTTTTGCACCCTGCTAAATCAAGCACT TCTTTTGACTTTCTGGAGCT1200
AAATTACAGTTTTGACAAAAATGCTGACACAACCATGAAA TGGCAGACAAAGGCATTTCC1260
AATAGTTGGGGAGCCTCTTCAGAAGCATCAAAGTTTGGAT TTGGGCTCTCTTTTGTTTGA1320
5 GGGATGTTCTAATTCTAAACCTGTAAATGCAGCAGGAAGA TATTTTAATTCAAAGGTGCC1380
O
AATAACACGGACCAAATCAACTCCTTTTGAATTGATACAG CAGAGAGAAACCAAGGAGGT1440
GGACAGCAAGGAAAACTTTTCTTATTTGGAATCTCAACCA CATGATTCTTGTTTTGTAGA1500
GATGCAGGCTCAAAAAGTAATGCATGTTTCTTCAGCAGAA CTGAATTATTCACTGCCATA1560
TGACTCTAAACACCAAATACGTAATGCCTCTAATGTAAAG CACCATGACTCTAGTGCTCT1620
5 TGGTGTATATTCTTACATACCTTTAGTGGAAAATCCTTAT TTTTCATCATGGCCTCCAAG1680
5
TGGTACCAGTTCTAAGATGTCTCTTGATTTACCTGAGAAG CAAGATGGAACTGTTTTTCC1740
TTCTTCTCTGTTGCCAACATCCTCTACATCCCTCTTCTCT TATTACAATTCACATGATTC1800
TTTATCACTGAATTCTCCAACCAATATTTCCTCACTATTG AACCAGGAGTCAGCTGTACT1860
AGCAACTGCTCCAAGGATAGATGATGAAATCCCCCCTCCA CTTCCTGTACGGACACCTGA1920
G ATCATTTATTGTGGTTGAGGAAGCTGGAGAATTCTCACCA AATGTTCCCAAATCCTTATC1980
O
CTCAGCTGTGAAGGTAAAAATTGGAACATCACTGGAATGG GGTGGAACATCTGAACCAAA2040
GAAATTTGATGACTCTGTGATACTTAGACCAAGCAAGAGT GTAAAACTCCGAAGTCCTAA2100
ATCAGAACTACATCAAGATCGTTCTTCTCCCCCACCTCCT CTCCCAGAAAGAACTCTAGA2160
GTCCTTCTTTCTTGCCGATGAAGATTGTATGCAGGCCCAA TCTATAGAAACATATTCTAC2220
6 TAGCTATCCTGACACCATGGAAAATTCAACATCTTCAAAA CAGACACTGAAGACTCCTGG2280
5
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AAAAAGTTTCACAAGGAGTAAGAGTTTGAA 2390
AATTTTGCGA
AACATGAAAA
AGAGTATCTG
TAATTCTTGCCCACCAAACAAGCCTGCAGAATCTGTTCAG TCAAATAACT CCAGCTCATT2400
TCTGAATTTTGGTTTTGCAAACCGTTTTTCAAAACCCAAA GGACCAAGGA ATCCACCACC2960
AACTTGGAATATTTAATAAAACTCCAGATTTATAATAATA TGGGCTGCAA GTACACCTGC2520
AAATAAAACTACTAGAATACTGCTAGTTAAAATAAGTGCT CTATATGCAT AATATCAAAT2580
ATGAAGATATGCTAATGTGTTAATAGCTTTTAAAAGAAAA GCAAAATGCC AATAAGTGCC2640
AGTTTTGCATTTTCATATCATTTGCATTGAGTTGAAAACT GCAAATAAAA GTTTGTCACT2700
TGAGCTTATGTACAGAATGCTATATGAGAAACACTTTTAG AATGGATTTA TTTTTCATTT2760
TTGCCAGTTATTTTTATTTTCTTTTACTTTTTTACATAAA CATAAACTTC AAAAGGTTTG2820
Z TAAGATTTGGATCTCAACTAATTTCTACATTGCCAGAATA TACTATAAAA AGTTAAP~AAA2880
O
AAACTTACTTTGTGGGTTGCAATACAAACTGCTCTTGACA ATGACTATTC CCTGACAGTT2990
ATTTTTGCCTAAATGGAGTATACCTTGTAAATCTTCCCAA ATGTTGTGGA AAACTGGAAT3000
ATTAAGAAAATGAGAAATTATATTTATTAGAATAAAATGT GCAAATAATG ACAATTATTT3060
GAATGTAACAAGGAATTCAACTGAAATCCTGATAAGTTTT AACCAAAGTC ATTAAATTAC3120
1 CAATTCTAGAAAAGTAATCAATGAAATATAATAGCTATCT TTTGGTAGCA AAAGATATAA3180
5
ATTGTATATGTTTATACAGGATCTTTCAGATCATGTGCAA TTTTTATCTA ACCAATCAGA3240
AATACTAGTTTAAAATGAATTTCTATATGAATATGGATCT GCCATAAGAA AATCTAGTTC3300
AACTCTAATTTTATGTAGTAAATAAATTGGCAGGTAATTG TTTTTACAAA GAATCCACCT3360
GACTTCCCCTAATGCATTAAAAATATTTTTATTTAAATAA CTTTATTTAT AACTTTTAGA3420
2 AACATGTAGTATTGTTTAAACATCATTTGTTCTTCAGTAT TTTTCATTTG GAAGTCCAAT3980
O
AGGGCAAATTGAATGAAGTATTATTATCTGTCTCTTGTAG TACAATGTAT CCAACAGACA3540
CTCAATAAACTTTTTGGTTGTTAAAAAAAAAA,AAAAAAAP. 3580
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
3 (A) LENGTH: 1598
O base
pairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS: single
iD) TOPOLOGY: linear
3 {xi) SEQUENCE
5 DESCRIPTION:
SEQ ID
NO: 2:
GCTCGCGGGCTCCCATGGCCCTCGGGCCCAGCGTGGTGACCCCGGGGGATGGAGCCGTTC60
CTCAGGAGGCGGCTGGCCTTCCTGTCCTTCTTCTGGGACAAGATCTGGCCGGCGGGCGGC120
GAGCCGGACCATGGCACCCCCGGGTCCCTGGACCCCAACACTGACCCAGTGCCCACGCTC180
4 CCCGCCGAGCCTTGCAGCCCCTTCCCTCAGCTCTTCCTTGCGCTCTATGACTTCACGGCG290
O
CGGTGTGGCGGGGAGCTGAGTGTCCGCCGCGGGGACAGGCTCTGTGCCCTCGAAGAGGGG300
GGCGGCTACATCTTCGCACGCAGGCTTTCGGGCCAGCCCAGCGCCGGGCTCGTGCCCATC360
ACCCACGTGGCCAAGGCTTCTCCTGAGACGCTCTCAGACCAACCCTGGTACTTTAGCGGG420
GTCAGTCGGACCCAGGCACAGCAGCTGCTCCTCTCCCCACCCAACGAACCAGGGGCCTTC980
4 CTCATCCGGCCCAGCGAGAGCAGCCTCGGGGGCTACTCACTGTCAGTCCGGGCCCAGGCC540
5
AAGGTCTGCCACTACCGGGTCTCCATGGCAGCTGATGGCAGCCTCTACCTGCAGAAGGGA600
CGGCTCTTTCCCGGCCTGGAGGAGCTGCTCACCTACTACAAGGCCAACTGGAAGCTGATC660
CAGAACCCCCTGCTGCAGCCCTGCATGCCCCAGAAGGCCCCGAGGCAGGACGTGTGGGAG720
CGGCCACACTCCGAATTCGCCCTTGGGAGGAAGCTGGGTGAAGGCTACTTTGGGGAGGTG780
5 TGGGAAGGCCTGTGGCTGGGCTCCCTGCCCGTGGCGATCAAGGTCATCAAGTCAGCCAAC840
O
ATGAAGCTCACTGACCTCGCCAAGGAGATCCAGACACTGAAGGGCCTGCGGCACGAGCGG900
CTCATCCGGCTGCACGCAGTGTGCTCGGGCGGGGAGCCTGTGTACATAGTCACGGAACTC960
ATGCGCAAGGGGAACCTGCAGGCCTTCCTGGGCACCCCCGAGGGCCGGGCCCTGCGTCTG1020
CCGCCACTCCTGGGCTTTGCCTGCCAGGTGGCTGAGGGCATGAGCTACCTGGAGGAGCAG1080
5 CGCGTTGTGCACCGGGACTTGGCCGCCCGGAACGTGCTCGTGGACGACGGCCTGGCCTGC1190
5
AAGGTGGCTGACTTCGGCCTGGCCCGGCTGCTCAAGGACGACATCTACTCCCCGAGCAGC1200
AGCTCCAAGATCCCGGTCAAGTGGACAGCGCCTGAGGCGGCCAATTATCGTGTCTTCTCC1260
CAGAAGTCAGACGTCTGGTCCTTCGGCGTCCTGCTGCACGAGGTTTTCACCTATGGCCAG1320
TGTCCCTATGAAGGGATGACCAACCACGAGACGCTGCAGCAGATCATGCGAGGGTACCGG1380
G CTGCCGCGCCCGGCTGCCTGCCCGGCGGAGGTCTACGTGCTCATGCTGGAGTGCTGGAGG1940
O
AGCAGCCCCGAGGAACGGCCCTCCTTTGCCACGCTGCGGGAGAAGCTGCACGCCATCCAC1500
AGATGCCACCCCTGAGTCCTCACGTGACCCAACGCTCTGGGCTCCAGC 1548
t _~_-_
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(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1785 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
1 O (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGTTATGTCTGACTCACTGCACTGGAGTTTGGCAAAAGCATCTCAGAAGTGGTTGTGCTT60
1 TTTTGAATGAAATGATCAATGGAGTGCTCCAGTTGTATGCTGGCCTCTGGATACTAACTA120
5
GACCTGCCTGACTCCAGGAACTAAGGCTCAGTATCTGCAGAAGCTTTTTGCCCATCTCAT180
TCCGGCTATGGGGACAACATGTCTTCACCCAGGAAGGTTAGAGGAAAAACTGGAAGAGAT290
AATGATGAAGAGGAGGGTAATTCAGGTAACCTGAATCTCCGCAACTCTTTGCCTTCATCG300
AGTCAGAAAATGACGCCTACGAAGCCGATTTTTGGGAATAAAATGAATTCAGAGAATGTA360
2 AAACCCTCCCATCACCTGTCATTCTCAGATAAGTATGAGCTTGTTTACCCAGAGCCTTTG920
O
GAAAGTGACACTGATGAGACTGTGTGGGATGTCAGTGACCGGTCTCTCAGAAACAGGTGG9$0
AACAGTATGGATTCAGAGACTGCAGGGCCGTCAAAGACTGTCTCCCCAGTGCTTTCTGGT540
AGTAGTAGGCTCTCAAAGGACACTGAAACATCTGTCTCTGAAAAGGAGCTAACTCAGTTG600
GCTCAGATTCGACCATTAATATTCAACAGTTCTGCACGGTCTGCTATGCGGGATTGTTTG660
2 AACACGCTTCAGAAAAAAGAAGAACTTGATATCATCCGTGAGTTTTTGGAGTTAGAACAA720
5
ATGACTCTGCCTGATGACTTCAATTCTGGGAATACACTACAGAACAGAGATAAGAACAGA780
TACCGAGATATTCTTCCATATGATTCAACACGTGTTCCTCTTGGAAAAAACAAGGACTAC890
ATCAACGCTAGTTATATTAGAATAGTAAATCATGAAGAAGAGTATTTTTATATTGCCACT900
CAAGGACCATTGCCAGAAACTATAGAAGACTTTTGGCAAATGGTTCTGGAAAATAATTGT960
3 AATGTTATTGCTATGATAACCAGAGAGATAGAATGTGGAGTTATCAAGTGTTACAGTTAC1020
O
TGGCCCATTTCTCTGAAGGAGCCTTTGGAATTCGAACACTTTAGTGTCTTTCTGGAGACC1080
TTTCATGTAACTCAATATTTCACCGTTCGAGTATTTCAGATTGTGAAGAAGTCCACAGGA1190
AAGAGCCAATGTGTAAAACACTTGCAGTTCACCAAGTGGCCAGACCATGGCACTCCTGCC1200
TCAGCAGATTTTTTCATAAAATATGTCCGTTATGTGAGGAAGAGCCACATTACAGGACCC1260
3 CTCCTTGTTCACTGCAGTGCTGGTGTAGGCCGAACAGGGGTGTTCATATGTGTGGATGTT1320
5
GTGTTCTCTGCCATCGAGAAGAACTACTCTTTTGACATTATGAACATAGTGACCCAGATG1380
AGAAAGCAGCGCTGTGGCATGATTCAAACCAAGGAGCAGTACCAGTTTTGTTATGAAATT1990
GTGCTTGAAGTTCTTCAGAACCTTCTGGCTTTGTATTAAGAGAGACTTCTGCGCCTGTCC1500
CTCGAGGTTACCGAGCAGCTTGGAGCCTGAGCCGTGCTGAAGCGTCTGCGGGCCGTGCAG1560
4 TCTGCCTTCTGATTTTTCTCTCTGAAAGTCCCTGAAGGTAGCACTACTGGGCACAGAGTG1620
O
AACTGTTTCCACTTGATCTTTCTGAACAAGAGCAAAATACCCTCCATGCCTTCTACGGAA1680
ACGGAAGTTGCATGAAACAACCTCCGCTTGGCTGTCTGGTTTGTGGTATTACAGAGCTTA1790
ATAAAAGACTTAGATGTGAAF~F~F1AAAAAAAAAAAAAAAAAAAAAA 1785
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
5O
(A) LENGTH: 1896 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
GGTTATGTCT GACTCACTGC ACTGGAGTTT GGCAAAAGCA TCTCAGAAGT GGTTGTGCTT 60
TTTTGAATGA AATGATCAAT GGAGTGCTCC AGTTGTATGC TGGCCTCTGG ATACTAACTA 120
6 O GACCTGCCTG ACTCCAGGAA CTAAGGCTCA GTATCTGCAG AAGCTTTTTG CCCATCTCAT 180
TCCGGCTATG GGGACAACAT GTCTTCACCC AGGAAGGTTA GAGGAAAAAC TGGAAGAGAT 240
AATGATGAAG AGGAGGGTAA TTCAGGTAAC CTGAATCTCC GCAACTCTTT GCCTTCATCG 300
AGTCAGAAAA TGACGCCTAC GAAGCCGGTA CAAAATAAAA ATCTCATGAA GTATGAAGAA 360
CACTTAGATA TATTGATGGT GTTTTTATTG ATAAAAACCA TATGGTATAA TGTCTTCAAA 420
TTATGGAAAG GCAAGCTTAT TTTTGGGAAT AAAATGAATT CAGAGAATGT AAAACCCTCC 480
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CATCACCTGTCATTCTCAGATAAGTATGAGCTTGTTTACCCAGAGCCTTTGGAAAGTGAC540
ACTGATGAGACTGTGTGGGATGTCAGTGACCGGTCTCTCAGAAACAGGTGGAACAGTATG600
GATTCAGAGACTGCAGGGCCGTCAAAGACTGTCTCCCCAGTGCTTTCTGGTAGTAGTAGG660
CTCTCAAAGGACACTGAAACATCTGTCTCTGAAAAGGAGCTAACTCAGTTGGCTCAGATT720
S CGACCATTAATATTCAACAGTTCTGCACGGTCTGCTATGCGGGATTGTTTGAACACGCTT780
CAGAAAAAAGAAGAACTTGATATCATCCGTGAGTTTTTGGAGTTAGAACAAATGACTCTG840
CCTGATGACTTCAATTCTGGGAATACACTACAGAACAGAGATAAGAACAGATACCGAGAT900
ATTCTTCCATATGATTCAACACGTGTTCCTCTTGGAAAAAACAAGGACTACATCAACGCT960
AGTTATATTAGAATAGTAAATCATGAAGAAGAGTATTTTTATATTGCCACTCAAGGACCA1020
1 TTGCCAGAAACTATAGAAGACTTTTGGCAAATGGTTCTGGAAAATAATTGTAATGTTATT1080
O
GCTATGATAACCAGAGAGATAGAATGTGGAGTTATCAAGTGTTACAGTTACTGGCCCATT1140
TCTCTGAAGGAGCCTTTGGAATTCGAACACTTTAGTGTCTTTCTGGAGACCTTTCATGTA1200
ACTCAATATTTCACCGTTCGAGTATTTCAGATTGTGAAGAAGTCCACAGGAAAGAGCCAA1260
TGTGTAAAACACTTGCAGTTCACCAAGTGGCCAGACCATGGCACTCCTGCCTCAGCAGAT1320
I TTTTTCATAAAATATGTCCGTTATGTGAGGAAGAGCCACATTACAGGACCCCTCCTTGTT1380
CACTGCAGTGCTGGTGTAGGCCGAACAGGGGTGTTCATATGTGTGGATGTTGTGTTCTCT1490
GCCATCGAGAAGAACTACTCTTTTGACATTATGAACATAGTGACCCAGATGAGAAAGCAG1500
CGCTGTGGCATGATTCAAACCAAGGAGCAGTACCAGTTTTGTTATGAAATTGTGCTTGAA1560
GTTCTTCAGAACCTTCTGGCTTTGTATTAAGAGAGACTTCTGCGCCTGTCCCTCGAGGTT1620
2 ACCGAGCAGCTTGGAGCCTGAGCCGTGCTGAAGCGTCTGCGGGCCGTGCAGTCTGCCTTC1680
O
TGATTTTTCTCTCTGAAAGTCCCTGAAGGTAGCACTACTGGGCACAGAGTGAACTGTTTC1740
CACTTGATCTTTCTGAACAAGAGCAAAATACCCTCCATGCCTTCTACGGAAACGGAAGTT1800
GCATGAAACAACCTCCGCTTGGCTGTCTGGTTTGTGGTATTACAGAGCTTAATAAAAGAC1860
TTAGATGTGAP~AAAAAAAAAF~~AAAAAAAAAAAAAA i
8
9
6
25
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1692 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
GGTTATGTCTGACTCACTGCACTGGAGTTTGGCAAAAGCATCTCAGAAGTGGTTGTGCTT60
TTTTGAATGAAATGATCAATGGAGTGCTCCAGTTGTATGCTGGCCTCTGGATACTAACTA120
4 GACCTGCCTGACTCCAGGAACTAAGGCTCAGTATCTGCAGAAGCTTTTTGCCCATCTCAT180
O
TCCGGCTATGGGGACAACATGTCTTCACCCAGGAAGGTTAGAGGAAAAACTGGAAGAGAT290
AATGATGAAGAGGAGGGTAATTCAGGTAACCTGAATCTCCGCAACTCTTTGCCTTCATCG300
AGTCAGAAAATGACGCCTACGAAGCCGATTTTTGGGAATAAAATGAATTCAGAGAATGTA360
AAACCCTCCCATCACCTGTCATTCTCAGATAAGTATGAGCTTGTTTACCCAGAGCCTTTG420
4 GAAAGTGACACTGATGAGACTGTGTGGGATGTCAGTGACCGGTCTCTCAGAAACAGGTGG480
5
AACAGTATGGATTCAGAGACTGCAGGGCCGTCAAAGACTGTCTCCCCAGTGCTTTCTGGT540
AGTAGTAGGCTCTCAAAGGACACTGAAACATCTGTCTCTGAAAAGGAGCTAACTCAGTTG600
GCTCAGATTCGACCATTAATATTCAACAGTTCTGCACGGTCTGCTATGCGGGATTGTTTG660
AACACGCTTCAGAAAAAAGAAGAACTTGATATCATCCGTGAGTTTTTGGAGTTAGAACAA720
S ATGACTCTGCCTGATGACTTCAATTCTGGGAATACACTACAGAACAGAGATAAGAACAGA780
O
TACCGAGATATTCTTCCATATGATTCAACACGTGTTCCTCTTGGAAAAAACAAGGACTAC840
ATCAACGCTAGTTATATTAGAATAGTAAATCATGAAGAAGAGTATTTTTATATTGCCACT900
CAAGGACCATTGCCAGAAACTATAGAAGACTTTTGGCAAATGGTTCTGGAAAATAATTGT960
AATGTTATTGCTATGATAACCAGAGAGATAGAATGTGGAGTTATCAAGTGTTACAGTTAC1020
5 TGGCCCATTTCTCTGAAGGAGCCTTTGGAATTCGAACACTTTAGTGTCTTTCTGGAGACC1080
5
TTTCATGTAACTCAATATTTCACCGTTCGAGTATTTCAGATTGTGAAGAAGTCCACAGGA1190
AAGAGCCAATGTGTAAAACACTTGCAGTTCACCAAGTGGCCAGACCATGGCACTCCTGCC1200
TCAGCAGATTTTTTCATAAAATATGTCCGTTATGTGAGGAAGAGCCACATTACAGGACCC1260
CTCCTTGTTCACTGCAGTGCTGGTGTAGGCCGAACAGGGGTGTTCATATGTGTGGATGTT1320
GO GTGTTCTCTGCCATCGAGAAGAACTACTCTTTTGACATTATGAACATAGTGACCCAGATG1380
AGAAAGCAGCGCTGTGGCATGATTCAAACCAAGGTTACCGAGCAGCTTGGAGCCTGAGCC1440
GTGCTGAAGCGTCTGCGGGCCGTGCAGTCTGCCTTCTGATTTTTCTCTCTGAAAGTCCCT1500
GAAGGTAGCACTACTGGGCACAGAGTGAACTGTTTCCACTTGATCTTTCTGAACAAGAGC1560
AAAATACCCTCCATGCCTTCTACGGAAACGGAAGTTGCATGAAACAACCTCCGCTTGGCT1620
65 GTCTGGTTTGTGGTATTACAGAGCTTAATAAAAGACTTAGATGTGAAAAAAAAAAAAAAA1680
_T _.._ _
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AAAAAAAAAA AA 1692
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 320 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
1 O (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAAAATAATT GTAATGTTAT TGCTATGATA ACCAGAGAGA TAGAAGGTGG AGTTATCAAG 60
1 5 TGTTGCAGTT ACTGGCCCGT TTCTCTGAAG GAGCCTTTGG AATTCAAACA CTTTCATGTC 120
CTTCTGGAGA ACTTTCAGAT AACTCAGTAT TTTGTCATCC GAATATTTCA AATTGTGAAG 180
AAGTCCACAG GAAAGAGTCA CTCTGTAAAA CACTTGCAGT TCATCAAATG GCCAGACCAT 240
GGCACTCCTG CCTCAGTAGA TTTTTTCATC AAATATGTCC GTTATGTGAG GAAGAGCCAC 300
ATTACAGGAC CCCTCCTTGT 320
(2) INFORMATION FOR SEQ ID N0: 7:
2 ~J (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9456 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
GGCACGAGAGGAGCAGCAGAAGTTCGGGGAGCGGGTTGCATACTTCCAGAGCGCCCTGGA60
3 CAAGCTCAATGAAGCCATCAAGTTGGCCAAGGGCCAGCCTGACACTGTGCAAGACGCGCT120
5
TCGCTTCACTATGGATGTCATTGGGGGAAAGTACAATTCTGCCAAGAAGGACAACGACTT180
CATTTACCATGAGGCTGTCCCAGCATTGACACCCTTCAGCCTGTAAAAGGAGCCCCCTTG240
GTGAAGCCCTTGCCAGTGAACCCCACAGACCCAGCTGTTACAGGCCCTGACATCTTTGCC300
AAACTGGTACCCATGGCTGCCCACGAGGCCTCGTCACTGTACAGTGAGGAGAAGGCCAAG360
9 CTGCTCCGGGAGATGATGGCCAAGATTGAGGACAAGAATGAGGTCCTGGACCAGTTCATG420
O
GATTCAATGCAGTTGGATCCCGAGACGGTGGACAACCTTGATGCCTACAGCCACATCCCA480
CCCCAGCTCATGGAGAAGTGCGCGGCTCTCAGCGTCCGGCCCGACACTGTCAGGAACCTT540
GTACAGTCCATGCAAGTGCTGTCAGGTGTGTTCACGGATGTGGAGGCTTCCCTGAAGGAC600
ATCAGAGATCTGTTGGAGGAGGATGAGCTGCTAGAGCAGAAGTTTCAGGAGGCGGTGGGC660
9 CAGGCAGGGGCCATCTCCATCACCTCCAAGGCTGAGCTGGCAGAGGTGAGGCGAGAATGG720
5
GCCAAGTACATGGAAGTCCATGAGAAGGCCTCCTTCACCAACAGTGAGCTGCACCGTGCC780
ATGAACCTGCACGTCGGCAACCTGCGCCTGCTCAGCGGGCCGCTTGACCAGGTCCGGGCT890
GCCCTGCCCACACCGGCCCTCTCCCCAGAGGACAAGGCCGTGCTGCAAAACCTAAAGCGC900
ATCCTGGCTAAGGTGCAGGAGATGCGGGACCAGCGCGTGTCCCTGGAGCAGCAGCTGCGT960
S GAGCTTATCCAGAAAGATGACATCACTGCCTCGCTGGTCACCACAGACCACTCAGAGATG1020
O
AAGAAGTTGTTCGAGGAGCAGCTGAAAAAGTATGACCAGCTGAAGGTGTACCTGGAGCAG1080
AACCTGGCCGCCCAGGACCGTGTCCTCTGTGCACTGACAGAGGCCAACGTGCAGTACGCA1190
GCCGTGCGGCGGGTACTCAGCGACTTGGACCAAAAGTGGAACTCCACGCTGCAGACCCTG1200
GTGGCCTCGTATGAAGCCTATGAGGACCTGATGAAGAAGTCGCAGGAGGGCAGGGACTTC1260
5 TACGCAGATCTGGAGAGCAAGGTGGCTGCTCTGCTGGAGCGCACGCAGTCCACCTGCCAG1320
5
GCCCGCGAGGCTGCCCGCCAGCAGCTCCTGGACAGGGAGCTGAAGAAGAAGCCGCCGCCA1380
CGGCCCACAGCCCCAAAGCCGCTGCTGCCCCGCAGGGAGGAGAGTGAGGCAGTGGAAGCA1940
GGAGACCCCCCTGAGGAGCTGCGCAGCCTCCCCCCTGACATGGTGGCTGGCCCACGACTG1500
CCTGACACCTTCCTGGGAAGTGCCACCCCGCTCCACTTTCCTCCCAGCCCCTTCCCCAGC1560
60 TCCACAGGCCCAGGACCCCACTATCTCTCAGGCCCCTTGCCCCCTGGTACCTACTCGGGC1620
CCCACCCAGCTGATACAGCCCAGGGCCCCAGGGCCCCATGCAATGCCCGTAGCACCTGGG1680
CCTGCCCTCTACCCAGCCCCTGCCTACACACCGGAGCTGGGCCTTGTGCCCCGATCCTCC1740
CCACAGCATGGCGTGGTGAGCAGTCCCTATGTGGGGGTAGGGCCGGCCCCACCAGTTGCA1800
GGTCTCCCCTCGGCCCCACCTCCTCAATTCTCAGGCCCCGAGTTGGCCATGGCGGTTCGG1860
65 CCAGCCACCACCACAGTAGATAGCATCCAGGCGCCCATCCCCAGCCACACAGCCCCACGG1920
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CCAAACCCCACCCCTGCTCCTCCCCCGCCCTGCTTCCCTGTGCCCCCACCGCAGCCACTG1980
CCCACGCCTTACACCTACCCTGCAGGGGCTAAGCAACCCATCCCAGCACAGCACCACTTC2040
TCTTCTGGGATCCCCACAGGTTTTCCAGCCCCAAGGATTGGGCCCCAGCCCCAGCCCCAT2100
CCTCAGCCCCATCCTTCACAAGCGTTTGGGCCTCAGCCCCCACAGCAGCCCCTTCCACTC2160
S CAGCATCCACATCTCTTCCCACCCCAGGCCCCAGGACTCCTACCCCCACAATCCCCCTAC2220
CCCTATGCCCCTCAGCCTGGGGTCCTGGGGCAGCCGCCACCCCCCCTACACACCCAGCTC2280
TACCCAGGTCCCGCTCAAGACCCTCTGCCAGCCCACTCAGGGGCTCTGCCTTTCCCCAGC2390
CCTGGGCCCCCTCAGCCTCCCCATCCCCCACTGGCATATGGTCCTGCCCCTTCTACCAGA2400
CCCATGGGCCCCCAGGCAGCCCCTCTTACCATTCGAGGGCCCTCGTCTGCTGGCCAGTCC2460
Z ACCCCTAGTCCCCACCTGGTGCCTTCACCTGCCCCATCTCCAGGGCCTGGTCCGGTACCC2520
O
CCTCGCCCCCCAGCAGCAGAACCACCCCCTTGCCTGCGCCGAGGCGCCGCAGCTGCAGAC2580
CTGCTCTCCTCCAGCCCGGAGAGCCAGCATGGCGGCACTCAGTCTCCTGGGGGTGGGCAG2640
CCCCTGCTGCAGCCCACCAAGGTGGATGCAGCTGAGGGTCGTCGGCCGCAGGCCCTGCGG2700
CTGATTGAGCGGGACCCCTATGAGCATCCTGAGAGGCTGCGGCAGTTGCAGCAGGAGCTG2760
1 GAGGCCTTTCGGGGTCAGCTGGGGGATGTGGGAGCTCTGGACACTGTCTGGCGAGAGCTG2820
S
CAAGATGCGCAGGAACATGATGCCCGAGGCCGTTCCATCGCCATTGCCCGCTGCTACTCA2880
CTGAAGAACCGGCACCAGGATGTCATGCCCTATGACAGTAACCGTGTGGTGCTGCGCTCA2990
GGCAAGGATGACTACATCAATGCCAGCTGCGTGGAGGGGCTCTCCCCATACTGCCCCCCG3000
CTAGTGGCAACCCAGGCCCCACTGCCTGGCACAGCTGCTGACTTCTGGCTCATGGTCCAT3060
2 GAGCAGAAAGTGTCAGTCATTGTCATGCTGGTTTCTGAGGCTGAGATGGAGAAGCAAAAA3120
O
GTGGCACGCTACTTCCCCACCGAGAGGGGCCAGCCCATGGTGCACGGTGCCCTGAGCCTG3180
GCATTGAGCAGCGTCCGCAGCACCGAAACCCATGTGGAGCGCGTGCTGAGCCTGCAGTTC3240
CGAGACCAGAGCCTCAAGCGCTCTCTTGTGCACCTGCACTTCCCCACTTGGCCTGAGTTA3300
GGCCTGCCCGACAGCCCCAGCAACTTGCTGCGCTTCATCCAGGAGGTGCACGCACATTAC3360
2 CTGCATCAGCGGCCGCTGCACACGCCCATCATTGTGCACTGCAGCTCTGGTGTGGGCCGC3420
S
ACGGGAGCCTTTGCACTGCTCTATGCAGCTGTGCAGGAGGTGGAGGCTGGGAACGGAATC3980
CCTGAGCTGCCTCAGCTGGTGCGGCGCATGCGGCAGCAGAGAAAGCACATGCTGCAGGAG3590
AAGCTGCACCTCAGGTTCTGCTATGAGGCAGTGGTGAGACACGTGGAGCAGGTCCTGCAG3600
CGCCATGGTGTGCCTCCTCCATGCAAACCCTTGGCCAGTGCAAGCATCAGCCAGAAGAAC3660
3 CACCTTCCTCAGGACTCCCAGGACCTGGTCCTCGGTGGGGATGTGCCCATCAGCTCCATC3720
O
CAGGCCACCATTGCCAAGCTCAGCATTCGGCCTCCTGGGGGGTTGGAGTCCCCGGTTGCC3780
AGCTTGCCAGGCCCTGCAGAGCCCCCAGGCCTCCCGCCAGCCAGCCTCCCAGAGTCTACC3890
CCAATCCCATCTTCCTCCCCACCCCCCCTTTCCTCCCCACTACCTGAGGCTCCCCAGCCT3900
AAGGAGGAGCCGCCAGTGCCTGAAGCCCCCAGCTCGGGGCCCCCCTCCTCCTCCCTGGAA3960
3 TTGCTGGCCTCCTTGACCCCAGAGGCCTTCTCCCTGGACAGCTCCCTGCGGGGCAAACAG9020
CGGATGAGCAAGCATAACTTTCTGCAGGCCCATAACGGGCAAGGGCTGCGGGCCACCCGG9080
CCCTCTGACGACCCCCTCAGCCTTCTGGATCCACTCTGGACACTCAACAAGACCTGAACA9140
GGTTTTGCCTACCTGGTCCTTACACTACATCATCATCATCTCATGCCCACCTGCCCACAC9200
CCAGCAGAGCTTCTCAGTGGGCACAGTCTCTTACTCCCATTTCTGCTGCCTTTGGCCCTG4260
4 CCTGGCCCAGCCTGCACCCCTGTGGGGTGGAAATGTACTGCAGGCTCTGGGTCAGGTTCT4320
O
GCTCCTTTATGGGACCCGACATTTTTCAGCTCTTTGCTATTGAAATAATAAACCACCCTG4380
TTCTGTGAAAP~P.AAAAAAAAAAAAAAAAAAAAAAAAAAAAF~~AAAAAAAAAAAAAAAAAA4490
AAAAAAP.AAAAAAAAA 9456
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1793 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CGGCCACACT GACTAGAGCC AACCGCGCACTTCAAAAGGGTGTCGGTGCCGCGCTCCCCT60
CCCGCGGCCC GGGAACTTCA AAGCGGGCCGTGCTGCCCCGGCTGCCTCGCTCTGCTCTGG120
C O GGCCTCGCAGCCCCGGCGCG GCCGCCTGGTGGCGATGACCCGGGCGCTCTGCTCAGCGCT180
CCGCCAGGCT CTCCTGCTGC TCGCAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGT240
ATGTCTTTTG TGTGATTCTT CAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATC300
AGTCATGCTA ACCAATGGAA AAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACT360
GAATGCTCAA GTCTTCTGTC ATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCAC420
C S AGATTTTTGCAACAACATAA CACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACT980
_ . ~ _ _._ _ i
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TGGACCCATGGAGCTGGCCATCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGC540
GATGCTGACAGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACC600
AAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCTGAAAGA660
TCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTACCTCTGTTGGTTCAAAG720
GACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGT780
GTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGA890
AAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACAT900
CCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGT960
ATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGT1020
I GGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGA1080
O
GATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATAT1190
CTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGA1200
TTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATAT1260
GGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGC1320
1 TGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGG1380
5
AATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGA1440
GGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCA1500
AAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGG1560
AGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGA1620
2 AGACTGCAAAGCCTAATGATGATAATTATGTTAAAAAGAAATCTCTCATAGCTTTCTTTT1680
O
CCATTTTCCCCTTTATGTGAATGTTTTTGCCATTTTTTTTTTGTTCTACCTCAAAGATAA1790
GACAGTACAGTATTTAAGTGCCCATAAGGCAGCATGAAAAGATAACTCTAAAG 1793
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
3 O (A) LENGTH: 807 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
3 5 (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met Asp Gln Arg Glu Ile Leu Gln Lys Phe Leu Asp Glu Ala Gln Ser
4O 1 5 10 15
Lys Lys Ile Thr Lys Glu Glu Phe Ala Asn Glu Phe Leu Lys Leu Lys
20 25 30
4 5 Arg Gln Ser Thr Lys Tyr Lys Ala Asp Lys Thr Tyr Pro Thr Thr Val
40 45
Ala Glu Lys Pro Lys Asn Ile Lys Lys Asn Arg Tyr Lys Asp Ile Leu
50 55 60
5O
Pro Tyr Asp Tyr Ser Arg Val Glu Leu Ser Leu Ile Thr Ser Asp Glu
65 70 75 80
Asp Ser Ser Tyr Ile Asn Ala Asn Phe Ile Lys Gly Val Tyr Gly Pro
55 85 90 95
Lys Ala Tyr Ile Ala Thr Gln Gly Pro Leu Ser Thr Thr Leu Leu Asp
100 105 110
6 O Phe Trp Arg Met Ile Trp Glu Tyr Ser Val Leu Ile Ile Val Met Ala
115 120 125
Cys Met Glu Tyr Glu Met Gly Lys Lys Lys Cys Glu Arg Tyr Trp Ala
130 135 140
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Glu ProGlyGluMetGlnLeuGluPheGlyProPheSerValSerCys
195 150 155 160
Glu AlaGluLysArgLysSerAspTyrIleIleArgThrLeuLysVal.
165 170 175
Lys PheAsnSerGluThrArgThrIleTyrGlnPheHisTyrLysAsn
180 185 190
1 Trp ProAspHisAspValProSerSerIleAspProIleLeuGluLeu
0
195 200 205
Ile TrpAspValArgCysTyrGlnGluAspAspSerValProIleCys
210 215 220
Ile HisCysSerAlaGlyCysGlyArgThrGlyValIleCysAlaIle
225 230 235 240
Asp TyrThrTrpMetLeuLeuLysAspGlyIleIleProGluAsnPhe
295 250 255
Ser ValPheSerLeuIleArgGluMetArgThrGlnArgProSerLeu
260 265 270
2 Val GlnThrGlnGluGlnTyrGluLeuValTyrAsnAlaValLeuGlu
5
275 280 285
Leu PheLysArgGlnMetAspValIleArgAspLysHisSerGlyThr
290 295 300
Glu SerGlnAlaLysHisCysIleProGluLysAsnHisThrLeuGln
305 310 315 320
Ala AspSerTyrSerProAsnLeuProLysSerThrThrLysAlaAla
325 330 335
Lys MetMetAsnGlnGlnArgThrLysMetGluIleLysGluSerSer
340 395 350
4 Ser PheAspPheArgThrSerGluIleSerAlaLysGluGluLeuVal
0
355 360 365
Leu HisProAlaLysSerSerThrSerPheAspPheLeuGluLeuAsn
370 375 380
Tyr SerPheAspLysAsnAlaAspThrThrMetLysTrpGlnThrLys
385 390 395 900
Ala PheProIleValGlyGluProLeuGlnLysHisGlnSerLeuAsp
405 410 415
Leu GlySerLeuLeuPheGluGlyCysSerAsnSerLysProValAsn
420 425 430
5 Ala AlaGlyArgTyrPheAsnSerLysValProIleThrArgThrLys
5
435 490 495
Ser ThrProPheGluLeuIleGlnGlnArgGluThrLysGluValAsp
950 455 460
Ser LysGluAsnPheSerTyrLeuGluSerGlnProHisAspSerCys
465 970 475 480
Phe ValGluMetGlnAlaGlnLysValMetHisValSerSerAlaGlu
485 990 495
__-~ __-___ .
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Leu Asn Tyr Ser Leu Pro Tyr Asp Ser Lys His Gln Ile Arg Asn Ala
500 505 510
Ser AsnVal LysHisHisAspSerSerAlaLeuGlyValTyrSerTyr
515 520 525
Ile ProLeu ValGluAsnProTyrPheSerSerTrpProProSerGly
530 535 590
a
Thr SerSer LysMetSerLeuAspLeuProGluLysGlnAspGlyThr
545 550 555 560
- Val PhePro SerSerLeuLeuProThrSerSerThrSerLeuPheSer
565 570 575
Tyr TyrAsn SerHisAspSerLeuSerLeuAsnSerProThrAsnIle
580 585 590
2 Ser SerLeu LeuAsnGlnGluSerAlaValLeuAlaThrAlaPro.Arg
~
595 600 605
Ile AspAsp GluIleProProProLeuProValArgThrProGluSer
610 615 620
Phe IleVal ValGluGluAlaGlyGluPheSerProAsnValProLys
625 630 635 640
Ser LeuSer SerAlaValLysValLysIleGlyThrSerLeuGluTrp
645 650 655
Gly GlyThr SerGluProLysLysPheAspAspSerValIleLeuArg
660 665 670
3 Pro SerLys SerValLysLeuArgSerProLysSerGluLeuHisGln
5
675 680 685
Asp ArgSer SerProProProProLeuProGluArgThrLeuGluSer
690 695 700
Phe PheLeu AlaAspGluAspCysMetGlnAlaGlnSerIleGluThr
705 710 715 720
Tyr SerThr SerTyrProAspThrMetGluAsnSerThrSerSerLys
725 730 735
Gln ThrLeu LysThrProGiyLysSerPheThrArgSerLysSerLeu
740 795 750
5 Lys IleLeu ArgAsnMetLysLysSerIleCysAsnSerCysProPro
~
755 760 765
Asn LysPro AlaGluSerValGlnSerAsnAsnSerSerSerPheLeu
770 775 780
Asn PheGly PheAlaAsnArgPheSerLysProLysGlyProArgAsn
785 790 795 800
Pro ProPro ThrTrpAsnIle
eo5
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(2) INFORMATION
FOR
SEQ
ID NO:
10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 488 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
1 (D) TOPOLOGY: linear
~
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:ID NO: 10:
SEQ
1 Met Glu Pro Phe Leu Arg Arg
5 Arg Leu Ala Phe
Leu Ser Phe Phe
Trp
1 5 10 15
Asp Lys Ile Trp Pro Ala Gly Gly Glu Pro Asp His Gly Thr Pro Gly
20 25 30
20
Ser LeuAspProAsnThrAspProValProThrLeuProAlaGluPro
35 90 95
Cys SerProPheProGlnLeuPheLeuAlaLeuTyrAspPheThrAla
25 50 55 60
Arg CysGlyGlyGluLeuSerValArgArgGlyAspArgLeuCysAla
65 70 75 80
3 Leu GluGluGlyGlyGlyTyrIlePheAlaArgArgLeuSerGlyGln
0
85 90 95
Pro SerAlaGlyLeuValProIleThrHisValAlaLysAlaSerPro
100 105 110
35
Glu ThrLeuSerAspGlnProTrpTyrPheSerGlyValSerArgThr
115 120 125
Gln AlaGlnGlnLeuLeuLeuSerProProAsnGluProGlyAlaPhe
40 130 135 140
Leu IleArgProSerGluSerSerLeuGlyGlyTyrSerLeuSerVal
145 150 155 160
4 Arg AlaGlnAlaLysValCysHisTyrArgValSerMetAlaAlaAsp
5
165 170 175
Gly SerLeuTyrLeuGlnLysGlyArgLeuPheProGlyLeuGluGlu
180 185 190
50
Leu LeuThrTyrTyrLysAlaAsnTrpLysLeuIleGlnAsnProLeu
195 200 205
Leu GlnProCysMetProGlnLysAlaProArgGlnAspValTrpGlu
55 210 215 220
Arg ProHisSerGluPheAlaLeuGlyArgLysLeuGlyGluGlyTyr
225 230 235 240
Phe GlyGluValTrpGluGlyLeuTrpLeuGlySerLeuProValAla
245 250 255
Ile LysValIleLysSerAlaAsnMetLysLeuThrAspLeuAiaLys
260 265 270
65
_.___-~_ __ __ _._ _. _. ~
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Glu Ile Gln Thr Leu Lys Gly Leu Arg His Glu Arg Leu Ile Arg Leu
275 280 285
His AlaValCysSerGlyGlyGluProValTyrIleValThrGluLeu
290 295 300
Met ArgLysGlyAsnLeuGlnAlaPheLeuGlyThrProGluGlyArg
305 310 315 320
Ala LeuArgLeuProProLeuLeuGlyPheAlaCysGlnValAlaGlu
325 330 335
Gly MetSerTyrLeuGluGluGlnArgValValHisArgAspLeuAla
340 395 350
Ala ArgAsnValLeuValAspAspGlyLeuAlaCysLysValAlaAsp
355 360 365
2 Phe GlyLeuAlaArgLeuLeuLysAspAspIleTyrSerProSerSer
0
370 375 380
Ser SerLysIleProValLysTrpThrAlaProGluAlaAlaAsnTyr
385 390 395 900
Arg ValPheSerGlnLysSerAspValTrpSerPheGlyValLeuLeu
405 410 415
His GluValPheThrTyrGlyGlnCysProTyrGluGlyMetThrAsn
420 425 430
His Glu Thr Leu Gln Gln Ile Met Arg Gly Tyr Arg Leu Pro Arg Pro
435 440 495
3 5 Ala Ala Cys Pro Ala Glu Val Tyr Val Leu Met Leu Glu Cys Trp Arg
450 455 960
Ser Ser Pro Glu Glu Arg Pro Ser Phe Ala Thr Leu Arg Glu Lys Leu
465 970 475 480
His Ala Ile His Arg Cys His Pro
485
4 (2) INFORMATION 11:
5 FOR
SEQ
ID
NO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 926 amino
acids
5 (B) TYPE: amino acid
0
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
55
(xi)SEQUENCE DESCRIPTION:SEQ ID NO:
11:
Met Ser Ser Pro Arg Lys Gly Lys Thr ArgAsp Asn
Val Arg Gly Asp
1 5 10 15
60
Glu Glu Glu Gly Asn Ser Leu Asn Leu AsnSer Leu
Gly Asn Arg Pro
20 25 30
Ser Ser Ser Gln Lys Met Thr Lys Pro PheGly Asn
Thr Pro Ile Lys
65 35 40 45
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Met Asn SerGluAsnValLysProSerHisHisLeuSerPheSer Asp
50 55 60
Lys Tyr GluLeuValTyrProGluProLeuGluSerAspThrAsp Glu
65 70 75 BO
Thr Val TrpAspValSerAspArgSerLeuArgAsnArgTrpAsn Ser
85 90 95
Met Asp SerGluThrAlaGlyProSerLysThrValSerProVal Leu
100 105 110
Ser Gly SerSerArgLeuSerLysAspThrGluThrSerValSer Glu
115 120 125
Lys Glu LeuThrGlnLeuAlaGlnIleArgProLeuIlePheAsn Ser
130 135 140
2 Ser Ala ArgSerAlaMetArgAspCysLeuAsnThrLeuGlnLys Lys
0
145 150 155 160
Glu Glu LeuAspIleIleArgGluPheLeuGluLeuGluGlnMet Thr
165 170 175
Leu Pro AspAspPheAsnSerGlyAsnThrLeuGlnAsnArgAsp Lys
180 185 190
Asn Arg TyrArgAspIleLeuProTyrAspSerThrArgValPro Leu
195 200 205
Gly Lys AsnLysAspTyrIleAsnAlaSerTyrIleArgIleVal Asn
210 215 220
3 His Glu GluGluTyrPheTyrIleAlaThrGlnGlyProLeuPro Glu
5
225 230 235 240
Thr Ile GluAspPheTrpGlnMetValLeuGluAsnAsnCysAsn Val
245 250 255
Ile Ala MetI1eThrArgGluIleGluCysGlyValIleLysCys Tyr
260 265 270
Ser Tyr TrpProIleSerLeuLysGluProLeuGluPheGluHis Phe
275 280 285
Ser Val PheLeuGluThrPheHisValThrGlnTyrPheThrVal Arg
290 295 300
5 Val Phe GlnIleValLysLysSerThrGlyLysSerGlnCysVal Lys
0
305 310 315 320
His Leu GlnPheThrLysTrpProAspHisGlyThrProAlaSer Ala
325 330 335
Asp Phe PheIleLysTyrValArgTyrValArgLysSerHisIle Thr
340 345 350
Gly Pro LeuLeuValHisCysSerAlaGlyValGlyArgThrGly Val
355 360 365
Phe Ile CysValAspValValPheSerAlaIleGluLysAsnTyr Ser
370 375 380
_ ___~ _ . ___-_~ __ _ _
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Phe Asp Ile Met Asn Ile Val Thr Gln Met Arg Lys Gln Arg Cys Gly
385 390 395 400
Met Ile Gln Thr Lys Glu Gln Tyr Gln Phe Cys Tyr Glu Ile Val Leu
405 410 415
Glu Val Leu Gln Asn Leu Leu Ala Leu Tyr
420 425
,10
(2) INFORMATION FORSEQIDN0:12:
(i) SEQUENCE ISTICS:
CHARACTER
(A)LENGTH: 963amino
acids
(B)TYPE: amino
acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULE peptide
TYPE:
(xi)SEQUENCE SEQID O:
DESCRIPTION: N 12:
2 Met Ser SerProArgLysValArgGlyLysThrGlyArgAspAsnAsp
5
1 5 10 15
Glu Glu GluGlyAsnSerGlyAsnLeuAsnLeuArgAsnSerLeuPro
20 25 30
Ser Ser SerGlnLysMetThrProThrLysProValGlnAsnLysAsn
35 90 45
Leu Met LysTyrGluGluHisLeuAspIleLeuMetValPheLeuLeu
50 55 60
Ile Lys ThrIleTrpTyrAsnValPheLysLeuTrpLysGlyLysLeu
65 70 75 80
4 Ile Phe GlyAsnLysMetAsnSerGluAsnValLysProSerHisHis
0
85 90 95
Leu Ser PheSerAspLysTyrGluLeuValTyrProGluProLeuGlu
100 105 110
Ser Asp ThrAspGluThrValTrpAspValSerAspArgSerLeuArg
115 120 125
Asn Arg TrpAsnSerMetAspSerGluThrAlaGlyProSerLysThr
130 135 140
Val Ser ProValLeuSerGlySerSerArgLeuSerLysAspThrGlu
145 150 155 160
5 Thr Ser ValSerGluLysGluLeuThrGlnLeuAlaGlnIleArgPro
5
165 170 175
Leu Ile PheAsnSerSerAlaArgSerAlaMetArgAspCysLeuAsn
180 185 190
Thr Leu GlnLysLysGluGluLeuAspIleIleArgGluPheLeuGlu
195 200 205
Leu Glu GlnMetThrLeuProAspAspPheAsnSerGlyAsnThrLeu
210 215 220
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Gln AsnArg AspLysAsnArgTyrArgAsp IleLeuPro TyrAspSer
225 230 235 240
Thr ArgVal ProLeuGlyLysAsnLysAsp TyrIleAsn AlaSerTyr
245 250 255
Ile ArgIle ValAsnHisGluGluGluTyr PheTyrIle AlaThrGln
260 265 270
Gly ProLeu ProGluThrIleGluAspPhe TrpGlnMet ValLeuGlu
275 280 285
Asn AsnCys AsnValIleAlaMetIleThr ArgGluIle GluCysGly
290 295 300
Val IleLys CysTyrSerTyrTrpProIle SerLeuLys GluProLeu
305 310 315 320
2 Glu PheGlu HisPheSerValPheLeuGlu ThrPheHis ValThrGln
O
325 330 335
Tyr PheThr ValArgValPheGlnIleVal LysLysSer ThrGlyLys
340 345 350
Ser GlnCys ValLysHisLeuGlnPheThr LysTrpPro AspHisGly
355 360 365
Thr ProAla SerAlaAspPhePheIleLys TyrValArg TyrVaIArg
370 375 380
Lys SerHis IleThrGlyProLeuLeuVal HisCysSer AlaGlyVal
385 390 395 900
3 Gly ArgThr GlyValPheIleCysValAsp ValValPhe SerAlaIle
5
405 410 415
Glu LysAsn TyrSerPheAspIleMetAsn IleValThr GlnMetArg
920 425 430
Lys GlnArg CysGlyMetIleGlnThrLys GluGlnTyr GlnPheCys
935 440 495
Tyr GluIle ValLeuGluValLeuGlnAsn LeuLeuAla LeuTyr
450 455 960
(2) INFORMATION FORSEQID 13:
NO:
5 (i)SEQUENCE
O CHARACTERISTICS:
(A) LENGTH: 405
amino
acids
(B) TYPE: amino
acid
(C) STRANDEDNESS: single
5 (D) TOPOLOGY: linear
5
(ii)MOLECULE peptide
TYPE:
(xi)SEQUENCE ID
DESCRIPTION: NO:
SEQ 13:
60
Met SerSer ProArgLysValArgGlyLys ThrGlyArg AspAsnAsp
1 5 10 15
Glu GluGlu GlyAsnSerGlyAsnLeuAsn LeuArgAsn SerLeuPro
65 20 25 30
_ ~ _.
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Ser SerSerGlnLysMetThrProThrLysProIlePheGlyAsnLys
35 40 45
Met AsnSerGluAsnValLysProSerHisHisLeuSerPheSerAsp
50 55 60
1 Lys TyrGluLeuValTyrProGluProLeuGluSerAspThrAspGlu
0
65 70 75 80
Thr ValTrpAspValSerAspArgSerLeuArgAsnArgTrpAsnSer
- 85 90 95
1 Met AspSerGluThrAlaGlyProSerLysThrValSerProValLeu
5
100 105 110
Ser GlySerSerArgLeuSerLysAspThrGluThrSerValSerGlu
115 120 125
20
Lys GluLeuThrGlnLeuAlaGlnIleArgProLeuIlePheAsnSer
130 135 140
Ser AlaArgSerAlaMetArgAspCysLeuAsnThrLeuGlnLysLys
. 145 150 155 160
2
5
Glu GluLeuAspIleIleArgGluPheLeuGluLeuGluGlnMetThr
165 270 175
3 Leu ProAspAspPheAsnSerGlyAsnThrLeuGlnAsnArgAspLys
0
180 185 190
Asn ArgTyrArgAspIleLeuProTyrAspSerThrArgValProLeu
195 200 205
35
Gly LysAsnLysAspTyrIleAsnAlaSerTyrIleArgIleValAsn
210 215 220
His GluGluGluTyrPheTyrIleAlaThrGlnGlyProLeuProGlu
4 225 230 235 240
0
Thr IleGluAspPheTrpGlnMetValLeuGluAsnAsnCysAsnVal
295 250 255
~ Ile AlaMetIleThrArgGluIleGluCysGlyValIleLysCysTyr
5
260 265 270
Ser TyrTrpProIleSerLeuLysGluProLeuGluPheGluHisPhe
275 280 285
50
Ser ValPheLeuGluThrPheHisValThrGlnTyrPheThrValArg
290 295 300
Val PheGlnIleValLysLysSerThrGlyLysSerGlnCysValLys
5 305 310 315 320
S
His LeuGlnPheThrLysTrpProAspHisGlyThrProAlaSerAla
325 330 335
6 Asp PhePheIleLysTyrValArgTyrValArgLysSerHisIleThr
0
390 345 350
Gly ProLeuLeuValHisCysSerAlaGlyValGlyArgThrGlyVal
355 360 365
65
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Phe Ile Cys Val Asp Val Val Phe Ser Ala Ile Glu Lys Asn Tyr Ser
370 375 380
Phe Asp Ile Met Asn Ile Val Thr Gln Met Arg Lys Gln Arg Cys Gly
385 390 395 900
Met Ile Gln Thr Lys
405
(2) INFORMATION IDNO:14:
FOR
SEQ
(i) SEQUENCE ISTICS:
CHARACTER
(A)LENGTH: 122 amin o
acids
(B)TYPE: amino id
ac
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULE peptide
TYPE:
(xi)SEQUENCE ON:SEQ ID 19:
DESCRIPTI NO:
2 Asp Phe TrpGly Met TrpGluAsn Asn Asn ValIleAlaMet
5 Met Cys
1 5 10 15
Ile Thr ArgGlu Ile GlyGlyVal Ile Cys CysSerTyrTrp
Glu Lys
20 25 30
Pro Val SerLeu Lys ProLeuGlu Phe His PheHisValLeu
Glu Lys
40 45
Leu Glu AsnPhe Gln ThrGlnTyr Phe Ile ArgIlePheGln
Ile Val
35 50 55 60
Ile Val LysLys Ser GIyLysSer His Val LysHisLeuGln
Thr Ser
65 70 75 80
4 Phe Ile LysTrp Pro HisGlyThr Pro Ser ValAspPhePhe
0 Asp Ala
85 90 95
Ile Lys TyrVal Arg ValArgLys Ser Ile ThrGlyProLeu
Tyr His
100 105 110
Leu Val HisCys Thr GlyValGly Arg
Ala
115 120
(2) INFORMATION
FOR
SEQ
ID NO:
15:
(i) SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 1274 amino acids
5
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
6 (ii) MOLECULE TYPE: peptide
0
(xi) SEQUENCE DESCRIPTION:ID NO: 15:
SEQ
Met Ala Ala His Glu Ala Ser Tyr Ser Glu Glu Lys
Ser Leu Ala Lys
65 1 5 to is
_ ~ ___ _.._
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Leu Leu Arg Glu Met Met Ala Lys Ile Glu Asp Lys Asn Glu Val Leu
20 25 30
Asp GlnPheMetAspSerMetGlnLeuAspProGluThrValAspAsn
35 40 45
Leu AspAlaTyrSerHisIleProProGlnLeuMetGluLysCysAla
50 55 60
Ala LeuSerValArgProAspThrValArgAsnLeuValGlnSerMet
65 70 75 80
' Gln ValLeuSerGlyValPheThrAspValGluAlaSerLeuLysAsp
1
5
85 90 95
Ile ArgAspLeuLeuGluGluAspGluLeuLeuGluGlnLysPheGln
100 105 110
2 Glu AlaValGlyGlnAlaGlyAlaIleSerIleThrSerLysAlaGlu
0
115 120 125
Leu AiaGluValArgArgGluTrpAlaLysTyrMetGluValHisGlu
130 135 140
25
Lys AlaSerPheThrAsnSerGluLeuHisArgAlaMetAsnLeuHis
145 150 155 160
Val GlyAsnLeuArgLeuLeuSerGlyProLeuAspGlnValArgAla
30 165 170 175
Ala LeuProThrProAlaLeuSerProGluAspLysAlaValLeuGln
180 185 190
3 Asn LeuLysArgIleLeuAlaLysValGlnGluMetArgAspGlnArg
5
195 200 205
Val SerLeuGluGlnGlnLeuArgGluLeuIleGlnLysAspAspIle
210 215 220
40
Thr AlaSerLeuValThrThrAspHisSerGluMetLysLysLeuPhe
225 230 235 240
Glu GluGlnLeuLysLysTyrAspGlnLeuLysValTyrLeuGluGln
4 245 250 255
5
Asn LeuAlaAlaGlnAspArgValLeuCysAlaLeuThrGluAlaAsn
260 265 270
5 Val GlnTyrAlaAlaValArgArgValLeuSerAspLeuAspGlnLys
0
275 280 285
Trp AsnSerThrLeuGlnThrLeuValAlaSerTyrGluAlaTyrGlu
290 295 300
55
Asp LeuMetLysLysSerGlnGluGlyArgAspPheTyrAlaAspLeu
305 310 315 320
Glu SerLysValAlaAlaLeuLeuGluArgThrGlnSerThrCysGln
60 325 330 335
Ala ArgGluAlaAlaArgGlnGlnLeuLeuAspArgGluLeuLysLys
390 345 350
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Lys Pro Pro Pro Arg Pro Thr Ala Pro Lys Pro Leu Leu Pro Arg Arg
355 360 365
Glu Glu Ser Glu Ala Val Glu Ala Gly Asp Pro Pro Glu Glu Leu Arg
370 375 380
Ser Leu Pro Pro Asp Met Val Ala Gly Pro Arg Leu Pro Asp Thr Phe
385 390 395 900
Leu Gly Ser Ala Thr Pro Leu His Phe Pro Pro Ser Pro Phe Pro Ser
905 410 915
1 Ser ThrGlyProGlyProHisTyrLeuSerGlyProLeuProPro Gly
5
420 425 930
Thr TyrSerGlyProThrGlnLeuIleGlnProArgAlaProGly Pro
435 940 945
His AlaMetProValAlaProGlyProAlaLeuTyrProAlaPro Ala
950 455 960
Tyr ThrProGiuLeuGlyLeuValProArgSerSerProGlnHis Gly
2 965 470 975 480
5
Val ValSerSerProTyrValGlyValGlyProAlaProProVal Ala
485 490 495
3 Gly LeuProSerAlaProProProGlnPheSerGlyProGluLeu Ala
0
500 505 510
Met AlaValArgProAlaThrThrThrValAspSerIleGlnAla Pro
515 520 525
35
Ile ProSerHisThrAlaProArgProAsnProThrProAlaPro Pro
530 535 540
Pro ProCysPheProValProProProGlnProLeuProThrPro Tyr
4 545 550 555 560
0
Thr TyrProAlaGlyAlaLysGlnProIleProAlaGlnHisHis Phe
565 570 575
4 Ser SerGlyIleProThrGlyPheProAlaProArgIleGlyPro Gln
5
580 585 590
Pro GlnProHisProGlnProHisProSerGlnAlaPheGlyPro Gln
595 600 605
50
Pro ProGlnGlnProLeuProLeuGlnHisProHisLeuPhePro Pro
610 615 620
Gln AlaProGlyLeuLeuProProGlnSerProTyrProTyrAla Pro
5 625 630 635 640
5
Gln ProGlyValLeuGlyGlnProProProProLeuHisThrGln Leu
645 650 655
6 Tyr ProGlyProAlaGlnAspProLeuProAlaHisSerGlyAla Leu
0
660 665 670
Pro PheProSerProGlyProProGlnProProHisProProLeu Ala
675 680 685
65
___ _.__ _. . _ _
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Tyr Gly Pro Ala Pro Ser Thr Arg Pro Met Gly Pro Gln Ala Ala Pro
690 695 700
Leu ThrI1eArgGlyProSerSerAlaGlyGlnSer ThrProSerPro
705 710 715 720
His LeuValProSerProAlaProSerProGlyPro GlyProValPro
725 730 735
Pro ArgProProAlaAlaGluProProProCysLeu ArgArgGlyAla
790 745 750
Ala AlaAlaAspLeuLeuSerSerSerProGluSer GlnHisGlyGly
755 760 765
Thr GlnSerProGlyGlyGlyGlnProLeuLeuGln ProThrLysVal
770 775 780
2 Asp AlaAlaGluGlyArgArgProGlnAlaLeuArg LeuIleGluArg
0
785 790 795 800
Asp ProTyrGluHisProGluArgLeuArgGlnLeu GlnGlnGluLeu
805 810 815
Glu AlaPheArgGlyGlnLeuGlyAspValGlyAla LeuAspThrVal
820 825 830
Trp ArgGluLeuGlnAspAlaGlnGluHisAspAla ArgGlyArgSer
835 840 895
Ile AlaIleAlaArgCysTyrSerLeuLysAsnArg HisGlnAspVal
850 855 860
3 Met ProTyrAspSerAsnArgValValLeuArgSer GlyLysAspAsp
5
865 870 875 880
Tyr IleAsnAlaSerCysValGluGlyLeuSerPro TyrCysProPro
885 890 895
Leu ValAlaThrGlnAlaProLeuProGlyThrAla AlaAspPheTrp
900 905 910
Leu MetValHisGluGlnLysValSerValIleVal MetLeuValSer
4 915 920 925
5
Glu AlaGluMetGluLysGlnLysValAlaArgTyr PheProThrGlu
930 935 940
5 Arg GlyGlnProMetValHisGlyAlaLeuSerLeu AlaLeuSerSer
0
995 950 955 960
Val ArgSerThrGluThrHisValGluArgValLeu SerLeuGlnPhe
965 970 975
55
Arg AspGlnSerLeuLysArgSerLeuValHisLeu HisPheProThr
980 985 990
Trp ProGluLeuGlyLeuProAspSerProSerAsn LeuLeuArgPhe
6 995 1000 1005
0
Ile GlnGluValHisAlaHisTyrLeuHisGlnArg ProLeuHisThr
1010 1015 1020
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Pro IleValHisCysSerSerGlyValGly ThrGlyAlaPhe
Ile Arg
1025 1030 1035 1040
Ala LeuTyrAlaAlaValGlnGluValGlu GlyAsnGlyIle
Leu Ala
1095 1050 1055
Pro LeuProGlnLeuValArgArgMetArg GlnArgLysHis
Glu Gln
1060 1065 1070
1 Met GlnGluLysLeuHisLeuArgPheCys GluAlaValVal
0 Leu Tyr
1075 1080 1085
Arg ValGluGlnValLeuGlnArgHisGly ProProProCys
His Val
1090 1095 1100
Lys LeuAlaSerAlaSerIleSerGlnLys HisLeuProGln
Pro Asn
1105 1110 1115 1120
Asp GlnAspLeuValLeuGlyGlyAspVal IleSerSerIle
Ser Pro
2 1125 1130 1135
0
Gln ThrIleAlaLysLeuSerIleArgPro GlyGlyLeuGlu
Ala Pro
1190 1145 1150
2 Ser ValAlaSerLeuProGlyProAlaGlu ProGlyLeuPro
5 Pro Pro
1155 1160 1165
Pro SerLeuProGluSerThrProIlePro SerSerProPro
Ala Ser
1170 1175 1180
30
Pro SerSerProLeuProGluAlaProGln LysGluGluPro
Leu Pro
1185 1190 1195 1200
Pro ProGluAlaProSerSerGlyProPro SerSerLeuGlu
Val Ser
3 1205 1210 1215
5
Leu AlaSerLeuThrProGluAlaPheSer AspSerSerLeu
Leu Leu
1220 1225 1230
4 Arg LysGlnArgMetSerLysHisAsnPhe GlnAlaHisAsn
0 Gly Leu
1235 1290 1295
Gly GlyLeuArgAlaThrArgProSerAsp ProLeuSerLeu
Gln Asp
1250 1255 1260
45
Leu ProLeuTrpThrLeuAsnLysThr
Asp
1265 1270
(2) 16:
INFORMATION
FOR
SEQ
ID
NO:
(i) SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 493 amino acids
5
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
6 (ii) MOLECULE TYPE: peptide
0
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 16:
Met Arg Ala Leu Cys Leu Arg Gln Ala Leu Leu
Thr Ser Ala Leu Leu
65 1 5 10 15
_ _____~. _ __ ~___. _
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Ala AlaAlaAlaGluLeuSerProGlyLeuLysCysValCysLeuLeu
20 25 30
Cys AspSerSerAsnPheThrCysGlnThrGluGlyAlaCysTrpAla
35 90 45
Ser ValMetLeuThrAsnGlyLysGluGlnValIleLysSerCysVal
50 55 60
Ser LeuProGluLeuAsnAlaGlnValPheCysHisSerSerAsnAsn
65 70 75 80
Val ThrLysThrGluCysCysPheThrAspPheCysAsnAsnIleThr
85 90 95
Leu HisLeuProThrAlaSerProAsnAlaProLysLeuGlyProMet
100 105 110
2 Glu LeuAlaIleIleIleThrValProValCysLeuLeuSerIleAla
0
115 120 12 5
Ala MetLeuThrValTrpAlaCysGlnGlyArgGlnCysSerTyrArg
130 135 140
Lys LysLysArgProAsnValGluGluProLeuSerGluCysAsnLeu
145 150 155 160
Val AsnAlaGlyLysThrLeuLysAspLeuIleTyrAspValThrAla
165 170 175
Ser GlySerGlySerGlyLeuProLeuLeuValGlnArgThrIleAla
180 185 190
3 Arg ThrIleValLeuGlnGluIleValGlyLysGlyArgPheGlyGlu
5
195 200 205
Val TrpHisGlyArgTrpCysGlyGluAspValAlaValLysIlePhe
210 215 220
Ser SerArgAspGluArgSerTrpPheArgGluAlaGluIleTyrGln
225 230 235 240
Thr ValMetLeuArgHisGluAsnIleLeuGlyPheIleAlaAlaAsp
4 245 250 255
5
Asn LysAspAsnGlyThrTrpThrGlnLeuTrpLeuValSerGluTyr
260 265 270
5 His GluGlnGlySerLeuTyrAspTyrLeuAsnArgAsnIleValThr
0
275 280 285
Val AlaGlyMetIleLysLeuAlaLeuSerIleAlaSerGlyLeuAla
290 295 300
55
His LeuHisMetGluIleValGlyThrGlnGlyLysProAlaIleAla
305 310 315 320
His ArgAspIleLysSerLysAsnIleLeuValLysLysCysGluThr
60 325 330 335
Cys AlaIleAlaAspLeuGlyLeuAlaValLysHisAspSerIleLeu
340 395 350
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Asn ThrIle Asp Ile AsnProLys Val Gly LysArgTyr
Pro Gln Thr
355 360 365
Met AlaPro Glu Met AspThrMet Asn Val IlePheGlu
Leu Asp Asn
370375 380
Ser PheLys Arg Ala TyrSerVal Gly Leu TyrTrpGlu
Asp Ile Val
385 390 395 900
1 Ile AlaArg Arg Cys GlyGlyIle Val Glu TyrGlnLeu
O Ser Val Glu
905 410 415
Pro TyrTyr Asp Met SerAspPro Ser Ile GluMetArg
Val Pro Glu
420 425 930
Lys ValVal Cys Asp PheArgPro Ser Ile AsnGlnTrp
Gln Lys Pro
435 440 445
GlriSerCys Glu Ala ValMetGly Arg Ile ArgGluCys
Leu Arg Met
950955 960
Trp TyrAla Asn Gly ArgLeuThr Ala Leu IleLysLys
Ala Ala Arg
965 970 475 980
2 Thr IleSer Gln Leu LysGluAsp Cys Lys
5 Cys Val Ala
485 990
3 (2) INFORMATION NO:17:
O FOR
SEQ
ID
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
3 (B) TYPE: nucleic acid
5
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix)FEATURE:
90
(D) OTHER INFORMATION : The letter T.
"Y" stands
for C or
The letter or
"V" stands
for A, C
G.
The letter G.
"R" stands
for A or
4 The letter G
5 "N" stands
for A, C,
or T.
(xi)SEQUENCE DESCRIPTION:SEQID NO: 17:
5 GAYTTYTGGV 23
O RNATGRTNTG
GGA
(2) INFORMATION NO:18:
FOR
SEQ
ID
55
(i)SEQUENCE CHARACTERISTI CS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
6 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(ix)FEATURE:
1 _
_. _..
CA 02288221 1999-10-27
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16'7
(D) OTHER INFORMATION:The letter "S" forC G.
stands or
The letter "Y" forC T.
stands or
The letter "N" forA, G
stands C,
or T.
The letter "W" forA T.
stands or
The letter "R" forA G.
stands or
(xi) SEQUENCE DESCRIPTION:ID NO: 18:
SEQ
,1 CGGCCSAYNC 23
O CNGCNSWRCA
RTG
(2)
INFORMATION
FOR
SEQ
ID
N0:
19:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
2 (D) OTHER INFORMATION:"Xaa" in positionsandstand
5 9 6
for an unspecified acid.
amino
"Xaa" in positiontandsfor
8 s
either Glu or
Asp.
3 (xi) SEQUENCE DESCRIPTION:iD NO: 19:
O SEQ
Asp Trp Xaa Met Xaa Trp
Phe Xaa
1 5
(2) INFORMATION
FOR
SEQ
ID NO:
20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
5 (D) OTHER INFORMATION:"Xaa"
O in
positions
3
and
6
stand
for unspecified amino
an acid.
(xi) SEQUENCE DESCRIPTION:ID 20:
SEQ N0:
5 His Cys Xaa Ala Gly Xaa Gly
5
1 5
6 O (2) INFORMATION FOR SEQ ID N0: 21:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 21:
1 O CACCGTTCGA GTATTTCAGA TTGTGAAGAA GTCC 34
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
2 O (A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
2 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 22:
GGACTTCTTC ACAATCTGAA ATACTCGAAC GGTG 39
(2) INFORMATION FOR SEQ ID
NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 pairs
base
3 (B) TYPE: nucleicacid
5
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 23:
SEQ N0:
CCGTTATGTG AGGAAGAGCC ACATTACAGGC 33
AC
4 (2) INFORMATION FOR SEQ ID
5 N0: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 pairs
base
5 (B) TYPE: nucleicacid
O
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 24:
SEQ NO:
55
GGTCCTGTAA TGTGGCTCTT CCTCACATAA 33
CGG
6 O (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
_ ....___~..-._._
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(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 25:
SEQ
GGCATGCATG GAGTATGAAA TGG 23
.10
(2) INFORMATION FOR SEQ ID
NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 26:
SEQ
CGTACATCCC AGATGAGCTC AAGAATAGGG 30
(2) INFORMATION FOR SEQ ID
NO: 27:
(i) SEQUENCE CHARACTERISTICS:
3 (A) LENGTH: 31 amino acids
0
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
3 (ii) MOLECULE TYPE: peptide
5
(xi) SEQUENCE DESCRIPTION: ID NO: 27:
SEQ
Ser Trp Pro Pro Ser Gly Thr Asp Asp Leu
Ser Ser Lys Met Ser Leu
40 1 5 IO 15
Pro Glu Lys Gln Asp Gly Thr Leu Pro
Val Phe Pro Ser Ser Leu
20 25 30
45
(2) INFORMATION FOR SEQ ID
NO: 28:
(i) SEQUENCE CHARACTERISTICS:
50
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
55
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: ID NO: 28:
SEQ
6 Tyr Ser Leu Pro Tyr Asp Ser Ala Ser Asn
0 Lys His Gln Ile Arg Asn
1 5 10 15
Val Lys His His Asp Ser Ser Tyr
Ala Leu Gly Val Tyr Ser
20 25 30
65
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(2) INFORMATION
FOR
SEQ
ID
NO:
29:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino
acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
1 (D) TOPOLOGY: linear
O
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION:ID NO: 29:
SEQ
His ThrLeu Gln Ala Asp Ser Pro Asn
Tyr Ser Leu Pro
Lys Ser
Thr
1 5 10 15
Thr LysAla Ala Lys Met Met Gln Arg
Asn Gln Thr Lys
Cys
20 25 30
(2) INFORMATION
FOR
SEQ
ID
NO:
30:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base
pairs
(B) TYPE: nucleic
acid
3 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(ix)FEATURE:
3 (D) OTHER INFORMATION:The letter stands for A,
5 "N" C, G
or T.
The letter stands for A or
"R" G.
The letter stands for C or
"Y" T.
4 (xi)SEQUENCE DESCRIPTION:ID NO: 30:
O SEQ
GGN CARTTYG 21
GNGANGTNTG
G
45
(2) INFORMATION
FOR
SEQ
ID
N0:
31:
(i)SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 24 base
O pairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
5 (ix)FEATURE:
5
(D) OTHER INFORMATION:The letter stands for A,
"N" C, G
or T.
The letter stands for C or
"Y" T.
60
(xi)SEQUENCE DESCRIPTION:ID N0: 31:
SEQ
CAGNGCNGCY 24
TCNGGNGCNG
TCCA
_ .__ _~ i
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(2) INFORMATION
FOR
SEQ
ID
NO:
32:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 aminoacids
(B) TYPE: amino id
ac
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(ix)FEATURE:
' (D) OTHER INFORMATION:"Xaa" position 5 stands for
1 in
5
eitherGlu or Asp.
(xi)SEQUENCE DESCRIPTION:ID 32:
SEQ NO:
2 Gly GlnPhe Gly Xaa Val Trp
O
1 5
2 (2) INFORMATION
5 FOR
SEQ
ID
NO:
33:
{i)SEQUENCE CHARACTERISTICS:
{A) LENGTH: 8 aminoacids
3 {B) TYPE: amino
O acid
{C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
35
(xi)SEQUENCE DESCRIPTION:ID 33:
SEQ NO:
Trp ThrAla Pro Glu Ala Leu
Leu
1 5
40
(2) INFORMATION
FOR
SEQ
ID
NO:
34:
4 (i)SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 19 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
5 (D) TOPOLOGY: linear
O
(xi)SEQUENCE DESCRIPTION:ID 34:
SEQ NO:
AGT GAGCTTC 19
ATGTTGGCT
55
{2) INFORMATION
FOR
SEQ
ID
NO:
35:
C (i)SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 18 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
6 (D) TOPOLOGY: linear
5
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
GGTAGAGGCT GCCATCAG 18
10
(2) INFORMATION FOR SEQ ID
N0: 36:
1 (i) SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 19 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
2 (D) TOPOLOGY: linear
O
(ix) FEATURE:
(D) OTHER INFORMATION: The
letter
"N"
stands
for
2 deoxythymidylate.
5
(xi) SEQUENCE DESCRIPTION: ID 36:
SEQ NO:
GACGATCGGA ATTCGCGAN 19
30
(2) INFORMATION FOR SEQ ID
N0: 37:
3 (i) SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 18 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
4 (D) TOPOLOGY: linear
O
(xi) SEQUENCE DESCRIPTION: ID 37:
SEQ NO:
GACGATCGGA ATTCGCGA 18
45
(2) INFORMATION FOR SEQ ID
NO: 38:
5 (i) SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 1? pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
55 (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 38:
SEQ NO:
CCCAGCCACA GGCCTTC 17
CO
(2) INFORMATION FOR SEQ ID
NO: 39:
6 (i) SEQUENCE CHARACTERISTICS:
5
_ _T. ___. _ _
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(A) LENGTH: 18 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 39:
SEQ N0:
CCACACCTCC CCAAAGTA 18
ZO
(2) INFORMATION FOR SEQ ID
N0: 40:
Z (i) SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 30 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
2 (D) TOPOLOGY: linear
O
(xi) SEQUENCE DESCRIPTION: ID 40:
SEQ NO:
TGGGAGCGGC CACACTCCGA ATTCGCCCTT 30
25
(2) INFORMATION FOR SEQ ID
NO: 41:
3 (i) SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 17 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
3 (D) TOPOLOGY: linear
5
(xi) SEQUENCE DESCRIPTION: ID 41:
SEQ NO:
GCCTGCGTGC GAAGATG 17
40
(2) INFORMATION FOR SEQ ID
NO: 92:
4 (i) SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 18 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
5 (D) TOPOLOGY: linear
O
(xi) SEQUENCE DESCRIPTION: ID 42:
SEQ NO:
CTTCGAGGGC ACAGAGCC 18
55
(2) INFORMATION FOR SEQ ID
NO: 43:
6 (i) SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 21 pairs
base
(B) TYPE: nucleicacid
iC) STRANDEDNESS: single
6 (D) TOPOLOGY: linear
5
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(xi) SEQUENCE DESCRIPTION:SEQID 43:
N0:
ATGGAGCCGT TCCTCAGGAG 21
G
(2) INFORMATION FOR SEQ 94:
ID NO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 pairs
base
(B) TYPE: nucleicacid
1 (C) STRANDEDNESS: single
5
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:SEQID 94:
N0:
2 TCACCCAGCT TCCTCCCAAG 21
O G
(2) INFORMATION FOR SEQ 45:
ID NO:
25
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 pairs
base
(B) TYPE: nucleicacid
3 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:SEQID 45:
NO:
3 AGGCCAACTG GAAGCTGATC 21
5 C
(2) INFORMATION FOR SEQ 46:
ID N0:
40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 pairs
base
(B) TYPE: nucleicacid
4 (C) STRANDEDNESS: single
5
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:SEQID 46:
NO:
5 GCTGGAGCCC AGAGCGTTGG 20
O
{2} INFORMATION FOR SEQ 97:
ID N0:
55
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 aminoacids
(B) TYPE: amino
acid
6 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
6 (ix) FEATURE:
5
__~ _ _.. -
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(D) OTHER INFORMATION: "Xaa" in position 6 stands
for an unspecified amino acid.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
His Arg Asp Leu Arg Xaa Ala Asn
1 5
(2) INFORMATION FOR SEQ ID
NO: 98:
(i) SEQUENCE CHARACTERISTICS:
'
(A) LENGTH: 8 aminoacids
(B) TYPE: amino id
ac
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
2 (D) OTHER INFORMATION: "Xaa" positions 6 stands
5 in
for unspecified amino acid.
an
(xi) SEQUENCE DESCRIPTION:ID 98:
SEQ N0:
3 His Arg Asp Leu Ala Xaa Arg
O Asn
1 5
3 (2) INFORMATION FOR SEQ ID
5 NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 pairs
base
4 (B) TYPE: nucleicacid
O
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:ID 49:
SEQ NO:
45
TCG CCAAGGA GATCCAGACA C 21
5 (2) INFORMATION FOR SEQ ID
O N0: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 pairs
base
5 (B) TYPE: nucleicacid
5
(C) STRANDEDNESS: single
' (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:ID 50:
SEQ NO:
60
GAAGTCAGCC 21
ACCTTGCAGG
C
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(2) INFORMATION FOR SEQ ID N0: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:ID 51:
SEQ NO:
1 GGATCCCCGG 13
5 ACC
(2) INFORMATION FOR SEQ ID
NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 acids
amino
(B) TYPE: amino id
ac
2 (C) STRANDEDNESS: single
5
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
3 (xi) SEQUENCE DESCRIPTION:ID 52:
O SEQ N0:
Met Arg Gly Ser His His His His
His His
1 5 10
35
(2) INFORMATION FOR SEQ ID
NO: 53:
(i) SEQUENCE CHARACTERISTICS:
40
(A) LENGTH: 30 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
45
(xi) SEQUENCE DESCRIPTION:ID 53:
SEQ NO:
ATGAGAGGAT 30
CGCATCACCA
TCACCATCAC
50
(2) INFORMATION FOR SEQ ID
NO: 59:
(i) SEQUENCE CHARACTERISTICS:
55
(A) LENGTH: 8 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
60
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
_ _.T __-_ _ _ .
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(D) OTHER INFORMATION:"Xaa" in positions 4
and 6 stand
for an unspecified amino
acid.
"Xaa" in position 8 stands
for
either Glu or Asp.
(xi)SEQUENCE DESCRIPTION:ID NO: 54:
SEQ
Asp Phe Trp Xaa Met Xaa Trp
Xaa
1 5
.
1O
(2) INFORMATION
~ FOR
SEQ
ID
N0:
55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
2 (xi)SEQUENCE DESCRIPTION:ID NO: 55:
5 SEQ
Tyr Pro Tyr Asp Val Pro Asp Ser
Tyr Ala
1 5 10
(2) INFORMATION
FOR
SEQ
ID
NO:
56:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION:ID NO: 56:
SEQ
4 His CysSer Ala Gly
5
1 5
5 (2) INFORMATION
O FOR
SEQ
ID
NO:
57:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
5 (B) TYPE: amino acid
5
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
60
(xi)SEQUENCE DESCRIPTION:ID NO: 57:
SEQ
Met SerSer Pro Arg Lys Val
Arg Gly Lys Thr
Gly Arg Asp Asn
Asp
1 5 10 15
65
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Glu GluGlu Gly Asn Ser LeuAsn Arg Asn
Gly Asn Leu
20 25
(2) INFORMATION 58:
FOR
SEQ
ID
NO:
(i)SEQUENCE CHARACTERISTICS:
1 (A) LENGTH: 29 aminoacids
O
(B) TYPE: amino
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1 (ii)MOLECULE TYPE: peptide
5
(xi)SEQUENCE DESCRIPTION:SEQID NO:
58:
Ser ProVal Leu Ser Gly ArgLeu Lys Asp Thr
Ser Ser Ser Glu Thr
20 1 5 10 15
Ser ValSer Glu Lys Glu GlnLeu Gln Ile
Leu Thr Ala
20 25
(2) INFORMATION
FOR
SEQ
ID
NO:
59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION:ID N0: 59:
SEQ
4 Trp Asp Val Ser Asp Arg Ser
O Leu Arg Asn Arg
Trp Asn Ser Met
Asp
1 5 10 15
Ser Glu Thr Ala Gly Pro Ser
Lys Thr Val Ser
Pro Val
20 25
(2) INFORMATION
FOR
SEQ
ID
NO:
60:
5 (i) SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
5 (D) TOPOLOGY: linear
5
(ix)FEATURE:
(D) OTHER INFORMATION:The letter "Y" stands for
C or T.
60 The letter "H" stands for
A, C or T.
The letter "M" stands for
A or C.
(xi)SEQUENCE DESCRIPTION:ID NO: 60:
SEQ
65 ATCCCCGGCT 26
CTGAYTAYAT
HMAYGC
___-.r_ __ _ _. ____.__
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(2) INFORMATION
FOR
SEQ
ID NO:
61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
1 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix FEATURE
)
(D) OTHER INFORMATION:"Xaa" in position 8
stands for
either Asn or His.
2 (xi) SEQUENCE DESCRIPTION:ID N0: 61:
O SEQ
Ile Pro Gly Ser Asp Tyr Ile
Xaa Ala
1 5
(2) INFORMATION
FOR
SEQ
ID
N0:
62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION:ID N0: 62:
SEQ
4 Met Glu Glu Leu Gln Asp Tyr Asn
O Glu Asp Met Met
Glu Glu
1 5 10
4 (2) INFORMATION
5 FOR
SEQ
ID
NO:
63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
5 (B) TYPE: amino acid
O
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
55
(xi)SEQUENCE DESCRIPTION:ID N0: 63:
SEQ
Tyr Gln Gln Gly Gln Asn Gln r Asn Glu Leu Gly
Leu Ty Leu Asn Arg
1 5 10 15
60
Arg Glu Glu Tyr Asp Val Leu s Arg Arg Asp
Asp Ly Gly Arg
20 25 30
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(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1 O (ii) MOLECULE TYPE: peptide
Z (ix) FEATURE:
5
(D) OTHER INFORMATION:"Xaa" in
positions
6 and 7
stand
for an unspecified
amino acid.
2 (xi) SEQUENCE DESCRIPTION:ID NO: 64:
O SEQ
His Arg Asp Leu Lys Xaa Xaa
Asn
1 5
25
(2) INFORMATION
FOR
SEQ
ID N0:
65:
(i) SEQUENCE CHARACTERISTICS:
30
(A) LENGTH: 23 base
pairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
35
(ix) FEATURE:
(D) OTHER INFORMATION:The letter standsfor A or
"R" G.
The letter standsfor Inosine.
"N"
40
(xi) SEQUENCE DESCRIPTION:ID NO: 65:
SEQ
GARRARGTNG 23
CNGTNAARRT
NTT
45
(2) INFORMATION
FOR
SEQ
ID NO:
66:
(i) SEQUENCE CHARACTERISTICS:
S (A) LENGTH: 29 base
O pairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
5 (ix) FEATURE:
5
(D) OTHER INFORMATION:The letter standsfor A or
"R" G.
The letter standsfor Inosine.
"N"
The letter standsfor G or
"K" T.
6 The letter standsfor A or
O "M" C.
The letter standsfor C or
"Y" T.
(xi) SEQUENCE DESCRIPTION:ID NO: 66:
SEQ
C TTRATRT CNC KRTGNGMNAT NGMNGGYTT 29
S
_ _~-_ ~_ _..
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(2) INFORMATION
FOR
SEQ
ID
N0:
67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
1 (C) STRANDEDNESS: single
O
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
. ( FEATURE
ix
)
(D) OTHER INFORMATION:"Xaa" in position for Lys
2 stands or
Glu. "Xaa" in positionstands
7 for
Val or Ile.
(xi)SEQUENCE DESCRIPTION:ID N0: 67:
SEQ
Glu Xaa Val Ala Val Lys Xaa
Phe
1 5
(2) INFORMATION
FOR
SEQ
ID
N0:
6B:
3 (i) SEQUENCE CHARACTERISTICS:
O
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
3 (D) TOPOLOGY: linear
5
(ii)MOLECULE TYPE: peptide
(ix)FEATURE:
40
(D) OTHER INFORMATION:"Xaa" in position for Ala
3 stands or
Ser. "Xaa" in positionstands
5 for
Ala or Ser.
4 (xi)SEQUENCE DESCRIPTION:ID NO: 68:
5 SEQ
Lys Pro Xaa Ile Xaa His Arg
Asp Ile Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 69:
AACTTTGGCT GGTATCTGAA TATC 24
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(2) INFORMATION FOR SEQ ID
NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 pairs
base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 70:
SEQ NO:
CCTTGTGTAC CAACAATCTC CATA 24
(2) INFORMATION FOR SEQ ID
N0: 71:
(i) SEQUENCE CHARACTERISTICS:
2 (A) LENGTH: 22 pairs
O base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
2 (xi) SEQUENCE DESCRIPTION: ID 71:
S SEQ NO:
CTCCAGAGAT GAGAGATCTT GG 22
30
(2) INFORMATION FOR SEQ ID
NO: 72:
(i) SEQUENCE CHARACTERISTICS:
3 (A) LENGTH: 22 pairs
5 base
(B) TYPE: nucleicacid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
4 (xi) SEQUENCE DESCRIPTION: ID 72:
O SEQ NO:
TTCCAGCCAC GGTCACTATG TT 22
4 {2) INFORMATION FOR SEQ ID
5 N0: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 pairs
base
S (B) TYPE: nucleicacid
O
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID 73:
SEQ NO:
55
CTTCGAAAGC TTGAAATCGG TACCATCGAT ACTTCGAA 48
TCTAGAGTTA
6 (2) INFORMATION FOR SEQ ID
O NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 pairs
base
65 (B) TYPE: nucleicacid
____-~_ _ _
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
CTCTAGAACG CGTTAAGGCG CGCCAATATC GATGAATTCT TCGAAGC 97
a,
1 (2) INFORMATION
5 FOR
SEQ
ID
NO:
75:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
2 (B) TYPE: amino acid
0
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
25
(xi)SEQUENCE DESCRIPTION:ID NO: 75:
5EQ
His CysSer Ser Gly
1 5
30
(2) INFORMATION
FOR
SEQ
ID
NO:
76:
3 (i)SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 13 amino
acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
4 (D) TOPOLOGY: linear
0
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION:ID NO: 76:
SEQ
4 Tyr ArgLys Lys Lys Arg Pro Glu Glu Pro
5 Asn Val Leu
