U.S. patent application number 09/895606 was filed with the patent office on 2003-11-06 for detection reagents for tpc2 and tpc3, two proteins that are coexpressed with telomerase.
Invention is credited to Adams, Robert R., Andrews, William H., Feng, Junli, Villeponteau, Bryant.
Application Number | 20030207404 09/895606 |
Document ID | / |
Family ID | 26671831 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207404 |
Kind Code |
A1 |
Villeponteau, Bryant ; et
al. |
November 6, 2003 |
Detection reagents for TPC2 and TPC3, two proteins that are
coexpressed with telomerase
Abstract
Purified and recombinant proteins TPC2 and TPC3 and recombinant
or synthetic oligonucleotides corresponding to those proteins or
fragments thereof can be used to detect regulators of telomere
length and telomerase activity in mammalian cells and for a variety
of related diagnostic and therapeutic purposes.
Inventors: |
Villeponteau, Bryant; (San
Carlos, CA) ; Feng, Junli; (San Carlos, CA) ;
Andrews, William H.; (Richmond, CA) ; Adams, Robert
R.; (Redwood City, CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
|
Family ID: |
26671831 |
Appl. No.: |
09/895606 |
Filed: |
June 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09895606 |
Jun 29, 2001 |
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09220157 |
Dec 23, 1998 |
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6300110 |
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09220157 |
Dec 23, 1998 |
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08710249 |
Sep 13, 1996 |
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5858777 |
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08710249 |
Sep 13, 1996 |
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08583808 |
Jan 5, 1996 |
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60003492 |
Sep 9, 1995 |
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Current U.S.
Class: |
435/70.21 ;
435/331; 435/7.92; 530/388.26 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4703 20130101 |
Class at
Publication: |
435/70.21 ;
435/331; 530/388.26; 435/7.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543; C12P 021/04; C07K 016/40 |
Claims
The invention claimed is:
1. A monoclonal or isolated polyclonal antibody that specifically
binds human TPC2 or TPC3 protein, as shown in SEQ. ID NO:2 or 4,
respectively.
2. The antibody of claim 1, which specifically binds human TPC2
protein (SEQ. ID NO:2).
3. The antibody of claim 1, which specifically binds human TPC3
protein (SEQ. ID NO:4).
4. A method for obtaining an antibody according to claim 1,
comprising collecting antiserum from a subject immunized with a
peptide comprising at least 10 contiguous amino acids of SEQ. ID
NO:2 or SEQ. ID NO:4.
5. A method for obtaining an antibody according to claim 1,
comprising collecting spleen cells from an animal immunized with a
peptide comprising at least 10 contiguous amino acids of SEQ. ID
NO:2 or SEQ. ID NO:4.
6. A method for obtaining an antibody according to claim 1,
comprising screening a phage antibody display library with a
peptide comprising at least 10 contiguous amino acids of SEQ. ID
NO:2 or SEQ. ID NO:4.
7. An antibody obtained according to the method of claim 4.
8. A host cell secreting a monoclonal antibody according to claim
1.
9. A composition effective for obtaining a TPC2-specific antibody
according to the method of claim 4, comprising at least 10
contiguous amino acids of SEQ. ID NO:2.
10. A composition effective for obtaining a TPC3-specific antibody
according to the method of claim 4, comprising at least 10
contiguous amino acids of SEQ. ID NO:4.
11. A method of determining a condition in a subject associated
with a high level of TPC2 (SEQ. ID NO:2), comprising combining the
antibody of claim 2 with a sample obtained from the subject, and
correlating protein detected by the antibody with the condition of
the subject.
12. A method of determining a condition in a subject associated
with a high level of TPC3 (SEQ. ID NO:4), comprising combining the
antibody of claim 3 with a sample obtained from the subject, and
correlating protein detected by the antibody with the condition of
the subject.
13. An isolated, recombinant or synthetic nucleic acid encoding a
peptide immunogenic for a TPC2-specific antibody according to claim
2, comprising at least 25 contiguous amino acids of SEQ. ID
NO:1.
14. The nucleic acid of claim 13, comprising at least 100
consecutive nucleotides of SEQ. ID NO:1.
15. A host cell containing a nucleic acid according to claim
13.
16. A method of obtaining an antibody according to claim 2,
comprising expressing the nucleic acid of claim 13 in a host
cell.
17. An isolated, recombinant or synthetic nucleic acid encoding a
peptide immunogenic for a TPC3-specific antibody according to claim
3, comprising at least 25 contiguous amino acids of SEQ. ID
NO:3.
18. The nucleic acid of claim 17, comprising at least 100
consecutive nucleotides of SEQ. ID NO:3.
19. A host cell containing a nucleic acid according to claim
17.
20. A method of obtaining an antibody according to claim 3,
comprising expressing the nucleic acid of claim 17 in a host cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/220,157, filed Dec. 23, 1998 (pending); which is a continuation
of 08/710,249, filed Sep. 13, 1996 (issued as U.S. Pat. No.
5,858,777 on Jan. 12, 1999); which is a continuation-in-part of
U.S. Ser. No. 08/583,808, filed Jan. 5, 1996 (now abandoned), and
claims the benefit of Provisional Application No. 60/003,492, filed
Sep. 8, 1995. All the priority documents are hereby incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention provides methods and reagents for
regulating telomere length and modulating telomerase activity in
mammalian cells as well as for detecting, diagnosing, and treating
related diseases and conditions in humans and other mammals. In an
important embodiment, the invention provides oligonucleotide probes
and primers, polynucleotide plasmids, peptides, proteins,
antibodies, and enzymes relating to genes and gene products that
regulate telomere length and telomerase activity in mammalian
cells. The invention has diverse applications and provides
important advances in the fields of molecular biology, chemistry,
pharmacology, and medical therapeutic and diagnostic
technology.
BACKGROUND OF THE INVENTION
[0003] The DNA at the ends of the telomeres of chromosomes in
mammalian cells consists of double- and single-stranded nucleic
acid composed of many tandem repeats of a simple nucleotide
sequence referred to as the telomeric repeat sequence. Telomeres
help maintain chromosome structure and function; the loss of
telomeric DNA can activate the cellular processes that detect and
control DNA damage and monitor and control cell proliferation and
senescence. The maintenance of telomeres and the regulation of
telomere length are vital cellular functions involved in
transmitting genetic information from generation to generation,
aging, the control of cell growth, and cancer. See Harley, 1991,
Mutation Research 256:271-282; and Blackburn, 1992, Annu. Rev.
Biochem. 61:113-129, each of which is incorporated herein by
reference (note: references cited herein are provided for
convenience; such citations are not to be construed as an admission
of prior invention).
[0004] The multi-component telomerase ribonucleoprotein enzyme
catalyzes the synthesis of the first strand of telomeric DNA
synthesized during telomere elongation, using the RNA component of
the enzyme as a template. Although the RNA component of human
telomerase (hTR) and other mammalian telomerase enzymes has been
identified, isolated, characterized, and described in the
scientific literature, the protein components of the telomerase
enzyme as well as most other cellular macromolecules involved in
telomere maintenance and the regulation of telomere length and
telomerase activity in mammalian cells have not. See Feng et al.,
1995, Science 269:1236-1241; PCT patent publication No. 96/01835;
and pending U.S. patent application Ser. Nos. 08/521,634, filed
Aug. 31, 1995, and 08/330,123, filed Oct. 27, 1994, each of which
is incorporated herein by reference.
[0005] Many useful methods and reagents relating to telomere and
telomerase biology have been described. See, e.g., U.S. Pat. No.
5,489,508; PCT patent publication Nos. 95/23572, 95/13381,
95/13382, and 95/13383; and U.S. patent application Ser. No.
08/632,662, filed Apr. 15, 1996, each of which is incorporated
herein by reference. Significant improvements to and new
opportunities for telomere- and telomerase-mediated therapies as
well as related assays, screens, diagnostic methods, and reagents
could be realized and obtained, however, if additional cellular
macromolecules involved in mammalian telomere maintenance and the
regulation of telomere length and telomerase activity could be
identified, characterized, and made available in pure or isolatable
form. In particular, the characterization of the nucleotide and
corresponding amino acid sequences of such macromolecules could
provide new and useful recombinant expression vectors and plasmids,
as well as related reagents useful in medical therapeutic and
diagnostic technology.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods and reagents for
regulating telomere length and modulating telomerase activity in
mammalian cells as well as for detecting, diagnosing, and treating
related diseases and conditions in humans and other mammals.
[0007] In one embodiment, the invention provides recombinant
mammalian host cells containing:
[0008] (i) a recombinant or synthetic nucleic acid comprising at
least about 10 to 15 to 25 to 100 or more contiguous nucleotides
corresponding to an open reading frame sequence of a human gene
TPC2 contained in a human DNA insert of an .about.3.5 kb
NotI-BstEII restriction fragment of plasmid pGRN109 (on deposit
with the American Type Culture Collection under the accession
number ATCC 97708); or
[0009] a synthetic or recombinant peptide or protein comprising at
least about 6 to 10 to 15 to 25 to 100 or more contiguous amino
acids corresponding to an amino acid sequence encoded by said open
reading frame sequence; and
[0010] (ii) a recombinant or synthetic nucleic acid comprising at
least about 10 to 15 to 25 to 100 or more contiguous nucleotides
corresponding to an open reading frame sequence of a human gene
TPC3 contained in a human DNA insert of an .about.1.4 kb
EcoRI-BamHI restriction fragment of plasmid pGRN92 (ATCC 97707);
or
[0011] a synthetic or recombinant peptide or protein comprising at
least about 6 to 10 to 15 to 25 to 100 or more contiguous amino
acids corresponding to an amino acid sequence encoded by said open
reading frame sequence of gene TPC3;
[0012] said TPC2 and TPC3 genes characterized in coding for
proteins that regulate telomere length or modulate telomerase
activity and are present in human or other mammalian cells that
express telomerase activity.
[0013] Other mammalian host cells provided by the invention include
those that comprise either or both TPC2- and TPC3-derived
recombinant or synthetic nucleic acids, peptides, or proteins.
Furthermore, the invention also provides such cells further
modified to contain a synthetic or recombinant nucleic acid
comprising at least about 10 to 15 to 25 to 100 or more contiguous
nucleotides corresponding to a contiguous nucleotide sequence of
human hTR located in an .about.2.5 kb HindIII-SacI restriction
fragment of pGRN33 (ATCC 75926).
[0014] The recombinant host cells of the invention have application
in many useful methods also provided by the invention. For example,
the invention provides recombinant host cells comprising novel
expression vectors with expression control sequences operatively
linked to nucleotide sequences encoding amino acids in a sequence
substantially identical to the amino acid sequences encoded by the
human TPC2 or TPC3 genes and, optionally, a recombinant hTR gene.
These recombinant host cells are useful for producing recombinant
human telomerase, for use in screens to identify agents that
modulate telomerase activity or regulate telomere length, as well
as for a variety of other purposes described more fully below. The
recombinant host cells of the invention can also be incorporated
into the germ line and/or somatic tissues of non-human transgenic
mammals, as well as be administered to mammals for therapeutic
purposes.
[0015] In another embodiment, the invention provides synthetic and
recombinant oligonucleotides and nucleic acids in a variety of
forms, i.e., isolatable, isolated, purified, or substantially pure,
and for a variety of purposes, i.e., as probes or primers, as
polynucleotide plasmids and vectors for introducing recombinant
gene products that regulate telomere length or modulate telomerase
activity in mammalian host cells, as restriction fragments for
creating useful nucleic acids, and as reagents for therapeutic,
diagnostic, and other applications. In particular, the invention
provides recombinant or synthetic nucleic acids comprising at least
about 10 to 15 to 25 to 100 or more contiguous nucleotides
substantially identical or complementary in sequence to a
contiguous nucleotide sequence located in either:
[0016] (i) an open reading frame sequence of a human gene TPC2
contained in a human DNA insert of an .about.3.5 kb NotI-BstEII
restriction fragment of plasmid pGRN109; or
[0017] (ii) an open reading frame sequence of a human gene TPC3
contained in a human DNA insert of an .about.1.4 kb EcoRI-BamHI
restriction fragment of plasmid pGRN92.
[0018] The novel oligonucleotide probes and primers of the
invention typically comprise nucleotides in a sequence
substantially identical or complementary to a sequence of
nucleotides in a TPC2 or TPC3 gene or gene product to allow
specific hybridization thereto in a complex mixture of nucleic
acids. Such probes and primers therefore have useful application in
a variety of diagnostic, therapeutic, and other applications.
[0019] The expression vectors of the invention typically comprise
expression control sequences operatively linked to a nucleotide
sequence encoding amino acids in a sequence identical to a sequence
of amino acids in a TPC2 or TPC3 protein gene product. Such
expression vectors have many useful applications, including in
therapeutic methods of the invention as gene therapy vectors for
modulating telomerase activity, either to activate or inhibit that
activity, or for regulating telomere length, either to increase or
decrease the length, in a target cell or tissue.
[0020] Gene therapy expression vectors of the invention also
include those that encode variants or "muteins" of the TPC2 and/or
TPC3 proteins, i.e., express proteins that differ from TPC2 and/or
TPC3 by deletion, substitution, and/or addition of one or more
amino acids. The gene therapy vectors of the invention may also,
however, encode useful nucleic acids, such as hTR, or antisense
nucleic acids or ribozymes that target the TPC2, TPC3, and/or hTR
gene products, i.e., mRNA and telomerase RNA. Such vectors are
useful in the therapeutic methods of the invention for treating or
preventing diseases or conditions in which modulation of telomerase
activity or telomere length can be of benefit. For example, in
telomerase positive cancer cells, inhibition of telomerase activity
can prevent telomere maintenance in those cells, inducing upon
continued proliferation telomere loss, cell crisis, and death. For
such purposes, the gene therapy vectors of the invention that
express a non-functional TPC2 or TPC3 mutein or variant protein or
other nucleic acid that can inhibit telomerase formation or
telomere elongation by telomerase activity in the cell, such as by
competing for RNA component or protein components, inhibition of
endogenous gene expression, or other means, are preferred.
[0021] In another embodiment, the present invention provides
peptides, proteins, antibodies, and enzymes, relating to genes and
gene products that regulate telomere length and telomerase activity
in mammalian cells. In particular, the invention provides synthetic
or recombinant peptides or proteins comprising at least about 6 to
10 to 15 to 25 to 100 or more contiguous amino acids identical in
sequence to an amino acid sequence encoded by an open reading frame
sequence of a human gene located in either:
[0022] (i) an .about.3.5 kb NotI-BstEII restriction fragment of
plasmid pGRN109; or
[0023] (ii) an .about.1.4 kb EcoRI-BamHI restriction fragment of
plasmid pGRN92.
[0024] The present invention provides the proteins encoded by the
TPC2 and TPC3 genes in isolatable form from host cells expressing
recombinant TPC2 and/or TPC3 protein, as well as in purified and
substantially pure form from synthesis in vitro or by purification
from recombinant host cells or by purification of the naturally
occurring proteins using antibodies or other reagents of the
invention. Such proteins have application in methods for
reconstituting in vitro telomerase or other enzymatic activities
that maintain telomeres and regulate telomere length. These methods
in turn have application in screens for therapeutic agents, for
diagnostic tests, and for other applications. In addition, peptides
corresponding to the amino acid sequences of TPC2 or TPC3 proteins
can also be used to regulate telomere length and telomerase
activity in mammalian cells.
[0025] The proteins and peptides of the invention can also be used
to generate antibodies specific for TPC2 or TPC3 proteins or for
particular epitopes on those proteins. Thus the invention provides
polyclonal and monoclonal antibodies that specifically bind to TPC2
or TPC3 proteins. These antibodies can in turn be used to isolate
TPC2 or TPC3 proteins from normal or recombinant cells and so can
be used to purify the proteins as well as other proteins associated
therewith. These antibodies also have important application in the
detection of cells comprising TPC2 or TPC3 proteins in complex
mixtures of cells. Such detection methods have application in
screening, diagnosing, and monitoring diseases and other
conditions, such as cancer, pregnancy, or fertility, because the
TPC2 and TPC3 proteins are present in most cells capable of
elongating telomeric DNA and expressing telomerase activity.
[0026] The immunogenic peptides and proteins of the invention can
also be used in therapeutic immunization and vaccination
procedures. See U.S. provisional patent application Ser. No.
60/008,949, filed Oct. 20, 1995, incorporated herein by reference.
The invention provides a method of immunizing a subject, as well as
vaccines useful in the method, against cells that maintain
telomeres and express telomerase activity that comprises
administering an immunostimulating amount of such peptides or
proteins of the invention.
[0027] In another embodiment, the invention provides a subtraction
hybridization differential display method to identify, isolate, and
clone expressed sequence tags (ESTs) of mRNA species encoding rare
proteins, such as those involved in telomere elongation and the
regulation of telomere length and telomerase activity. This method
comprises the steps of:
[0028] (i) obtaining mRNA from a first population of mammalian
cells which contain said rare protein, i.e., a protein component of
telomerase, and from a second population of mammalian cells which
do not contain said rare protein;
[0029] (ii) subjecting such mRNA to reverse-transcription and
second-strand synthesis to form first and second cDNA preparations,
said first and second cDNA preparations differing from one another
with respect to presence or absence of cDNA molecules encoding said
rare protein and a label incorporated into one of said first and
second cDNA preparations;
[0030] (iii) combining said cDNA preparations under conditions such
that complementary strands of cDNA from said first and second cDNA
preparations anneal to form a mixture of double-stranded and
single-stranded cDNA; and
[0031] (iv) separating cDNA comprising said label from cDNA that
does not, thereby forming an isolated preparation of cDNA from said
first population that has been depleted from complementary cDNA in
said second population and enriched for said cDNA encoding said
rare protein. Steps (iii) and (iv) of the above method can be
repeated as often as desired, and the cDNA isolated after
completion of step (iv) can be amplified by PCR, to provide cDNA
preparations greatly enriched for the desired cDNA.
[0032] These and other embodiments of the invention will be
described in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1, in parts A, B, and C, is a bar graph showing the
results of RT-PCR analysis using primers specific for TPC2 (FIG.
1A) or TPC3 (FIG. 1B) cDNA. In this and the other bar graphs, the
number over each bar is the numerical result obtained; for RT-PCR
results, this number was generated by scanning autoradiograms or
Phosphorlmager.TM. screens (Molecular Dynamics) of the RT-PCR
products after gel electrophoresis. Under these test conditions,
TPC2 and TPC3 mRNA is absent or detectable only at very low levels
in the telomerase negative cell lines tested (labeled "Mortal" in
the Figure) and detectable in all (most at clearly detectable
levels) telomerase positive cell lines tested (labeled "Immortal"
in the Figure). FIG. 1C shows TPC3 mRNA levels normalized to GAPDH
levels and illustrates the difference in TPC3 mRNA levels between
mortal and immortal cells (the spaces marked "0.0" are provided
merely as breaks in the graphed data). GAPDH mRNA was used as a
control; due to its greater abundance, the RT-PCR of the GAPDH
samples was allowed to complete fewer cycles of PCR than used for
the TPC2 or TPC3 samples.
[0034] FIG. 2, in parts A, B, and C, is a bar graph showing the
results of an RT-PCR analysis of hTR RNA and TPC2 and TPC3 mRNA
levels as well as telomerase activity in a variety of cell lines.
FIG. 2A shows TPC2 and TPC3 mRNA levels normalized to GAPDH mRNA
levels in various cell lines, all of which are telomerase positive
except IMR-90, and demonstrates a correlation in the levels of
these two telomere length and telomerase activity regulatory
proteins. FIG. 2B shows how TPC3 mRNA levels correlate with
telomerase activity (as measured using the TRAP assay) in a variety
of cell lines. The IMR90, HTB-153, WI-38 VA13, KMSF, and T0
(unactivated T cells; note that T7 represents activated T cells)
express no or only very low levels of telomerase activity. FIG. 2C
shows how hTR RNA levels correlate with telomerase activity levels
in a variety of cell lines. Taken together, these results show that
TPC2 and TPC3 mRNA levels correlate with hTR levels and with
telomerase activity levels in a variety of mortal and immortal
cells lines.
[0035] FIG. 3 shows a restriction site and function map of the
.about.7.2 kb plasmid pGRN109, which contains an .about.3.5 kb
NotI-BstEII restriction fragment that contains an .about.3.3 kb
open reading frame encoding the TPC2 protein (labeled "ORF" and
"TPC2").
[0036] FIG. 4 lists portions of the nucleotide (SEQ ID NO: 1)
sequence and deduced amino acid sequence (SEQ ID NO:2) of the TPC2
open reading frame corresponding to the human TPC2 gene, mRNA, and
protein products. In the Figure, as well as throughout the
specification and Figures, nucleotides and amino acids are
represented using standard abbreviations and designations; however,
ambiguous nucleotides are represented as shown in the key at the
bottom of FIG. 4. The initiating methionine codon is believed to be
at nucleotides 16-18 of the sequence; the termination codon is
marked with "---".
[0037] FIG. 5 shows a restriction site and function map of the
.about.8 kb plasmid pGRN92, which contains an .about.1.4 kb
EcoRI-BamHI restriction fragment that contains an .about.1.1 kb
open reading frame encoding the TPC3 protein (labeled "ORF" and
"TPC3").
[0038] FIG. 6 lists the nucleotide sequence (SEQ ID NO: 4) and
deduced amino acid sequence (SEQ ID NO: 4) of the TPC3 open reading
frame corresponding to the human TPC3 gene, mRNA, and protein
products. The initiating methionine codon is marked with "***" and
the stop codon with "---".
[0039] FIG. 7 shows the results of an analysis of telomerase
activity levels in stable recombinant HeTe7 clones expressing the
sense or antisense mRNA of gene TPC3 or a control vector. The
recombinant sense TPC3 mRNA reduced telomerase activity markedly in
these cells.
[0040] FIG. 8 shows the results of an analysis of telomere length
in stable recombinant HeTe7 clones expressing the sense or
antisense mRNA of gene TPC3 or a control vector. The recombinant
TPC3 sense mRNA decreased telomere length (mean TRF) in the
cells.
[0041] FIG. 9 lists the nucleotide sequence (SEQ ID NO: 5) of the
hTR gene and corresponding RNA transcript; the sequence shown is
that of one strand of an .about.1 kb PstI restriction fragment that
can be isolated from plasmid pGRN33. The sequence of the mature hTR
transcript, which serves as the template in the telomerase
ribonucleoprotein, is masked with ***--the 3' end of the transcript
is marked with an ">".
[0042] These Figures are discussed in more detail below, where a
variety of preferred embodiments of the invention are
described.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The present invention provides methods and reagents for
regulating telomere length and modulating telomerase activity in
mammalian cells as well as for detecting, diagnosing, and treating
related diseases and conditions in humans and other mammals. To
facilitate understanding and practice of the invention in its many
and diverse applications, this description is organized as shown
below.
[0044] i. DEFINITIONS
[0045] i. CLONING AND CHARACTERIZATION OF THE TPC2 AND TPC3
GENES
[0046] III. RECOMBINANT HOST CELLS
[0047] IV. OLIGONUCLEOTIDES AND NUCLEIC ACIDS
[0048] V. PEPTIDES AND PROTEINS
[0049] VI. ANTIBODIES
[0050] VII. METHODS
[0051] VIII. EXAMPLES
[0052] I. Definitions
[0053] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For purposes of the present invention, the following terms are
defined below.
[0054] "Antibody" refers to naturally occurring and recombinant
polypeptides and proteins encoded by immunoglobulin genes, or
fragments thereof, that specifically bind to or "recognize" an
analyte or "antigen". Immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. An antibody
can exist as an intact immunoglobulin or as any one of a number of
well characterized fragments, e.g., Fab' and F(ab)'.sub.2
fragments, produced by various means, including recombinant
methodology and digestion with various peptidases.
[0055] "cDNA" refers to deoxyribonucleic acids produced by
reverse-transcription and typically second-strand synthesis of mRNA
or other RNA produced by a gene; if double-stranded, a cDNA
molecule has both a coding or sense and a non-coding or antisense
strand. "Complementary to" refers to a polynucleotide sequence that
can hybridize specifically to another polynucleotide sequence; for
example, a nucleic acid comprising nucleotides in the sequence
"5'-TATAC" is complementary to a nucleic acid comprising
nucleotides in the sequence "5'-GTATA".
[0056] "Corresponds to" or "corresponding to" refers to (i) a
polynucleotide having a nucleotide sequence that is substantially
identical or complementary to all or a portion of a reference
polynucleotide sequence or encoding an amino acid sequence
identical to an amino acid sequence in a peptide or protein; or
(ii) a peptide or polypeptide having an amino acid sequence that is
substantially identical to a sequence of amino acids in a reference
peptide or protein.
[0057] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a nucleic acid, such as a gene in a
chromosome or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having a
defined sequence of nucleotides (i.e., rRNA, tRNA, other RNA
molecules) or amino acids and the biological properties resulting
therefrom. Thus a gene encodes a protein, if transcription and
translation of mRNA produced by that gene produces the protein in a
cell or other biological system. Both the coding strand, the
nucleotide sequence of which is identical to the mRNA sequence and
is usually provided in sequence listings, and non-coding strand,
used as the template for transcription, of a gene or cDNA can be
referred to as encoding the protein or other product of that gene
or cDNA. A nucleic acid that encodes a protein includes any nucleic
acids that have different nucleotide sequences but encode the same
amino acid sequence of the protein due to the degeneracy of the
genetic code. Nucleic acids and nucleotide sequences that encode
proteins may include introns.
[0058] "Expression control sequence" refers to nucleotide sequences
in nucleic acids that regulate the expression (transcription and/or
translation) of a nucleotide sequence operatively linked thereto.
Expression control sequences can include, for example and without
limitation, sequences of promoters, enhancers, transcription
terminators, a start codon (i.e., ATG), splicing signals for
introns, and stop codons.
[0059] "Immunoassay" refers to an assay that utilizes an antibody
to bind an analyte specifically. An immunoassay is characterized by
the use of specific binding properties of a particular antibody to
isolate, target, and/or quantify the amount of an analyte.
[0060] "Label" or "labeled" refers to a detectable marker and to
the incorporation of such a marker into a nucleic acid, protein, or
other molecule. The label may be detectable directly, i.e., the
label can be a radioisotope (e.g., .sup.3H, .sup.14C., .sup.35S,
.sup.125I, .sup.131I) or a fluorescent or phosphorescent molecule
(e.g., FITC, rhodamine, lanthanide phosphors), or indirectly, i.e.,
by enzymatic activity (e.g., horseradish peroxidase,
beta-galactosidase, luciferase, alkaline phosphatase) or ability to
bind to another molecule (e.g., streptavidin, biotin, an epitope).
Incorporation of a label can be achieved by a variety of means,
i.e., by use of radiolabeled or biotinylated nucleotides in
polymerase-mediated primer extension reactions, epitope-tagging, or
binding to an antibody. Labels can be attached directly or via
spacer arms of various lengths to reduce steric hindrance.
[0061] "Naturally occurring" refers to a substance, typically an
amino acid, nucleotide, nucleic acid, or protein, that exists in
nature without human intervention. For example, deoxyribonucleic
acid or DNA is naturally occurring.
[0062] "Oligonucleotide" refers to a polymer composed of a
multiplicity of nucleotide units (ribonucleotides or
deoxyribonucleotides or related structural variants or synthetic
analogs thereof linked via phosphodiester bonds (or related
structural variants or synthetic analogs thereof. Thus, while the
term "oligonucleotide" typically refers to a nucleotide polymer in
which the nucleotides and the linkages between them are naturally
occurring; the term also refers to various analogs, such as, for
example and without limitation, peptide-nucleic acids (PNAs),
phosphoramidates, phosphorothioates, methyl phosphonates,
2-O-methyl ribonucleic acids, and the like. An oligonucleotide
typically rather short in length, generally from about 10 to 30
nucleotides, but the term can refer to molecules of any length,
although the term "polynucleotide" or "nucleic acid" is typically
used for large oligonucleotides.
[0063] "Open reading frame" refers to a nucleotide sequence that
encodes a polypeptide or protein and is bordered on the 5'-end by
an initiation codon (ATG) or another codon that does not encode a
stop codon and on the 3'-end by a stop codon but otherwise does not
contain any in-frame stop codons between the codons at the
5'-border and the 3'-border.
[0064] "Pharmaceutical composition" refers to a composition
suitable for pharmaceutical use in a mammal. A pharmaceutical
composition comprises a pharmacologically effective amount of an
active agent and a pharmaceutically acceptable carrier.
"Pharmacologically effective amount" refers to that amount of an
agent effective to produce the intended pharmacological result.
"Pharmaceutically acceptable carrier" refers to any of the standard
pharmaceutical carriers, buffers, and excipients, such as a
phosphate buffered saline solution, 5% aqueous solution of
dextrose, and emulsions, such as an oil/water or water/oil
emulsion, and various types of wetting agents and/or adjuvants.
Suitable pharmaceutical carriers and formulations are described in
Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.,
Easton, 1995). Preferred pharmaceutical carriers depend upon the
intended mode of administration of the active agent. Typical modes
of administration include enteral (i.e., oral) or parenteral (i.e.,
subcutaneous, intramuscular, or intravenous intraperitoneal
injection; or topical, transdermal, or transmucosal
administration).
[0065] "Physiological conditions" refer to temperature, pH, ionic
strength, viscosity, and like biochemical parameters that are
compatible with a viable organism and/or that typically exist
intracellularly in a viable mammalian cell. For example, the
intracellular conditions in a mammalian cell grown under typical
laboratory culture conditions are physiological conditions.
Suitable in vitro reaction conditions for PCR and many
polynucleotide enzymatic reactions and manipulations are generally
physiological conditions. In general, in vitro physiological
conditions comprise 50-200 mM NaCI or KCI, pH 6.5-8.5, 20-45
degrees C., and 0.001-10 mM divalent cation (e.g., Mg++, Ca++);
preferably about 150 mM NaCI or KCI, pH 7.2-7.6, 5 mM divalent
cation, and, often, including 0.01-1.0 percent nonspecific protein
(e.g., BSA). A non-ionic detergent (Tween, NP -40 Triton X-100) can
also be present, usually at about 0.001 to 2%, typically 0.05-0.2%
(v/v). Particular aqueous conditions may be selected by the
practitioner according to conventional methods. For general
guidance, the following buffered aqueous conditions may be
applicable: 10-250 mM NaCI, 5-50 mM Tris HCI, pH 5-8, with optional
addition of divalent cation(s) and/or metal chelators and/or
nonionic detergents and/or membrane fractions and/or antifoam
agents and/or scintillants.
[0066] "Polynucleotide" or "nucleic acid" refers to an
oligonucleotide and is typically used to refer to oligonucleotides
greater than 30 nucleotides in length. Conventional notation is
used herein to portray polynucleotide sequences: the left-hand end
of single-stranded polynucleotide sequences is the 5'-end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5'-direction. The direction of 5' to 3' addition
of nucleotides to nascent RNA transcripts is referred to as the
transcription direction; the DNA strand having the same sequence as
an mRNA is referred to as the "coding strand"; sequences on the DNA
strand having the same sequence as an mRNA transcribed from that
DNA and which are located 5' to the 5'-end of the RNA transcript
are referred to as "upstream sequences"; sequences on the DNA
strand having the same sequence as the RNA and which are 3' to the
3' end of the coding RNA transcript are referred to as "downstream
sequences". Polynucleotides and recombinantly produced protein, and
fragments or analogs thereof, may be prepared according to methods
known in the art and described in Maniatis et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., (1989), Cold Spring Harbor,
N.Y., and Berger and Kimmel, Methods in Enzymology, Volume 152,
Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,
San Diego, Calif., which are incorporated herein by reference.
[0067] "Polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
and to variants and synthetic analogs of the same. Thus, these
terms apply to amino acid polymers in which one or more amino acid
residues is a synthetic non-naturally occurring amino acid, such as
a chemical analog of a corresponding naturally occurring amino
acid, as well as to naturally occurring amino acid polymers.
Conventional notation is used herein to portray polypeptide
sequences: the left-hand end of polypeptide sequences is the
amino-terminus; the right-hand end of polypeptide sequences is the
carboxy-terminus. The term "recombinant protein" refers to a
protein that is produced by expression of a recombinant DNA
molecule that encodes the amino acid sequence of the protein. Terms
used to describe sequence relationships between two or more
polynucleotides or polypeptides include "reference sequence",
"comparison window", "sequence identity", "percentage of sequence
identity", and "substantial identity". A "reference sequence" is a
defined sequence used as a basis for a sequence comparison and may
be a subset of a larger sequence, i.e., a complete cDNA, protein,
or gene sequence. Generally, a reference sequence is at least 12
but frequently 15 to 18 and often at least 25 nucleotides (or other
monomer unit) in length. Because two polynucleotides may each
comprise (1) a sequence (i.e., only a portion of the complete
polynucleotide sequence) that is similar between the two
polynucleotides, and (2) a sequence that is divergent between the
two polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison window"
refers to a conceptual segment of typically at least 12 contiguous
residues that is compared to a reference sequence; the comparison
window may comprise additions or deletions (i.e., gaps) of about 20
percent or less as compared to the reference sequence (which does
not comprise additions or deletions) for optimal alignment of the
two sequences. Optimal alignment of sequences for aligning a
comparison window may be conducted by computerized implementations
of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.) or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by any of the various methods
is selected.
[0068] "Primer" refers to an oligonucleotide, i.e., a purified
restriction fragment or a synthetic oligonucleotide, that is
capable of acting as a point of initiation of synthesis when placed
under conditions in which synthesis of a primer extension product
complementary to a nucleic acid strand (the "template") is induced,
i.e., in the presence of nucleotides and an agent for
polymerization such as DNA polymerase and at a suitable temperature
and pH. The primer is preferably single-stranded for maximum
efficiency in amplification but may alternatively be
double-stranded. If double stranded, the primer may need to be
treated to separate its strands before being used to prepare
extension products. Primers are typically
oligodeoxyribonucleotides, but a wide variety of synthetic and
non-naturally occurring oligonucleotide primers can be used for
various applications. A primer must be sufficiently long to prime
the synthesis of extension products in the presence of the agent
for polymerization. The length of a primer depends on many factors,
including application, temperature to be employed, template,
reaction conditions, other reagents, and source of primers. For
example, depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-25 or more
nucleotides, although it may contain fewer nucleotides. Short
primer molecules generally require cooler temperatures to form
stable hybrid complexes with template. Primers can be large
polynucleotides, such as from about 200 nucleotides to several
kilobases or more. A primer must be substantially complementary to
the sequence on the template to which it is designed to hybridize
to serve as a site for the initiation of synthesis but need not
reflect the exact sequence of the template. For example,
non-complementary nucleotides may be attached to the 55'-end of the
primer, with the remainder of the primer sequence being
complementary to the template. Alternatively, non-complementary
nucleotides or longer sequences can be interspersed into a primer,
provided that the primer sequence has sufficient complementarity
with the sequence of the template to hybridize therewith and
thereby form a template for synthesis of the extension product of
the primer.
[0069] "Probe" refers to a molecule that binds to a specific
sequence or subsequence or other moiety of another molecule. Unless
otherwise indicated, the term "probe" typically refers to an
oligonucleotide probe that binds to another nucleic acid, often
called the "target nucleic acid", through complementary base
pairing. Probes may bind target nucleic acids lacking complete
sequence complementarity with the probe, depending upon the
stringency of the hybridization conditions. Probes can be directly
or indirectly labeled.
[0070] "Recombinant" refers to methods and reagents in which
nucleic acids synthesized or otherwise manipulated in vitro are
used to produce gene products encoded by those nucleic acids in
cells or other biological systems. For example, an amplified or
assembled product polynucleotide may be inserted into a suitable
DNA vector, such as a bacterial plasmid, and the plasmid can be
used to transform a suitable host cell. The gene is then expressed
in the host cell to produce the recombinant protein. The
transformed host cell may be prokaryotic or eukaryotic, including
bacterial, mammalian, yeast, Aspergillus, and insect cells. A
recombinant polynucleotide may serve a non-coding function (e.g.,
promoter, origin of replication, ribosome-binding site, etc.) as
well.
[0071] "Recombinant host cell" refers to a cell that comprises a
recombinant nucleic acid molecule, typically a recombinant plasmid
or other expression vector. Thus, for example, recombinant host
cells can express genes that are not found within the native
(non-recombinant) form of the cell.
[0072] "Selected from" refers, in connection with sequences, to one
sequence sharing identity with another sequence.
[0073] "Sequence identity" refers to sequences that are identical
(i.e., on a nucleotide-by-nucleotide or amino acid-by-amino acid
basis) over the window of comparison. The term "percentage of
sequence identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, U, or I) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity.
[0074] "Specifically binds to" refers to the ability of one
molecule, typically a macromolecule such as an antibody or
oligonucleotide, to contact and associate with another specific
molecule even in the presence of many other diverse molecules. For
example, a single-stranded nucleic acid can "specifically bind to"
a single-stranded oligonucleotide that is complementary in
sequence, and an antibody "specifically binds to" or "is
specifically immunoreactive with" its corresponding antigen. Thus,
under designated immunoassay conditions, an antibody binds
preferentially to a particular protein and not in a significant
amount to other proteins present in the sample. Specific binding to
a protein under such conditions requires an antibody selected for
its specificity for a particular protein. To select antibodies
specifically immunoreactive with a particular protein, one can
employ a variety of means, i.e., solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See Harlow and Lane (1988),
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
N.Y.
[0075] "Specific hybridization" refers to the formation of hybrids
between a probe polynucleotide (e.g., a polynucleotide of the
invention which may include substitutions, deletions, and/or
additions) and a specific target polynucleotide (e.g., a
polynucleotide having the sequence of a TPC2 or TPC3 gene or gene
product), wherein the probe preferentially hybridizes to the
specific target and not to other polynucleotides in the mixture
that do not share sequence identity with the target.
[0076] "Substantial identity" or "substantially identical" denotes
a characteristic of a polynucleotide or polypeptide that comprises
a sequence that is at least 80 percent identical, preferably at
least 85 percent and often 90 to 95 percent identical, more usually
at least 99 percent identical, to a reference sequence over a
comparison window of at least 20 nucleotide positions, frequently
over a window of at least 25 to 50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence, which may
include deletions or additions that total 20 percent or less of the
reference sequence, over the window of comparison. The reference
sequence may be a subset of a larger sequence.
[0077] "Stringent conditions" refer to temperature and ionic
conditions used in nucleic acid hybridization. The stringency
required is nucleotide sequence dependent and also depends upon the
various components present during hybridization. Generally,
stringent conditions are selected to be about 5 to 20 degrees C.
lower than the thermal melting point T.sub.m) for the specific
sequence at a defined ionic strength and pH. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
target sequence hybridizes to a complementary probe.
[0078] "Substantially pure" means an object species is the
predominant species present (i.e., on a molar basis, more abundant
than any other individual macromolecular species in the
composition), and a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition means that about 80 to
90 percent or more of the macromolecular species present in the
composition is the purified species of interest. The object species
is purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods) if
the composition consists essentially of a single macromolecular
species. Solvent species, small molecules (<500 Daltons),
stabilizers (e.g., BSA), and elemental ion species are not
considered macromolecular species for purposes of this
definition.
[0079] "Suitable reaction conditions" are those conditions suitable
for conducting a specified reaction using commercially available
reagents. Such conditions are known or readily established by those
of skill in the art for a variety of reactions. For example,
suitable polymerase chain reaction (PCR) conditions include those
conditions specified in U.S. Pat. Nos. 4,683,202; 4,683,195;
4,800,159; and 4,965,188, each of which is incorporated herein by
reference. As one example and not to limit the invention, suitable
reaction conditions can comprise: 0.2 mM each dNTP, 2.2 mM
MgCI.sub.2, 50 mM KCI, 10 mM Tris-HCI, pH 9.0, and 0.1% Triton
X-100.
[0080] "telomere length regulatory protein" and "telomerase
regulatory protein" refers to polypeptides involved in telomere
metabolism and telomerase activity. Such proteins include
telomerase, the protein components of telomerase, proteins that
selectively bind nucleic acids containing telomere repeat sequences
or telomeric ends, proteins required for telomere repair,
maintenance, and/or elongation, and proteins necessary for
expression or formation of active telomerase enzyme. Although the
present invention relates to such proteins generally, mammalian
telomerase, and particularly human telomerase, and related proteins
are provided as preferred embodiments.
[0081] "Telomerase activity" refers to the ability of telomerase
protein components to associate with one another and the RNA
component of telomerase either in vivo or in vitro into a
multi-component enzyme that can elongate telomeric DNA. A preferred
assay method for detecting telomerase activity is the TRAP assay.
See PCT patent publication No. 95/13381, supra. This assay measures
the amount of radioactive nucleotides incorporated into elongation
products, polynucleotides, formed by nucleotide addition to a
telomerase substrate or primer. The radioactivity incorporated can
be measured as a function of the intensity of a band on a
Phosphorlmager.TM. screen exposed to a gel on which the radioactive
products are separated. A test experiment and a control experiment
can be compared by visually using the Phosphorlmager.TM. screens.
See also the commercially available TRAP-eze.TM.. telomerase assay
kit (Oncor); and Morin, 1989, Cell 59:521-529.
[0082] II. Cloning and Characterization of the TPC2 and TPC3
Genes
[0083] The present invention provides methods and reagents for
regulating telomere length and modulating telomerase activity in
mammalian cells as well as for detecting, diagnosing, and treating
related diseases and conditions in humans and other mammals. The
present invention arose in part out of an effort to clone the
protein components of telomerase and other protein components of
macromolecules that regulate telomere length and telomerase
activity in human and other mammalian cells. These rare proteins
and the mRNAs that encode these proteins are present in very low
abundance in mammalian cells, necessitating the use of a novel mRNA
isolation and identification method called "subtraction
hybridization differential display."
[0084] In brief, this method involves obtaining mRNA from a first
population of mammalian cells which contain the rare or low
abundant protein of interest and from a second population of
mammalian cells that contain 10- to 100-fold lower levels of the
rare protein. The two mRNA populations are then individually used
to generate cDNA preparations by reverse-transcription and
second-strand synthesis to form first and second cDNA preparations.
A detectable label is incorporated as well into the second cDNA
preparation. The two cDNA preparations are then denatured and
combined under conditions such that complementary strands of cDNA
from the two cDNA preparations anneal to form a mixture of
double-stranded and single-stranded cDNA. The mixture of cDNAs is
then separated into two different populations, one comprising the
label and one that does not, thereby forming an isolated, unlabeled
preparation of cDNA that has been enriched for cDNA encoding the
rare protein of interest. The steps of hybridization and separation
can be repeated as often as desired, and the cDNA isolated after
the separation step can be amplified by PCR, to provide cDNA
preparations greatly enriched for the desired cDNA. Typically after
two cycles of subtraction, cDNAs corresponding to abundant
transcripts are depleted more than 100-fold and low abundant
transcripts are enriched in the subtracted cDNA libraries. The
reproducibility of the method is excellent, and the method can be
used to identify low abundant gene products such as those encoding
telomere length and telomerase regulatory proteins.
[0085] To isolate cDNAs corresponding to telomere length and
telomerase regulatory proteins, cDNA libraries were prepared from
six different cells lines or tissues, three of which were
"telomerase positive" (i.e., the cells express telomerase activity;
the IDH4 and 293 cell lines, and testes tissue), and three of which
were "telomerase negative" (i.e., the cells do not express
telomerase activity; the HUVEC, BJ, and IMR-90 cell lines). These
cDNA libraries were subjected to subtraction hybridization against
the telomerase negative HUVEC cDNA library. Then, differential
display was performed by first replicating each of the six
subtracted cDNA libraries with either a single 5'-arbitrary primer
or in a PCR with a 5'-arbitrary primer and a 3'-polydT primer,
separating the replication products by gel electrophoresis, and
identifying and isolating the differentially expressed products
(identified visually as bands on a gel).
[0086] This process generated a number of differentially expressed
cDNAs. Two of these cDNAs that were present in the cDNA libraries
generated from the telomerase positive cell lines but not present
(or present at much lower levels) in the telomerase negative cell
lines, and that were later identified as originating from the
3'-ends of mRNA produced by the TPC2 and TPC3 genes, were isolated,
cloned, and characterized by DNA sequence analysis. The DNA
sequence analysis was used to design oligonucleotide primers that,
in turn, were used to perform reverse-transcription and PCR
(RT-PCR) on mRNA prepared from each of the same panel of six cell
lines used to prepare the subtracted cDNA libraries. This RT-PCR
experiment was designed to confirm that the mRNA corresponding to
the putatively differentially expressed cDNAs is expressed at much
higher levels in telomerase positive cell lines. The results were
as predicted: the RT-PCR generated products of the predicted size;
for the primers specific for the TPC2 mRNA, a substantial amount of
product was generated using IDH4 mRNA, while lower amounts of
product were generated using 293 and testes mRNA, and product was
almost undetectable in mRNA prepared from HUVEC, BJ, and IMR-90
cells; for the primers specific for the TPC3 mRNA, product was
generated only using mRNA from the telomerase positive cell
lines.
[0087] To extend the analysis of the expression pattern of TPC2 and
TPC3 in various cell lines and tissues, RT-PCR with primers
specific for nucleotide sequences in the cDNAs corresponding to the
differentially expressed TPC2 and TPC3 mRNAs was performed on a
variety of cell lines. As a control, RT-PCR with primers specific
for nucleotide sequences in GAPDH mRNA (GAPDH is a "house-keeping"
enzyme present in both telomerase positive and telomerase negative
cell lines) was performed as well. In brief, the primers used for
TPC2 were:
1 the primers used for TPC2 were: tpc-p1
5'-ATGGGGATTCCAGGGTGGAGCT-3', and (SEQ ID NO: 6) tpc-p4
5'-ACCTGCTCTCAGGGCCCACMGT-3', (SEQ ID NO: 7) and the primers used
for TPC3 were: tpc-p13 5'-TAAGACAAAGAACAGGTCACMCA-3', and (SEQ ID
NO: 8) tpc-p14 5'-ATTTGTGCTTAGAGGTCGTGCCAG-3'. (SEQ ID NO: 9)
[0088] The RT-PCR was performed by making first strand cDNA made
from total RNA with random hexamer primers and then PCR-amplifying
the single-stranded cDNA with one of the two primer sets above,
following the protocol of 16 to 28 cycles of PCR amplification
(typically, 16 cycles for GAPDH mRNA, 25 cycles for TPC2 mRNA, and
27 cycles for TPC3 mRNA), with each cycle consisting of a step at
94 degrees C. for 45 sec., 65 degrees C. for 45 sec., and 72
degrees C. for 90 sec. Other illustrative RT-PCR primers and
conditions are shown in Parts C and D of the Examples below.
[0089] FIG. 1, in parts A, B, and C, shows the results of RT-PCR
analysis using primers specific for the TPC2 (FIG. 1A) or TPC3
(FIG. 1B) cDNA. Under these test conditions, TPC2 and TPC3 mRNA is
absent or detectable only at very low levels in the telomerase
negative cell lines tested (labeled "Mortal" in the Figure) and
detectable in all (most at clearly detectable levels) telomerase
positive cell lines tested (labeled "Immortal" in the Figure).
These results, which show that TPC2 and TPC3 mRNA is present in
testes tissue as well as most tumor cell lines but absent or
present at lower abundance in normal cell lines, demonstrate how
the methods of the invention for detecting and quantitating TPC2
and/or TPC3 gene products can be used to detect immortal cells,
especially telomerase positive cancer cells, and so to diagnose
cancer and other diseases and conditions in humans and other
mammals. FIG. 1C shows TPC3 mRNA levels normalized to GAPDH levels
and illustrates the clear difference in TPC3 mRNA levels between
mortal and immortal cells. This RT-PCR analysis also indicated
that, as expected, the TPC2 and TPC3 mRNA is present in very low
abundance even in telomerase positive cells TPC2 or TPC3 mRNA
amplification products detected after .about.25 cycles; GAPDH or
HPRT detected after .about.15 or .degree.20 cycles, respectively).
Confirmatory evidence for the low abundance of TPC2 mRNA in
telomerase positive cells was obtained in the cloning of a cDNA
corresponding to one-half of the full length TPC2 mRNA, where a
primary screen of a lambda GT11 cDNA library from telomerase
positive 293 cells showed that only one of .about.1.4 million
plaques was positive, indicating a very rare transcript.
[0090] FIG. 2, in parts A, B, and C, is a bar graph showing the
results of an RT-PCR analysis of hTR RNA and TPC2 and TPC3 mRNA
levels as well as telomerase activity in a variety of cell lines.
FIG. 2A shows TPC2 and TPC3 mRNA levels normalized to GAPDH mRNA
levels in various cell lines, all of which are telomerase positive
except IMR-90, and demonstrates a correlation in the levels of
these two telomere length and telomerase activity regulatory
proteins. FIG. 2B shows how TPC3 mRNA levels correlate with
telomerase activity levels in a variety of cell lines. The IMR90,
HTB-153, WI-38 VA13, KMSF, and TO (unactivated T cells; note that
T7 represents activated T cells) express no or only very low levels
of telomerase activity. FIG. 2C shows how hTR RNA levels correlate
with telomerase activity levels in a variety of cell lines. The
RT-PCR protocol for hTR RNA is described in Part D of the Examples;
the nucleotide sequence of the hTR gene and transcribed RNA is
shown in FIG. 9.
[0091] Taken together, these FIGS. show that TPC2 and TPC3 mRNA
levels as well as hTR levels correlate with telomerase activity
levels in a variety of mortal and immortal cells lines. These
results demonstrate how the methods of the invention for detecting
TPC2 or TPC3 gene products can be used to detect immortal cells,
especially telomerase positive cancer cells, and so to diagnose
cancer and other diseases and conditions in humans and other
mammals. These results also demonstrate the utility of the methods
of the invention in which the detection or quantitation of TPC2 or
TPC3 gene products, together with measurements of other factors,
shTR levels, can bength, telomerase activity, or hTR levels, can be
used to identify immortal cells, such as cancer cells, or to
evaluate the proliferative capacity of a cell.
[0092] The absence or very low abundance of the TPC2 and TPC3 gene
products in telomerase negative mortal cells and the low but
clearly detectable abundance of those gene products in telomerase
positive immortal cells demonstrate the utility of the methods and
reagents of the invention for detecting the presence gene products
that encode proteins such as the protein components of telomerase
and other proteins that regulate telomere length and telomerase
activity in mamnmalian cells. A comparison of telomere length by
mean terminal restriction fragment (mean TRF) analysis of immortal
cell lines with TPC2 mRNA levels indicates that TPC2 mRNA levels
are inversely related to telomere length. In one test, ten immortal
cell lines with relatively high TPC2 mRNA levels had mean TRFs of
.about.2.5 to 5.0 kb, whereas two immortal cell lines with very low
TPC2 mRNA levels had mean-TRFs of .about.17.5 to 35 kb (probability
of this difference arising by chance is less than 1%). In general,
TPC2 mRNA levels also correlate well with telomerase activity
levels in most cell lines tested.
[0093] Tests such as those described above can also be used to
determine the mechanism of action by which the TPC2 and TPC3 gene
products serve to regulate telomere length and telomerase activity.
The tests on TPC2 provide some indication that the TPC2 gene
product functions, at least in part, by acting as an indicator of
telomere length, much like the yeast EST1 protein. TPC2 is
up-regulated in most tumor cell lines and in testes cells and
down-regulated in normal cell lines. However, some cell lines with
apparently high levels of telomerase activity and very long
telomeres have low levels of TPC2 mRNA. As noted above, however,
telomerase positive cell lines that have relatively low TPC2 levels
also have relatively high mean TRFs, i.e., skin melanoma LOX
(.about.35.2 kb TRF), testes embryonic carcinoma Tera-1
(.about.27.0 kb), and lung carcinoma NCI-H23 (.about.17.5 kb). In
contrast, skin melanoma lines SK MEL2 (.about.2.3 kb), SK MEL28
(.about.15.7 kb), SK MEL5 (.about.4.0 kb), and testes tissue
(.about.15 kb) have relatively lower mean TRFs and relatively
higher TPC2 mRNA levels. Because all of these cell lines have
relatively high telomerase activity and high hTR levels, the tests
indicate that cell lines with relatively long telomeres in general
have low TPC2 mRNA levels, suggesting that the TPC2 protein may
encode a protein with a telomere-sensing function. The analysis of
TPC3 mRNA levels and telomerase activity in the same cell lines
indicates that the TPC3 gene product may act as a core component of
the telomerase enzyme.
[0094] Significant additional information regarding the mechanism
of action of the TPC2 and TPC3 gene products in the regulation of
telomere length and telomerase activity can be derived by analysis
of the nucleotide sequence and corresponding amino acid sequence of
the open reading frames of the corresponding genes. The subtraction
hybridization differential display identification and cloning
generated only cDNAs corresponding to the 3'-ends of the TPC2 and
TPC3 mRNA gene products, but the nucleotide sequence information
generated from those cDNAs provided a means to attempt to identify
and isolate clones in cDNA libraries prepared from telomerase
positive cell lines that comprise additional portions of the
mRNA.
[0095] Full length cDNA for the TPC2 and TPC3 gene products was
obtained by a variety of methods, including the screening of
subtracted and other specialized libraries and the use of 5'-RACE.
Initially, a lambda GT11 cDNA library containing human cDNA from
293 cells (a telomerase positive human-transformed kidney cell line
available from ATCC) was screened to identify lambda clones that
hybridized to the short TPC2 and TPC3 cDNAs obtained by subtraction
hybridization differential display. Then, after screening
additional cDNA libraries and combining fragments from various
subclones, full length open reading frames and genes were assembled
into the plasmids pGRN92 (comprises the open reading frame of the
TPC3 gene) and pGRN109 (comprises the open reading frame of the
TPC2 gene).
[0096] For example, for TPC2, cDNA inserts in lambda clones were
identified by screening with TPC2-specific probes and subcloned
into plasmid pGEX and derivative vectors (Pharmacia) to yield
plasmids that contained TPC2 cDNA in various reading frames to test
expression products and obtain partial nucleotide sequence and
deduced amino acid sequence information about the open reading
frame of the TPC2 mRNA. In the case of TPC3, for example, cDNA
fragments were cloned into pBluescript IIsk vector (Stratagene) to
generate vectors for sequencing and analysis.
[0097] FIG. 3 shows a restriction site and function map of the
.about.7.2 kb plasmid pGRN109, which contains an .about.3.5 kb
NotI-BstEII restriction fragment that contains an .about.3.3 kb
open reading frame encoding the TPC2 protein (labeled "ORF" and
"TPC2"). FIG. 4 lists portions of the nucleotide sequence and
deduced amino acid sequence of the TPC2 open reading frame
corresponding to the human TPC2 gene, mRNA, and protein products.
FIG. 5 shows a restriction site and function map of the .about.8 kb
plasmid pGRN92, which contains an .about.1.4 kb EcoRI-BamHI
restriction fragment that contains an .about.1.1 kb open reading
frame encoding the TPC3 protein (labeled "ORF" and "TPC3"). FIG. 6
lists the nucleotide sequence and deduced amino acid sequence of
the TPC3 open reading frame corresponding to the human TPC3 gene,
mRNA, and protein products. The initiating methionine codon is
marked with "***" and the stop codon with "---". Plasmid pGRN92
does not comprise nucleotides 1-82 shown in FIG. 6.
[0098] Neither the TPC2 nor the TPC3 open reading frame or other
gene sequences show significant homology to sequences in public
databases other than to ESTs; however, both have motif signatures.
TPC2 contains two WW domains and one L22 signature domain; TPC3
contains a homeobox domain. The "homeobox" is a protein domain of
60 amino acids (see Gehring, 1992, Trends Biochem. Sci. 17:277-280)
first identified in a number of Drosophila homeotic and
segmentation proteins and since found to be extremely well
conserved in many animals, including vertebrates. This domain binds
DNA through a helix-turn-helix type of structure. Proteins that
contain homeobox domains are likely to play a role in development;
most are known to be sequence specific DNA-binding transcription
factors. Recent publications suggest that homeobox domains can bind
RNA as well. See Dubnau and Struhl, Feb. 22, 1996, Nature 379:694.
The homeobox domain in TPC3 is: LAMCTNLPEARVQVWFKNRRAKFR (SEQ ID
NO: 10).
[0099] TPC2 contains two WW domains and an L22 ribosomal RNA
signature domain. The ribosomal protein L22 is a protein component
of the large ribosomal subunit that, in E. coli, binds 23S rRNA;
the protein belongs to a family of ribosomal proteins. See Gantt et
al., 1991, EMBO J. 10:3073-3078. For TPC2, this domain is:
SSSKVHSFGKRDQAIRRNPNVPVVV (SEQ ID NO: 11). The WW domain, also
known as rsp5 or WWP, is a short conserved region in a number of
unrelated proteins, among them dystrophin, responsible for Duchenne
muscular dystrophy. The domain spans about 35 residues, can be
repeated up to 4 times in some proteins, and has been shown to bind
proteins with particular proline-motifs,
>AP!-P-P->AP!-Y>A/P!-P-P->A/P! (SEQ ID NO: 12), and so
somewhat resembles SH3 domains. The WW domain is frequently
associated with other proteins in signal transduction processes and
appears to contain beta-strands grouped around four conserved
aromatic positions, generally Trp; the name WWP derives from the
presence of these conserved Trp and Pro residues. For TPC2, this
domain is represented by three amino acid residue sequences:
2 WSYGVCRDGRVFFINDQLRCTTWLHP; (SEQ ID NO: 13)
WFVLADYCLFYYKAEKKRSSXSIP (SEQ ID NO: 14) and
WEEGFTEEGASYFIDHNQQTTAFRHP. (SEQ ID NO: 15)
[0100] The availability of plasmids encoding the TPC2 and TPC3 open
reading frames provides a wide variety of benefits, including the
benefit of recombinant host cells that express recombinant gene
products comprising TPC2 and/or TPC3 open reading frame sequences
or sequences encoding products that react specifically with TPC2
and/or TPC3 gene products.
[0101] III. Recombinant Host Cells
[0102] In one embodiment, the invention provides recombinant
mammalian host cells containing:
[0103] (i) a recombinant or synthetic nucleic acid comprising at
least about 10 to 15 to 25 to 100 or more contiguous nucleotides
corresponding to an open reading frame sequence of a human gene
TPC2 contained in a human DNA insert of an .about.3.5 kb
NotI-BstEII restriction fragment of plasmid pGRN109; or
[0104] a synthetic or recombinant peptide or protein comprising at
least about 6 to 10 to 15 to 25 to 100 or more contiguous amino
acids corresponding to an amino acid sequence encoded by said open
reading frame sequence; and
[0105] (ii) a recombinant or synthetic nucleic acid comprising at
least about 10 to 15 to 25 to 100 or more contiguous nucleotides
corresponding to an open reading frame sequence of a human gene
TPC3 contained in a human DNA insert of an .about.1.4 kb
EcoRI-BamHI restriction fragment of plasmid pGRN92; or
[0106] a synthetic or recombinant peptide or protein comprising at
least about 6 to 10 to 15 to 25 to 100 or more contiguous amino
acids corresponding to an amino acid sequence encoded by said open
reading frame sequence of gene TPC3;
[0107] said TPC2 and TPC3 genes characterized in coding for
proteins that regulate telomere length or modulate telomerase
activity and are present in human or other mammalian cells that
express telomerase activity.
[0108] Other mammalian host cells provided by the invention include
those that comprise either or both TPC2- and TPC3-derived
recombinant or synthetic nucleic acids, peptides, or proteins.
Furthermore, the invention also provides such cells further
modified to contain a synthetic or recombinant nucleic acid
comprising at least about 10 to 15 to 25 to 100 or more contiguous
nucleotides corresponding to a contiguous nucleotide sequence of
human hTR located in an .about.2.5 kb HindIII-SacI restriction
fragment of pGRN33 (ATCC 75926).
[0109] The recombinant host cells of the invention have application
in many useful methods also provided by the invention. For example,
the invention provides recombinant host cells comprising novel
expression vectors with expression control sequences operatively
linked to nucleotide sequences encoding amino acids in a sequence
substantially identical to the proteins encoded by the human TPC2
or TPC3 genes, optionally with a recombinant hTR gene as well.
These recombinant host cells are useful for producing recombinant
human telomerase, for use in screens to identify agents that
modulate telomerase activity or regulate telomere length, as well
as for a variety of other purposes described below. The recombinant
host cells of the invention can also be incorporated into the germ
line and/or somatic tissues of non-human transgenic mammals, as
well as be administered to mammals for therapeutic purposes.
[0110] Thus, genomic clones of a gene that regulates telomere
length or telomerase activity, such as the human TPC2 or TPC3 gene,
or recombinant versions thereof, including versions that encode
mutein TPC2 or TPC3 gene products, may be used to construct
homologous targeting constructs for generating cells and transgenic
nonhuman animals having at least one functionally disrupted (or
otherwise altered) allele. Guidance for construction of homologous
targeting constructs may be found in the art, including: Rahemtulla
et al., 1991, Nature 353:180; Jasin et al., 1990, Genes Devel.
4:157; Koh et al., 1992, Science 256:1210; Molina et al., 1992,
Nature 357:161; Grusby et al., 1991, Science 253:1417; and Bradley
et al., 1992, Bio/Technology 10:534. See also U.S. Pat. Nos.
5,464,764 and 5,487,992. Transgenic cells and/or transgenic
non-human animals may be used to screen for antineoplastic agents
and/or to screen for potential carcinogens, as inappropriate
expression of a protein that regulates telomere length or
telomerase activity may result in a pre-neoplastic or neoplastic
state or other disease state or condition. Homologous targeting can
be used to generate so-called "knockout" mice, which are
heterozygous or homozygous for an inactivated allele. Such mice may
be sold commercially as research animals for investigation of
immune system development, neoplasia, spermatogenesis, or as pets,
or for animal products (foodstuff), or other purposes.
[0111] Chimeric transgenic mice are derived according to Hogan et
al., 1988, Manipulating the Mouse Embryo: A Laboratory Manual, Cold
Spring Harbor Laboratory, and Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, E. J. Robertson, ed., IRL Press,
Washington, D.C. (1987). Embryonic stem cells are manipulated
according to published procedures (Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL Press,
Washington, D.C. (1987); PCT patent publication No. 96/22362;
Zjilstra et al., 1989, Nature 342:435; and Schwartzberg et al.,
1989, Science 246:799, each of which is incorporated herein by
reference).
[0112] Additionally, a TPC2 or TPC3 cDNA or genomic clone may be
used to construct transgenes for expressing polypeptides at high
levels and/or under the transcriptional control of transcription
control sequences which do not naturally occur adjacent to the gene
(or vice-versa, i.e., the promoter of the TPC2 or TPC3 gene is
positioned in front of a reporter gene for use in screening or
other use). For example but not limitation, a constitutive promoter
(e.g., an HSV-tk or pgk (phosphoglycerate kinase) promoter) or a
cell-lineage specific transcriptional regulatory sequence (e.g., an
CD4 or CD8 gene promoter/enhancer) may be operably linked to a
protein encoding polynucleotide sequence to form a transgene
(typically in combination with a selectable marker such as a neo
gene expression cassette). Such transgenes can be introduced into
cells (e.g., ES cells, hematopoietic stem cells, cancer cells), and
transgenic cells, cell lines, and transgenic nonhuman animals may
be obtained according to conventional methods therewith.
[0113] The recombinant host cells of the invention are often
prepared using, or serve as a source of, valuable oligonucleotide
and nucleic acid reagents provided by the present invention, such
as the expression control vectors noted above. These nucleic acid
reagents are described in more detail in the following section.
[0114] IV. Oligonucleotides and Nucleic Acids
[0115] In another embodiment, the invention provides synthetic and
recombinant oligonucleotides and nucleic acids in a variety of
forms, i.e., isolatable, isolated, purified, or substantially pure,
and for a variety of purposes, i.e., as probes or primers, as
polynucleotide plasmids and vectors for introducing recombinant
gene products that regulate telomere length or modulate telomerase
activity in mammalian host cells, as restriction fragments for
creating useful nucleic acids, and as reagents for therapeutic,
diagnostic, and other applications. Isolated or purified
polynucleotides of the invention typically are less than .about.10
kb in size. In particular, the invention provides recombinant or
synthetic nucleic acids comprising at least about 10 to 15 to 25 to
100 or more contiguous nucleotides substantially identical or
complementary in sequence to a contiguous nucleotide sequence
located in either:
[0116] (i) an open reading frame sequence of a human gene TPC2
contained in a human DNA insert of an .about.3.5 kb NotI-BstEII
restriction fragment of plasmid pGRN109; or
[0117] (ii) an open reading frame sequence of a human gene TPC3
contained in a human DNA insert of an .about.1.4 kb EcoRI-BamHI
restriction fragment of plasmid pGRN92.
[0118] The novel oligonucleotide probes and primers of the
invention typically comprise nucleotides in a sequence
substantially identical or complementary to a sequence of
nucleotides in a TPC2 or TPC3 gene or gene product to allow
specific hybridization thereto in a complex mixture of nucleic
acids. Nucleotide substitutions, deletions, and additions may be
incorporated into the polynucleotides of the invention. Nucleotide
sequence variation may result from sequence polymorphisms of
various alleles, minor sequencing errors, and the like. The minimum
length of a polynucleotide required for specific hybridization to a
target sequence depends on several factors: G/C content,
positioning of mismatched bases (if any), degree of uniqueness of
the sequence as compared to the population of target
polynucleotides, and chemical nature of the polynucleotide (e.g.,
methylphosphonate backbone, polyamide nucleic acid,
phosphorothioate, etc.), among others.
[0119] The probes and primers of the invention have useful
application in a variety of diagnostic, therapeutic, and other
applications. Because they are expressed differentially between
immortal human cells lines, TPC2 and TPC3 genes and gene products
serve as telomerase activity and tumor cell markers.
[0120] Oligonucleotides corresponding to unique TPC2 or TPC3 gene
sequences can be used as primers or probes, may be attached to
other nucleic acids, proteins, labels, etc., and are useful for a
variety of purposes, including, for example, as diagnostic probes
for tumor cells in clinical specimens. The oligonucleotides of the
invention can be used as hybridization probes or PCR primers to
detect the presence of TPC2 or TPC3 gene products, to diagnose a
neoplastic disease characterized by the presence of an elevated or
reduced TPC2 or TPC3 mRNA level in cells, to perform tissue typing
(i.e., identify tissues characterized by the expression of
telomerase or TPC2 or TPC3 mRNA), and the like. Probes can be used
to detect TPC2 or TPC3-specific nucleotide sequences in a DNA
sample, such as for forensic DNA analysis or for diagnosis of
diseases characterized by amplification, alteration, and/or
rearrangements of the TPC2 or TPC3 genes. Certain preferred
oligonucleotides of the invention typically comprise at least 8 to
10 to 15 to 25 to 99 to 250 to 1000 or more contiguous nucleotides
capable of hybridizing under stringent hybridization conditions to
nucleic acids corresponding to a nucleotide sequence in the
.about.3.5 kb NotI-BstEIII insert of pGRN109 or the .about.1.4 kb
EcoRI-BamHI insert of pGRN92 and are useful as probes, primers,
antisense therapeutics, and ribozyme therapeutics, for example.
[0121] Where expression of a polypeptide is not desired,
polynucleotides of this invention need not encode a functional
protein. Polynucleotides of this invention may serve as
hybridization probes and/or PCR primers and/or LCR oligomers for
detecting RNA or DNA sequences. Alternatively, polynucleotides of
this invention may serve as hybridization probes or primers for
detecting RNA or DNA sequences of related genes, for example, genes
that encode structurally or evolutionarily related proteins. For
such hybridization and other applications, such as those involving
PCR, the polynucleotides of the invention need not encode a
functional polypeptide. Thus, certain polynucleotides of the
invention may contain substantial deletions, additions, nucleotide
substitutions, and/or transpositions, so long as the ability of
specific hybridization to or specific amplification of a TPC2 or
TPC3 gene or mRNA gene product is retained.
[0122] As one example, antisense polynucleotides can include
nucleotide substitutions, additions, deletions, or transpositions,
so long as specific hybridization to the relevant target sequence,
typically an mRNA, is retained as a functional property of the
polynucleotide. Complementary antisense polynucleotides include
soluble antisense RNA or DNA oligonucleotides that can hybridize
specifically to mRNA species and genes and so prevent either
transcription of the gene to produce the mRNA and/or translation of
the mRNA. Antisense polynucleotides of various lengths may be used,
although such antisense polynucleotides typically comprise a
sequence of at least about 25 consecutive nucleotides that are
substantially identical to a naturally occurring TPC2 or TPC3 gene
sequence. Antisense polynucleotides may be produced from a
heterologous expression cassette in a transfectant cell or
transgenic cell, such as a transgenic pluripotent hematopoietic
stem cell used to reconstitute all or part of the hematopoietic
stem cell population of an individual. Alternatively, the antisense
polynucleotides may comprise soluble oligonucleotides that are
administered to the external milieu, either in the culture medium
in vitro or in the circulatory system or interstitial fluid in
vivo. Soluble antisense polynucleotides present in the external
milieu have been shown to gain access to the cytoplasm and inhibit
translation of specific mRNA species. In some embodiments the
antisense polynucleotides comprise methylphosphonate or other
synthetic moieties. For general methods relating to antisense
polynucleotides, see Antisense RNA and DNA (1988), D. A. Melton,
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
[0123] The inhibitory nucleic acid also can be a so-called "sense"
or other nucleic acid, i.e., a triplex-forming nucleic acid. As one
example, expression of recombinant TPC3 mRNA in a cancer cell line
resulted in the inhibition of telomerase activity by over 90%. In
this example, the entire .about.1.1 kb coding sequence of the TPC3
gene was isolated as an EcoRI fragment (.about.2.1 kb) from vector
pTATPC3.9 and inserted into the EcoRI site of mammalian expression
vector pBBS212 to give rise to two vectors: pGRN111, in which the
sense strand of the TPC3 gene is operatively linked to the myelo
proliferative sarcoma virus (MPSV) promoter, and pGRN112, in which
the antisense strand is operatively linked to the MPSV promoter.
Vector pTATPC3.9 was constructed by ligation of TPC3 5'-RACE
product (.about.2.1 kb) into pCRII vector (Invitrogen). The sense
and antisense vectors, as well as control vector pBBS212, were used
to transform HeTe7 cells by electroporation. The medium was changed
to selection medium containing hygromycin (300 _g/ml) and puromycin
(0.2 _g/ml) for four weeks to obtain individual clones. The
individual clones were then isolated, expanded, and assayed for the
expression of sense or antisense TPC3 gene product and vector
transcription by RT-PCR. The positive clones were then assayed for
telomerase activity using the TRAP assay, and mean TRF values were
measured at different time points.
[0124] FIG. 7 shows the results of the analysis of telomerase
activity levels in recombinant HeTe7 cells expressing the sense or
antisense mRNA of gene TPC3 or a control vector. As noted above,
presence of the recombinant sense mRNA reduced telomerase activity
markedly in these cells. FIG. 8 shows the results of the analysis
of telomere length in recombinant HeTe7 cells expressing the sense
or antisense mRNA of gene TPC3 or a control vector. The recombinant
TPC3 sense mRNA decreased the mean TRF in the cells. Thus, the
recombinant TPC3 gene product can regulate not only telomerase
activity but also telomere length in these cells. This experiment
shows how the recombinant nucleic acids of the invention can be
expressed by transfecting the cell with an expression vector
comprising expression control sequences operatively linked thereto.
Fragments or analogs of TPC2 or TPC3 can also be expressed and
function to compete with other active components of enzymes that
regulate telomere length or telomerase activity. Assembly of
ribonucleoproteins or other macromolecules with non-functional
components results in non-functional complexes and subsequent
decrease in associated activity, i.e., telomerase activity,
telomere maintenance, and telomere length.
[0125] The expression vectors of the invention typically comprise
expression control sequences operatively linked to a nucleotide
sequence encoding amino acids in a sequence identical to a sequence
of amino acids in a TPC2 or TPC3 protein gene product. The operably
linked nucleotide sequence typically encodes at least 5 to 9 amino
acids, or encodes all of or at least an active portion of the TPC2
or TPC3 proteins, or encode from 15 to 20 to 25 to 100 or more
contiguous amino acids in a sequence selected from the amino acid
sequences of TPC2 or TPC3, or variant but related sequences
thereto. For example, useful TPC2 and TPC3 variant proteins include
fusion proteins, in which all or a portion of the TPC2 or TPC3
protein is fused to peptide or polypeptide that imparts some useful
feature, such as a binding site for use in affinity purification,
i.e., a polyhistidine tag of about six histidine residues or the
maltose binding protein. Preferably, these amino acid sequences
occur in the given order of the naturally occurring proteins (in
the amino-terminal to carboxy-terminal orientation) but may
comprise other intervening and/or terminal sequences; generally
such polypeptides are less than 1000 amino acids in length, more
usually less than about 500 amino acids in lengths, and frequently
about 200 amino acids in length. The degeneracy of the genetic code
gives a finite set of polynucleotide sequences encoding these amino
acid sequences; this set of degenerate sequences may be readily
generated by hand or by computer using commercially available
software (Wisconsin Genetics Software Package Release 7.0). These
and other expression vectors of the invention have many useful
applications, including in therapeutic methods of the invention as
gene therapy vectors for modulating telomerase activity, either to
activate or inhibit that activity, or for regulating telomere
length, either to increase or decrease the length, in a target cell
or tissue.
[0126] Thus, the gene therapy expression vectors of the invention
include those that encode variants or "muteins" of the TPC2 and/or
TPC3 proteins, i.e., express proteins that differ from TPC2 and/or
TPC3 by deletion, substitution, and/or addition of one or more
amino acids. The gene therapy vectors of the invention may also,
however, encode other useful nucleic acids, such as hTR, or
antisense nucleic acids or ribozymes that target the TPC2, TPC3,
and/or hTR gene products, i.e., mRNA and telomerase RNA. The
vectors of the invention can also code for the expression of a
protein which, when presented as an immunogen, elicits the
production of an antibody that specifically binds to TPC2 or TPC3
proteins or cells expressing those proteins. Such vectors can also
code for a structurally-related protein, such as a TPC2 or TPC3
protein fragment or analog. These vectors are useful in the
therapeutic methods of the invention for treating or preventing
diseases or conditions in which modulation of telomerase activity
or telomere length can be of benefit. For example, in telomerase
positive cancer cells, inhibition of telomerase activity can
prevent telomere maintenance in those cells, inducing upon
continued proliferation telomere loss, cell crisis, and death. For
such purposes, the gene therapy vectors of the invention that
express a non-functional TPC2 or TPC3 mutein or variant protein or
other nucleic acid (i.e., over expression of TPC3 mRNA) that can
inhibit telomerase formation or telomere elongation by telomerase
activity in the cell, such as by competing for RNA component or
protein components, inhibition of endogenous gene expression, or
other means, are preferred.
[0127] Expression vectors of the invention comprise expression and
replication signals compatible with the host cell of interest,
i.e., sequences that facilitate transcription and translation
(expression sequences) of the coding sequences, such that the
encoded polypeptide product is produced. Construction of such
polynucleotides is well known in the art and is described further
in Maniatis et al., supra. For example, but not for limitation,
such polynucleotides can include a promoter, a transcription
termination site (polyadenylation site in eukaryotic expression
hosts), a ribosome binding site, and, optionally, an enhancer for
use in eukaryotic expression hosts, and, optionally, sequences
necessary for replication of a vector. A typical eukaryotic
expression cassette will include a polynucleotide sequence encoding
a polypeptide linked downstream (i.e., in translational reading
frame orientation; polynucleotide linkage) of a promoter such as
the HSV, tk, pgk, metallothionein, or any of a wide variety of
other promoters suitable for use in mammalian cells, optionally
linked to an enhancer and a downstream polyadenylation site (e.g.,
an SV40 large T Ag poly A addition site). Expression vectors useful
for expressing the recombinant TPC2, TPC3, and other proteins of
this invention include viral vectors such as retroviruses,
adenoviruses and adeno-associated viruses, i.e., for therapeutic
methods, plasmid vectors such as pcDNA1 (Invitrogen, San Diego,
Calif.), in which the expression control sequence comprises the CMV
promoter, cosmids, liposomes, and the like. Viral and plasmid
vectors are often preferred for transfecting mammalian cells.
[0128] The nucleic acid reagents of the invention also include
reagents useful in identifying, isolating, and cloning nucleic
acids that encode proteins that interact with TPC2 and TPC3 gene
products as well as mammalian (i.e., mouse) homologs of human TPC2
and TPC3 genes. Homologous DNA can be readily identified by
screening a genomic or cDNA clone library prepared from the
mammalian cells of interest, such as a mouse, rat, rabbit, or other
cells, i.e., in yeast artificial chromosomes, cosmids, or
bacteriophage lambda (e.g., Charon 35), with a polynucleotide probe
comprising a sequence of about at least 24 (or in the range of 15
to 30 or more) contiguous nucleotides (or their complement) of the
cDNA sequences of TPC2 or TPC3 disclosed herein. Typically,
hybridization and washing conditions are performed at varying
degrees of stringency according to conventional hybridization
procedures. Positive clones are isolated and sequenced. For
illustration and not for limitation, a full length polynucleotide
corresponding to the open reading frame sequences of the TPC2 and
TPC3 genes can be labeled and used as a hybridization probe to
isolate genomic clones from a murine or other mammalian genomic
clone or cDNA library (i.e., those available from Promega
Corporation, Madison, Wis.).
[0129] The nucleic acids of the invention can also be employed to
isolate and identify gene products that interact with or bind to
TPC2 and/or TPC3 gene products. The yeast "two-hybrid" system (see
Chien et al., 1991, Proc. Natl. Acad. Sci. (U.S.A.) 88:9578)
utilizes expression vectors that encode the predetermined
polypeptide sequence as a fusion protein and is used to identify
protein-protein interactions in vivo through reconstitution of a
transcriptional activator (see Fields and Song, 1989, Nature
340:245). Usually the yeast Gal4 transcription protein, which
consists of separable domains responsible for DNA-binding and
transcriptional activation, serves as the transcriptional
activator. Polynucleotides encoding two hybrid proteins, one
consisting of the yeast Gal4 DNA-binding domain fused to a
polypeptide sequence of a first protein and the other consisting of
the Gal4 activation domain fused to a polypeptide sequence of a
second protein (either the first or second protein typically is a
number of different proteins to be screened for ability to interact
specifically with the other protein), are constructed and
introduced into a yeast host cell. Intermolecular binding, if any,
between the two fusion proteins reconstitutes the Gal4 DNA-binding
domain with the Gal4 activation domain, which leads to the
transcriptional activation of a reporter gene (e.g., lacZ, HIS3)
operably linked to the Gal4 binding site. Typically, the twohybrid
method is used to identify novel polypeptide sequences which
interact with a known protein.
[0130] The invention also provides two- and three-hybrid systems,
typically in the form of polynucleotides encoding a first hybrid
protein comprising either TPC2 or TPC3, a second hybrid protein,
and a reporter gene, wherein said polynucleotide(s) are either
stably replicated or introduced for transient expression. The host
organism can be a yeast cell (e.g., Saccharomyces cervisiae) in
which the reporter gene transcriptional regulatory sequence
comprises a Gal4-responsive promoter (binding site). Yeast cells
comprising (1) an expression cassette encoding a Gal4 DNA binding
domain (or Gal4 activator domain) fused to a binding fragment of
TPC2 or TPC3 protein; (2) an expression cassette encoding a Gal4
DNA activator domain (or Gal4 binding domain, respectively) fused
to a member of a cDNA library; and (3) a reporter gene (e.g.,
betagalactosidase) comprising a cis-linked Gal4 transcriptional
response element, can be used to screen cDNAs to identify those
that encode polypeptides that bind to TPC2 and/or TPC3 proteins
specifically. Yeast two-hybrid systems may be used to screen a
mammalian (typically human) cDNA expression library, such as, for
example, a cDNA library produced from human mature B cell line
(Namalwa) mRNA (see Ambrus et al., 1993, Proc. Natl. Acad. Sci.
(U.S.A.)). Once cDNAs encoding such interacting polypeptides are
identified, the resulting polypeptides can be cloned,
characterized, and used to screen compounds to identify compounds
that can inhibit the binding interaction.
[0131] Notwithstanding the many and diverse application of the
oligonucleotide and nucleic acid reagents of the invention, one
important application relates to the production of recombinant
peptides and proteins of the invention, as discussed in the
following section.
[0132] V. Peptides and Proteins
[0133] In another embodiment, the present invention provides
peptides, proteins, antibodies, and enzymes relating to genes and
gene products that regulate telomere length and telomerase activity
in mammalian cells. In particular, the invention provides synthetic
or recombinant peptides or proteins comprising at least about 6 to
10 to 15 to 25 to 100 or more contiguous amino acids identical in
sequence to an amino acid sequence encoded by an open reading frame
sequence of a human gene located in either:
[0134] (i) an .about.3.5 kb NotI-BstEII restriction fragment of
plasmid pGRN109; or
[0135] (ii) an .about.1.4 kb EcoRI-BamHI restriction fragment of
plasmid pGRN92.
[0136] The present invention provides the peptides and proteins
encoded by the TPC2 and TPC3 genes, as well as fragments and
analogs thereof, in isolatable form from eukaryotic or prokaryotic
host cells expressing recombinant TPC2 and/or TPC3 protein, or from
an in vitro translation system, as well as in purified and
substantially pure form from synthesis in vitro or by purification
from recombinant host cells or by purification of the naturally
occurring proteins using antibodies or other reagents of the
invention. Methods for expression of heterologous proteins in
recombinant hosts, chemical synthesis of polypeptides, and in vitro
translation are well known in the art and are described further in
Maniatis et al. and Berger and Kimmel, supra. Such proteins have
application in methods for reconstituting in vitro telomerase or
other enzymatic activities that maintain telomeres and regulate
telomere length. These methods in turn have application in screens
for therapeutic agents, for diagnostic tests, and for other
applications.
[0137] Because they are expressed differentially between immortal
human cells lines, TPC2 and TPC3 genes and gene products serve as
telomerase activity and tumor cell markers. Polypeptides having the
full or partial amino acid sequence of TPC2 or TPC3 proteins are
useful, for example, in the production of antibodies against TPC2
or TPC3 proteins and that are useful in the detection of TPC2 or
TPC3 proteins in tumor cells. The invention provides purified TPC2
and TPC3 proteins having an amino acid sequence substantially
identical to the amino acid sequences encoded by the open reading
frames of the TPC2 and TPC3 genes. Such genes include human allelic
variants or mammalian cognate genes that can be obtained in
accordance with and using the reagents provided by the present
invention.
[0138] The invention also provides TPC2 and TPC3 protein analogs,
non-naturally occurring polypeptides comprising at least 5 to 10 to
15 to 20 to 25 to 100 or more amino acids in a contiguous sequence
selected from the amino acid sequences of the TPC2 and TPC3
proteins but include one or more deletions or additions of amino
acids, either at the amino- or carboxy-termini, or internally, or
both; analogs may further include sequence transpositions. Analogs
may also comprise amino acid substitutions, preferably conservative
substitutions. Analogs include active fragments as well as various
muteins. For example, single or multiple amino acid substitutions
(preferably conservative amino acid substitutions) may be made in
the naturally occurring sequence. Preferred amino acid
substitutions include those that: (1) reduce susceptibility to
proteolysis, (2) reduce susceptibility to oxidation, (3) alter
post-translational modification of the analog, possibly including
phosphorylation, and (4) confer or modify other physicochemical or
functional properties of such analogs. TPC2 or TPC3 protein analogs
can be immunogenic for TPC2 or TPC3 proteins, i.e., when presented
as an immunogen, the analog elicits the production of an antibody
that specifically binds to TPC2 or TPC3 proteins. Active fragments
can be identified empirically by generating fragments of the full
length protein by deletion from either the amino-terminus or the
carboxy-terminus or both, and testing the resulting fragments for
activity.
[0139] Conservative amino acid substitution is a substitution of an
amino acid by a replacement amino acid which has similar
characteristics (e.g., those with acidic properties: Asp and Glu).
A conservative (or synonymous) amino acid substitution does not
substantially change the structural characteristics of the parent
protein (e.g., a replacement amino acid should not tend to break a
helix that occurs in the parent sequence, or disrupt other types of
secondary structure that characterizes the parent sequence).
Examples of art-recognized polypeptide secondary and tertiary
structures are described in Proteins, Structures and Molecular
Principles (1984), Creighton (ed.), W. H. Freeman and Company, New
York; Introduction to Protein Structure (1991), C. Branden and J.
Tooze, Garland Publishing, New York, N.Y.; and Thornton et al.,
1991, Nature 354:105; which are incorporated herein by reference.
The following six groups each contain amino acids that are
conservative substitutions for one another: (1) Alanine (A), Serine
(S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3)
Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0140] Analogs may include heterologous sequences generally linked
at the amino- or carboxy-terminus, wherein the heterologous
sequence(s) confer a functional property to the resultant analog
not shared by the native protein. Such analogs are referred to as
fusion proteins and for purposes of the present invention typically
comprise a TPC2 or TPC3 protein or analog and an additional peptide
or protein moiety. Fusion proteins usefully combine properties of
two different polypeptides or proteins, and can be used, for
example, to confer a label, such as a polyhistidine polypeptide or
a maltose binding protein, useful in affinity isolation of the
fusion protein or to protect the fusion protein from degradation
inside a cell. The fusion protein may comprise a linker peptide
with desired properties, for example, a peptidase site that renders
the TPC2 or TPC3 protein or analog cleavable from the remainder of
the fusion protein. The fusion protein can also confer an antigenic
epitope to the TPC2 or TPC3 protein of interest; antibodies that
bind the epitope could then be used to immunoprecipitate the fusion
protein for purification or to identify associated proteins.
[0141] Thus, the invention provides recombinant fusion proteins in
which all or a portion of the TPC2 or TPC3 protein is fused to
another polypeptide or protein of interest. For example, plasmids
pGRN103, pGRN104, pGRN106, and pGRN110 are expression plasmids of
the invention that code for the expression of novel fusion proteins
of the invention that comprise a portion of either TPC2 or TPC3
protein and maltose binding protein (MBP). These vectors were
created using the commercially available pMALc2 expression vector
and system (New England Biolabs). Plasmid pGRN103 encodes a fusion
protein comprising the amino-terminal portion of TPC3 protein and
MBP and was prepared by replacing the XmnI-PstI restriction
fragment of plasmid pMALc2 with the PvuII-PstI restriction fragment
of plasmid pGRN92. Plasmid pGRN104 encodes a fusion protein
comprising the carboxy-terminal portion of TPC3 protein and MBP and
was prepared by replacing the EcI136II-BamHI restriction fragment
of plasmid pMALc2 with the BspEI (treated with Klenow in the
presence of dCTP and dGTP only)-BamHI restriction fragment of
plasmid pGRN92. Plasmid pGRN106 encodes a fusion protein comprising
the amino-terminal portion of TPC2 protein and MBP and was prepared
by replacing the SaII-PstI restriction fragment of plasmid pMALc2
with a SaII-Sse8387I restriction fragment that can be isolated from
plasmid pGRN109. Plasmid pGRN110 encodes a fusion protein
comprising the carboxy-terminal portion of TPC2 protein and MBP and
was prepared by inserting a restriction fragment containing the
carboxy-terminal portion of the open reading frame of TPC2 into
plasmid pMALc2 such that the fusion protein shown below (SEQ ID NO:
16) results from expression of the plasmid in E. coli W3110 cells
(only the ends of the MBP and TPC2 proteins at the junction region
are shown): ##STR1## These and other fusion proteins of the
invention can be isolated in accordance with standard procedures
and then used to immunize animals, i.e., mouse and rabbits, for the
production of polyclonal antisera and monoclonal antibodies, as
described in the following section.
[0142] TPC2 or TPC3 proteins, analogs, peptides, and polypeptides
can also be prepared by chemical synthesis using well known
methods. For example, various peptides with amino acid sequences
corresponding to sequences of the TPC2 and TPC3 proteins can be
chemically synthesized in vitro and used to generate antibodies
that specifically bind to TPC2 and/or TPC3 proteins. Illustrative
peptides of the invention include RGLKRQSDERKRDRE (SEQ ID NO: 17)
and KVTSPLQSPTKAKPK (SEQ ID NO: 18), which have been chemically
synthesized in vitro and used to immunize animals to generate
antibodies specific for TPC3 protein. Such peptides may correspond
to structural and functional domains identified by comparison of
the nucleotide and/or amino acid sequence data of a gene or protein
to public or other sequence databases. Computerized comparison
methods can be used to identify sequence motifs or predicted
protein conformation domains that occur in other proteins of known
structure and/or function. See Proteins, Structures and Molecular
Principles (1984), Creighton (ed.), W. H. Freeman and Company, New
York, incorporated herein by reference. Methods to identify protein
sequences that fold into a known three-dimensional structure are
known. See Bowie et al., 1991, Science 253:164. Recognized sequence
motifs and structural conformations may be used to define
structural and functional domains. Computer programs GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package
(Genetics Computer Group, 575 Science Dr., Madison, Wis.) can be
used to identify sequences in databases, such as GenBank/EMBL, that
have regions of homology. Neural network methods, whether
implemented in hardware or software, may be used to: (1) identify
related protein sequences and nucleotide sequences, and (2) define
structural or functional domains in polypeptides. See Brunak et
al., 1991, J. Mol. Biol. 220:49, incorporated herein by
reference.
[0143] Thus, one class of preferred peptides and proteins of the
invention are fragments of the TPC2 or TPC3 proteins having amino-
and/or carboxy-termini corresponding to amino acid positions near
functional domain borders. Alternative fragments may also be
prepared. The choice of the amino- and carboxy-termini of such
fragments rests with the discretion of the practitioner and is
based on considerations such as ease of construction, stability to
proteolysis, thermal stability, immunological reactivity, amino- or
carboxyl-terminal residue modification, or other
considerations.
[0144] The immunogenic peptides and proteins of the invention can
be used in therapeutic immunization and vaccination procedures. See
U.S. provisional patent application Ser. No. 60/008,949, filed Oct.
20, 1995, incorporated herein by reference. The invention therefore
provides a method of immunizing a subject, as well as vaccines
useful in the method, against cells that maintain telomeres and
express telomerase activity, such as cancer cells, that comprise
administering an immunostimulating amount of such peptides or
proteins of the invention.
[0145] Peptides and proteins of the invention are suitably obtained
in substantially pure form if at least about 50 percent (w/w) or
more pure and substantially free of interfering proteins and
contaminants. Preferably, these polypeptides are isolated or
synthesized in a purity of at least 80 percent (w/w) or, more
preferably, in at least about 95 percent (w/w), and are
substantially free of other proteins or contaminants.
[0146] One important application of the peptides and proteins of
the invention is the generation of antibodies that specifically
bind to TPC2 or TPC3 proteins, as discussed in the following
section.
[0147] VI. Antibodies
[0148] The proteins and peptides of the invention can also be used
to generate antibodies specific for TPC2 or TPC3 proteins, or for
particular epitopes on those proteins. TPC2 or TPC3 proteins,
fragments thereof, or analogs thereof, can be used to immunize an
animal for the production of specific antibodies. For example, but
not for limitation, a recombinantly produced fragment of a TPC2 or
TPC3 protein or a fusion protein can be injected into a mouse along
with an adjuvant following immunization protocols known to those of
skill in the art so as to generate an immune response.
Alternatively, or in combination with a recombinantly produced
polypeptide, a chemically synthesized peptide having an amino acid
sequence corresponding to a TPC2 or TPC3 protein may be used as an
immunogen to raise antibodies which bind a TPC2, TPC3, or another
telomere-or telomerase-related protein. Immunoglobulins that bind
the target protein with a binding affinity of at least about
1.times.10.sup.6 M.sup.-1 can be harvested from the immunized
animal as an antiserum, and may be further purified by
immunoaffinity chromatography or other means.
[0149] Additionally, spleen cells can be harvested from the
immunized animal (typically rat or mouse) and fused to myeloma
cells to produce a bank of monoclonal antibody-secreting hybridoma
cells. The bank of hybridomas can be screened for clones that
secrete immunoglobulins that bind the protein of interest
specifically, i.e., with an affinity of at least 1.times.10.sup.7
M.sup.-1. Animals other than mice and rats may be used to raise
antibodies; for example, goats, rabbits, sheep, and chickens may
also be employed to raise antibodies reactive with a TPC2 or TPC3
protein. Transgenic mice having the capacity to produce
substantially human antibodies also may be immnunized and used for
a source of antiserum and/or for making monoclonal antibody
secreting hybridomas.
[0150] Thus, the invention provides polyclonal and monoclonal
antibodies that specifically bind to TPC2 or TPC3 proteins.
Bacteriophage antibody display libraries may also be screened for
phage able to bind peptides and proteins of the invention
specifically. Combinatorial libraries of antibodies have been
generated in bacteriophage lambda expression systems and may be
screened as bacteriophage plaques or as colonies of lysogens. For
general methods to prepare antibodies, see Antibodies: A Laboratory
Manual (1988), E. Harlow and D. Lane, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., incorporated herein by
reference.
[0151] These antibodies can in turn be used to isolate TPC2 or TPC3
proteins from normal or recombinant cells and so can be used to
purify the proteins as well as other proteins associated therewith.
Such antibodies are useful in the detection of TPC2 or TPC3
proteins in samples and in the detection of cells comprising TPC2
or TPC3 proteins in complex mixtures of cells. Such detection
methods have application in screening, diagnosing, and monitoring
diseases and other conditions, such as cancer, pregnancy, or
fertility, because the TPC2 and TPC3 proteins are present in most
cells capable of elongating telomeric DNA and expressing telomerase
activity and are present in those cells at levels significantly
higher than the levels observed in telomerase negative cells.
[0152] For some applications of the antibodies of the invention,
such as identifying immuno-crossreactive proteins, the desired
antiserum or monoclonal antibody(ies) is/are not monospecific. In
these or other instances, it may be preferable to use a synthetic
or recombinant fragment of a TPC2 or TPC3 protein as an antigen
rather than the entire protein. More specifically, where the object
is to identify immuno-crossreactive polypeptides that comprise a
particular structural moiety, such as a DNA-binding domain, it is
preferable to use as an antigen a fragment corresponding to part or
all of a commensurate structural domain in the TPC2 or TPC3
protein.
[0153] Cationized or lipidized antibodies reactive with TPC2 or
TPC3 can be used therapeutically to treat or prevent diseases of
excessive or inappropriate expression (e.g., neoplasia) of these
proteins and the processes regulated thereby. Other methods of the
invention are discussed in the following section.
[0154] VII. Methods
[0155] The various reagents of the invention described above have a
wide variety of applications. The provision of polynucleotides
capable of hybridizing to TPC2 or TPC3 cDNA and antibodies that
specifically bind to TPC2 or TPC3 proteins allows one to detect
expression of TPC2 and TPC3 in cells. The detection of TPC2 or TPC3
gene expression in cells suspected of being cancerous is useful in
the diagnosis of cancer. Accordingly, this invention provides
methods of detecting TPC2 or TPC3 mRNA or protein in a cell by
hybridization or immunoassay methods. Hybridization methods can
involve any of the routine methods including Northern blotting;
Southern hybridization; amplification of target or probe nucleic
acids by PCR, b-DNA, antibodies labeled with enzymes, LCR, Q-beta
replicase, or 3SR; and the like, may also be used.
[0156] The polynucleotide sequences of the present invention can be
used for forensic identification of individual humans, such as for
identification of decedents, determination of paternity, criminal
identification, and the like. The invention also provides TPC2 or
TPC3 polynucleotide probes for diagnosis of disease states (e.g.,
neoplasia or pre-neoplasia) by detection of a TPC2 or TPC3 mRNA or
rearrangements or amplification of the TPC2 or TPC3 gene in cells
explanted from a patient, or detection of a pathognomonic TPC2 or
TPC3 allele. Cells which contain an altered amount of TPC2 or TPC3
mRNA as compared to non-neoplastic or non-diseased cells of the
same cell type(s) can be identified as candidate diseased cells in
accordance with the methods of the invention. Similarly, the
detection of pathognomonic rearrangements or amplification of the
TPC2 or TPC3 gene locus or closely linked loci in a cell sample
will identify the presence of a pathological condition or a
predisposition to developing a pathological condition (e.g.,
cancer, genetic disease).
[0157] The isolation of three telomerase-related and telomere
length regulatory components, TPC2, TPC3, and hTR, allows the
production of recombinant telomerase comprising one or more of
these components. In one method, recombinant telomerase is produced
by expressing a TPC2 or TPC3 protein or active TPC2 or TPC3 analog
and/or recombinant hTR in a cell. In another, telomerase is
re-constituted in vitro. The recombinant RNA component of
telomerase can be, for example, an RNA molecule derived from the
sequence encoded by the .about.2.5 kb HindIII-SacI insert of pGRN33
(ATCC 75926). Recombinant telomerase is useful, for example for
screening assays to determine whether a compound modulates
telomerase activity.
[0158] Telomerase- and telomere length-modulating agents which
reduce a cell's capacity to repair telomere DNA damage (e.g., by
inhibiting endogenous naturally occurring telomerase) are candidate
antineoplastic agents. Candidate antineoplastic agents are then
tested further for antineoplastic activity in assays which are
routinely used to predict suitability for use as human
antineoplastic drugs. Examples of these assays include, but are not
limited to, assays to measure the ability of the candidate agent
(1) to inhibit anchorage-independent transformed cell growth in
soft agar, (2) to reduce tumorigenicity of transformed cells
transplanted into nu/nu mice, (3) to reverse morphological
transformation of transformed cells, (4) to reduce growth of
transplanted tumors in nu/nu mice, (5) to inhibit formation of
tumors or pre-neoplastic cells in animal models of spontaneous or
chemically-induced carcinogenesis, and (6) to induce a more
differentiated phenotype in transformed cells.
[0159] Administration of an efficacious dose of an agent capable of
specifically inhibiting telomere-maintenance or telomerase activity
to a patient can be used as a therapeutic or prophylactic method
for treating pathological conditions (e.g., cancer, inflammation,
lymphoproliferative diseases, autoimmune disease, neurodegenerative
diseases, and the like), which are effectively treated by
modulating telomerase activity and telomere length. Additional
embodiments directed to modulation of neoplasia or cell death
include methods that employ specific inhibitory nucleic acids,
e.g., sense or antisense polynucleotides corresponding to
nucleotide sequences encoding TPC2, TPC3, or a cognate mammalian
TPC2 or TPC3 protein.
[0160] The foregoing description of the preferred embodiments of
the present invention has been presented for purposes of
illustration and description and is not intended to be exhaustive
or to limit the invention to the precise form disclosed but instead
to illuminate the many modifications and variations possible in
light of the invention and description and to include such
modifications and variations as may be apparent to a person skilled
in the art in light of this description within the scope of this
invention and the claims thereto. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
VII. EXAMPLES
[0161] The following examples are given to illustrate but not limit
the invention. Generally, the nomenclature used herein and many of
the laboratory procedures in cell culture, molecular genetics, and
nucleic acid chemistry and hybridization described below are those
well known and commonly employed in the art. All percentages given
throughout the specification and examples are based upon weight
unless otherwise indicated. All protein molecular weights are based
on mean average molecular weights unless otherwise indicated.
[0162] A. Methods In Molecular Genetics
[0163] Standard techniques are used for recombinant nucleic acid
methods, polynucleotide synthesis, in vitro polypeptide synthesis,
microbial culture and transformation (e.g., electroporation), and
the like. Generally enzymatic reactions and purification steps
using commercially available starting materials are performed
according to the manufacturer's specifications. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references (see, generally,
Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed.
(1989); Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., incorporated herein by reference) referenced herein.
[0164] Oligonucleotides can be synthesized on an Applied Bio
Systems or other commercially available oligonucleotide synthesizer
according to specifications provided by the manufacturer.
Polynucleotide primers may be prepared using any suitable method,
such as, for example, the phosphotriester and phosphodiester
methods, or automated embodiments thereof. In one such automated
embodiment, diethylphosphoramidites are used as starting materials
and may be synthesized as described by Beaucage et al., 1981,
Tetrahedron Letters 22:1859, and U.S. Pat. No. 4,458,066.
[0165] Methods for PCR amplification are known in the art (PCR
Technology: Principles and Applications for DNA Amplification, Ed.
Erlich, Stockton Press, New York, N.Y. (1989); PCR Protocols: A
Guide to Methods and Applications, eds. Innis, Gelfland, Sninsky,
and White, Academic Press, San Diego, Calif. (1990); Mattila et
al., 1991, Nucleic Acids Res. 19:4967; Eckert and Kunkel, 1991, PCR
Methods and Applications 1:17; and the U.S. Patents noted above.
Optimal PCR and hybridization conditions will vary depending upon
the sequence composition and length(s) of the targeting
polynucleotide(s) primers and target(s) employed, and the
experimental method selected by the practitioner. Various
guidelines may be used to select appropriate primer sequences and
hybridization conditions (see, Sambrook et al., supra). Generally
PCR is carried out in a buffered aqueous solution, preferably at a
pH of 7-9, most preferably about 8. The deoxyribonucleoside
triphosphates dATP, dCTP, dGTP, and TTP are also added to the
synthesis mixture in adequate amounts, and the resulting solution
is heated to about 85-100 degrees C. for about 1 to 10 minutes,
preferably from 1 to 4 minutes. After this heating period, the
solution is allowed to cool to about 20-40 degrees C., for primer
hybridization. To the cooled mixture is added an agent for
polymerization, and the reaction is allowed to occur under
conditions known in the art. This synthesis reaction may occur at
from room temperature up to a temperature just over which the agent
for polymerization no longer functions efficiently. Thus, for
example, if a heat-labile DNA polymerase is used as the agent for
polymerization, the synthesis temperature is generally no greater
than about 45 degrees C. The agent for polymerization may be any
compound or system that will function to accomplish the synthesis
of primer extension products, including enzymes. Suitable enzymes
for this purpose include, for example, E. coli DNA polymerase I or
the Klenow fragment thereof, Taq DNA polymerase, and other
available DNA polymerases.
[0166] The newly synthesized strand and its complementary nucleic
acid strand form double-stranded molecules used in the succeeding
steps of the process. In the next step, the strands of the
double-stranded molecule are separated using any of the procedures
described above to provide single-stranded molecules. The steps of
strand separation and extension product synthesis can be repeated
as often as needed to produce the desired quantity of the specific
nucleic acid sequence. The amount of the specific nucleic acid
sequence produced will accumulate in an exponential fashion.
[0167] B. Subtractive Hybridization Differential Display
[0168] Both the subtractive hybridization method and the
differential display method have disadvantages for isolating rare
mRNAs that are differentially expressed. Subtractive hybridization
can be useful for enriching a pool of non-abundant cDNA species,
but conventional screening of the resultant library (ies), even if
PCR amplified, is biased in favor of identifying species that are
still abundant within the selected non-abundant cDNA pool, making
difficult the isolation of very rare cDNA species with a
conventional subtractive hybridization enrichment protocol.
Differential display of mRNA amplified by PCR is biased by the
initial abundance of the various mRNA species and often
under-represents or fails to detect rare mRNA species among the
many mRNA species that are more abundant and not substantially
differentially expressed.
[0169] The present invention provides a subtractive hybridization
differential display method that is particularly preferred for
isolating rare mRNAs, such as those expressed by the TPC2 and TPC3
genes. In brief, this method comprises the steps of: (1) one or
more cycles of subtractive hybridization of two cDNA populations to
generate a population of subtracted cDNA that is selectively
enriched for cDNA species of low abundance mRNAs that are present
at higher levels in one of the two cDNA populations, and (2)
differential display of the cDNA on an electrophoretic gel and
recovery of individual differentially expressed cDNAs by recovery
from the gel. PCR amplification, under suitable PCR conditions, of
said subtracted cDNA population with a 5' primer of arbitrary
nucleotide sequence and optionally with a 3' primer comprising
poly(dT) and/or poly(dT) and two or more arbitrary nucleotides at
the 3' end to generate PCR products is typically used to replicate
or amplify a subtracted library.
[0170] To accomplish the initial step(s) of subtractive
hybridization, RNA prepared by conventional methods from a first
cell population and RNA from a second cell population are
separately reverse-transcribed and second-strand synthesized to
form two pools of double-stranded cDNA, a tester pool comprising
sequences of the mRNA species(s) for which enrichment is desired,
and a driver pool comprising the sequences to be subtracted from
the tester pool. The two pools may be fragmented by endonuclease
digestion (restriction endonuclease or non-specific endonuclease)
if desired to degrade cDNA consisting of tandem repeated sequences
and to enhance hybridization efficiency. The driver pool is
labeled, such as by photobiotinylation or attachment of another
suitable recoverable label. The driver pool and tester pool are
denatured and mixed together in a reaction mixture under
hybridization conditions and incubated for a suitable hybridization
period. The reaction mixture is contacted with a ligand which binds
the recoverable label on the driver cDNA and which can be readily
recovered from the reaction mixture (e.g., using avidin attached to
magnetic beads), such that a substantial fraction of the driver
cDNA and any tester cDNA hybridized thereto is selectively removed
from the reaction mixture.
[0171] The remaining reaction mixture is enriched for tester cDNA
species that are preferentially expressed in the first cell
population as compared to the second cell population. The enriched
(subtracted) tester cDNA pool may be subjected to one or more
additional rounds of subtractive hybridization with a pool of
labeled driver cDNA, which may be substantially identical to the
initial pool of driver cDNA or which may represent a different cell
population having mRNA species which are desired to be subtracted
from the subtracted tester cDNA pool. A variety of means for
accomplishing the subtractive hybridization(s) and suitable
methodological guidance are available to the artisan. See Lee et
al., 1991, Proc. Natl. Acad. Sci. (U.S.A.) 88:2825; Milner et al.,
1995, Nucleic Acids Res. 23:176; Luqmani et al., 1994, Anal.
Biochem. 222:102; Zebrowski et al., 1994, Anal. Biochem. 35
222:285; Robertson et al., 1994, Genomics 23:42; Rosenberg et al.,
1994, Proc. Natl. Acad. Sci. (U.S.A.) 91:6113; Li et al., 1994,
Biotechniques 16:722; Hakvoort et al., 1994, Nucleic Acids Res.
22:878; Satoh et al., 1994, Mutat. Res. 316:25; Marechal et al.,
1993, Anal. Biochem. 208:330; El-Deiry et al., 1993, Cell 75:817;
Hara et al., 1991, Nucleic Acids Res. 19:7097; and Herfort and
Garber, 1991, Biotechniques 11:598, each of which is incorporated
herein by reference.
[0172] After the subtractive hybridization is completed, the
subtracted tester cDNA is subjected to differential display. The
general strategy involves amplification of cDNAs from the
subtracted tester cDNA pool by PCR using one or a set of arbitrary
sequence primers. Arbitrary primers are selected according to
various criteria at the discretion of the practitioner so that each
will amplify only a fraction of the DNAs in the subtracted cDNA
pool so that the amplification products can be resolved and
individually recovered on a separation system, such as a
polyacrylamide gel. In part because the number and complexity of
cDNA species represented in any particular subtracted tester pool
may vary considerably depending upon the nature and complexity of
the driver and tester pools, the selection of arbitrary primers and
their sequence(s) is determined by the practitioner with reference
to the literature. See U.S. patent application Ser. No. 08/235,180,
filed Apr. 29, 1994; Linskens et al., 1995, Nucleic Acids Res. 23
(16): 3244-3251; Liang et al., 1993, Nucleic Acids Res. 21:3269;
Utans et al., 1994, Proc. Natl.
[0173] Acad. Sci. (U.S.A.) 91:6463; Zimmermann et al., 1994, Proc.
Natl. Acad. Sci. (U.S.A.) 91:5456; Fischer et al., 1995, Proc.
Natl. Acad. Sci. (U.S.A.) 92:5331; Lohmann et al., 1995,
Biotechniques 18:200; Reeves et al., 1995, Biotechniques 18:18; and
Maser et al., 1995, Semin. Nephrol. 15:29, each of which is
incorporated herein by reference.
[0174] The subtracted tester cDNA pool and a separate cDNA pool
prepared in the same way from a cell line or tissue that does not
express (or expresses at lower levels) the rare protein is
amplified with suitable arbitrary primer(s) (i.e., primers having a
predetermined sequence that is selected without reference to a
sequence of a desired differentially expressed mRNA) for a suitable
number of amplification cycles to generate sufficient amplification
product for display and recovery of desired species, as can be
determined empirically. The primer(s) may comprise 5'-terminal
sequences which serve to anchor other PCR primers (distal primers)
and/or which comprise a restriction site or half-site or other
ligatable end. The amplified products are usually labeled and are
typically resolved by electrophoresis on a polyacrylamide gel; the
location(s) where label is present in the subtracted tester cDNA
but not present (or present at much lower levels) in the control
cDNA are excised, and the labeled product(s) is (are) recovered
from the gel portion, typically by elution.
[0175] The resultant recovered product species (typically an
expressed sequence tag or EST cDNA) can be subcloned into a
replicable vector with or without attachment of linkers, amplified
further, and/or sequenced directly. Once the EST(s) is recovered,
it can be used to obtain a substantially full length cDNA from a
cDNA library. The EST(s) can be sequenced and the sequence
information used to generate a primer for primer extension
(5'-RACE), or the EST can be labeled and used as a hybridization
probe to identify larger cDNA clones from a cDNA library. Genomic
or full length cDNA clones corresponding to ESTs can be isolated
from clone libraries (e.g., available from Clontech, Palo Alto,
Calif.) using the labeled EST (e.g., by nick-translation or
end-labeling) or other hybridization probes with nucleotide
sequences corresponding to those identified in the EST in
conventional hybridization screening methods.
[0176] Thus, double stranded cDNA is made from total RNA purified
by CsCI gradient centrifugation. In general, mix 5 _g of total RNA,
0.5 _g oligo dT (12 to 18 bases), and water (deionized water is
routinely used) in a total of 7 .sub.13 I, denature RNA at 95
degrees C. for 5 to 10 minutes, and placed on ice. The denatured
RNA and oligo dT is then added to a tube containing 4 _I of
5.times.first strand synthesis buffer (BRL), 2 _I of 0.1 M DTT
(BRL), 1 _I of dNTP (10 mM each), and 1 _I of RNAsin (Pharmacia),
and warmed for 2 minutes at 42 degrees C. About 5 _I of Superscript
II.TM. reverse transcriptase (BRL) is added to the reaction
mixture, and first strand cDNA synthesis is performed at 42 degrees
C. for 60 minutes. Then, the reaction mixture is placed on ice and
is ready for the synthesis of second strand. The first strand cDNA
is added to a tube containing 111.1 _I of water, 16 _I of
10.times.E. coli DNA ligase buffer, 3 _I of dNTP (10 mM each), 1.5
_I of E. coli DNA ligase (15 units, BRL), 7.7 _I E. coli DNA
polymerase (40 units, Pharmacia), and 0.7 _I of E. coli RNAse H
(BRL). The reaction mixture is incubated for two hours at 16
degrees C., and then 1 _I of T4 DNA polymerase (10 units,
Pharmacia) is added. The incubation continues for 5 more minutes at
the same temperature, and the reaction is stopped by the addition
of 2 _I of 0.5 M EDTA and phenol/chloroform extraction, usually
performed twice. The double-stranded cDNA is precipitated with
ethanol and resuspended in 12 _I of TE buffer.
[0177] The cDNA is then modified by the addition of linkers. Mix 10
_I of cDNA prepared as above with 4 _I of 10.times.buffer for RsaI,
21 _I of water, and 5 _I of RsaI (25-50 units), and incubate the
mixture for two hours at 37 degrees C. Four _I is removed and
checked on an agarose gel (1%) along with the uncut cDNA for
completion of digestion. The restriction enzyme is then inactivated
for 10 min. at 65 degrees C.
[0178] The linkers are prepared as double stranded oligonucleotides
by mixing 10 _g of each of:
3 (SEQ ID NO: 19) NotA (5'-pATAGCGGCCGCAAGAATTCA-NH.sub.2-- 3'; and
(SEQ ID NO: 20) NotB (5'-TGAATTCTTGCGGCCGCTAT-3'; or (SEQ ID NO:
21) Ancol (5'-pCAGAAGCTTGGTTGGATCCAGCAAG-NH.sub.2-3'; and (SEQ ID
NO: 22) PCR02 (5'-CTTGCTGGATCCACCAAGCTTCTG-3'- ,
[0179] PCRO2 (5'-CTTGCTGGATCCAACCAAGCTTCTG-3'(SEQ ID NO: 22), with
5.6 _I 10.times.buffer (One for all .TM., Pharmacia) and water to a
final volume of 56 _I. Heat the mixture at 68 degrees C. for 5
minutes, then 55 degrees C. for 5 minutes, and then 45 degrees C.
for 10 minutes. Add 55 _I of double stranded oligonucleotide NotAB
to the tube containing the digested tester cDNA (HUVEC, BJ, IMR90,
IDH4, 293 or testes tissue--the telomerase negative cell lines are
used as controls). Add 55 _I of double stranded oligonucleotide
Ancol-PCR02 to the tube containing the digested driver cDNA
(HUVEC). To both tubes add 2 _I of 100 mM ATP, 3.3 _I of
10.times.lipase buffer (Pharmacia), 1 _I of T4 DNA ligase
(Pharmacia), and water to 100 _I. The reaction mixture is incubated
at 15 degrees C. overnight. The reaction mixture is then removed
from a 15 degrees C. water bath to room temperature and incubated
for another two hours. The ligated cDNA is extracted with
phenol/chloroform twice and ethanol precipitated. The pellet is
resuspended in 12 _I of TE buffer. Half of the product is loaded on
a 1.4% low melting point agarose gel, and DNA with a size range
from 100 to 1600 base pairs is excised.
[0180] PCR amplification of the tester and driver cDNA libraries is
carried out by taking about 1 _I of each gel slice isolated as
above (melted at 65 degrees C. before use) and mixing with 10 _I of
NotB (for testers--this oligonucleotide serves as both the 5' and
3' primers) or PCR02 (for the driver), 5 _I of 10.times.PCR buffer,
6 _I dNTP (2.5 mM each), 1 unit of Taq polymerase (Boehringer
Mannheim or Perkin Elmer), 1 unit of Pfu polymerase (Stratagene),
0.2 _g of gene 32 protein (Boehringer Mannheim), and water to 50
_I. PCR is performed for 20 cycles at 94 degrees C. for 45 sec., 60
degrees C. for 45 sec., and 72 degrees C. for 2 min., with a 5 min.
extension at 72 degrees C. after completion of the last cycle. The
driver is PCR amplified in multiple reactions to make enough DNA
for photobiotinylation.
[0181] Photobiotinylation of the driver cDNA is conveniently
accomplished as follows. About 100 _g of driver cDNA in 1 mM EDTA
is mixed with 100 _I of photo biotin (Vector). This mixture is
placed on ice with the lid open and irradiated for 15 min. with a
light source located about 10 cm away from the tube. After the
irradiation, 30 _I of 1 M Tris-CI (pH 9.1) is added to the tube,
and the biotinylated DNA is extracted with water-saturated butanol
several times (4.times.) until the orange color disappears from the
aqueous phase. The extraction process is repeated once, and the
biotinylated DNA is precipitated with ethanol and resuspended in TE
buffer to a final concentration of 1 _g/_I.
[0182] Subtraction hybridization is conveniently accomplished as
follows. Mix 8 _g of biotinylated driver DNA with 0.4 _g of tester
DNA (concentrations estimated by OD measurement and ethidium
bromide staining of the gel). The mixed DNA is precipitated with
ethanol and resuspended in 10 _I of HE buffer (10 mM HEPES, pH
7.3,1 mM EDTA). The DNA is denatured at 100 degrees C. for 4 min.
and transferred to ice. About 10 _I of 2.times.hybridization
solution containing 1.5 M NaCI, 50 mM HEPES, pH 7.3,10 mM EDTA, and
0.2% SDS is then added to the tube. Two drops of mineral oil are
added, and the DNA is denatured again at 100 degrees C. for 4 min.
and transferred to a water bath at 68 degrees C. The hybridization
is performed at this temperature for 22 hours. Biotinylated DNA is
removed with streptavidin MagneSphere.TM. Paramagnetic Particles
(Promega), and the tester DNA remaining is recovered.
[0183] A second subtraction is performed by mixing recovered tester
DNA (about 80 _I) with 8 _I (8 _g) of biotinylated driver DNA and
then precipitation with ethanol. The precipitated DNA pellet is
resuspended in 10 _I of HE buffer. The denaturation, hybridization,
and recovery are performed as above; however, the second
hybridization is performed for only 2 hours at 68 degrees C. PCR
amplify the recovered DNA (0.3 _I) for 18 cycles in a reaction
mixture containing 2 _I of 10.times.Pfu polymerase buffer, 2.5 _I
of 2.5 mM dNTP, 0.2 _I of Taq polymerase (1 unit), 0.4 _I of Pfu
polymerase (1 unit), 0.04 _I of T4 gene 32 protein, and water to 20
_I. The products are checked on a 1% agarose gel to confirm
relative concentrations. The subtraction hybridization can be
repeated on these samples. The final subtracted samples are PCR
amplified (18 cycles) and diluted (1 to 10 or 1 to 15) and used for
enhanced differential display.
[0184] Enhanced differential display of subtracted cDNA involves
PCR amplification with 5' arbitrary primer(s) and a 3' oligo dT
primer with two randomized bases at the 3' end, recovery of bands
identified as containing cDNA corresponding to differentially
expressed mRNAs, and PCR amplification, sequencing, and/or cloning
of the bands identified. Add 1 _I of one 5' primer (20 _M stock) or
two 5' primers (half of each) or 1.2 _I of one 5' primer (1 _I) and
one 3' primer (0.2 _I) to the tube. Add 1 _I of subtracted DNA to
the same tube. To this mixture, add 8 _I of cocktail mix containing
1 _I of 10.times.. PCR buffer for Pfu polymerase (commercially
available), 1 _I of dNTP (2.5 mM each), 0.3 mM alpha-.sup.32P-dATP,
0.1 _I of Taq polymerase, 0.2 _I of Pfu polymerase (Stratagene),
0.02 _I of T4 gene 32 protein (Boehringer Mannheim), and 5.38 _I
water. Overlay one drop of mineral oil, and PCR amplify for 4
cycles at 94 degrees C. for 45 sec., 39 degrees C. for 1 min., and
72 degrees C. for 1 min., and then 22 cycles at 94 degrees C. for
45 sec., 60 degrees C. for 1 min., and 72 degrees C. for 1 min.,
with a final extension for 5 min. at 72 degrees C. About 5 _I of
formamide dye is added to the PCR product, and the products are
denatured at 95 degrees C. for 2-3 min. and loaded onto a prewarmed
6% polyacrylamide sequencing gel, which is run at 1900 to 2000
constant voltage (do not allow current to reach 50 mA) until the
xylene cyanol dye is one inch from the bottom of the gel. The gel
is dried under vacuum at 80 degrees C. for 45 min. and exposed to
Phosphorlmager.TM.. screen (for notebook record) and/or then to
X-ray film at room temperature for one or two days (tape the gel to
the film and punch three holes at the three corner of the gel and
film for easy identification of bands).
[0185] Differentially expressed gene fragments appear as bands on
the screen or film that are present in the lanes on the gel
corresponding to the cDNA of the tester cells but present at lower
levels or absent from the lanes corresponding to the cDNA of the
control lanes. The bands can be recovered from the gel by first
aligning the gel with the film or screen (based on the three holes
and marks) and then excising the bands of interest with a razor
blade and transferring the gel slice to an Eppendorf.TM. tube.
Rinse the razor blade between each cutting operation to avoid cross
contamination. To remove the urea and paper backing used with
sequencing gels without substantial loss of the desired DNA, add
about 900 _I of TE buffer to the tube containing the gel slice,
incubate the tube at room temperature for 10 min., and then remove
and discard the paper and TE buffer. To prepare a solution of the
desired DNA from the gel slice, the gel slice is suspended in 40 _I
of TE buffer containing 100 mM NaCI and heated for 10 min. at 95-98
degrees C. The liquid is collected (a short centrifugation collects
the liquid at the bottom of the tube) and serves as a source of the
desired DNA.
[0186] This DNA can be PCR-amplified by placing 1-3 _I of recovered
DNA in a 50 _I total reaction volume in a reaction mixture
containing 6 _I of total primer(s), 5 _I of 10.times.. PCR buffer
for Pfu polymerase, 6 _i of dNTP (2.5 mM each), 0.25 _I of Taq
polymerase, 0.5 _I of Pfu polymerase, 0.05 _I of T4 gene 32
protein, and water. The PCR is performed for 25 cycles at 94
degrees C. for 45 sec., 60 degrees C. for 1 min., and 72 degrees C.
for 1 min., with a 5 min. extension at 72 degrees C. at the end of
the last cycle. The PCR products can be stored or further
processed, i.e., subcloned and sequenced.
[0187] The availability of plasmids comprising restriction
fragments corresponding to the open reading frames of the TPC2 and
TPC3 genes makes possible the efficient isolation of these gene and
gene products from other mammalian cells as well as the chemical
synthesis in vitro of these genes and gene products and related
reagents, i.e., peptides, oligonucleotides, antibodies, and
antibody fragments.
[0188] C. RT-PCR Protocol for TPC3
[0189] Cell extracts are prepared using CHAPS, as described for the
TRAP assay (TRAP-eze.TM. kit, Oncor). About 2 _I of cell extract
are used per assay; typically 30-35 cycles of PCR are performed.
Total RNA is prepared using the TRIzol.TM. RNA extraction method
(Life Technologies) on cell pellets or CHAPS extracts. Each PCR
tube contains: 15 _I of water; 2.5 _I of 25 _M Mn(OAc).sub.2; 5.5
_I of 5.times.EZ buffer (Perkin Elmer); 0.3 _I of 25 _M dNTPs; 1 _I
of rTth DNA polymerase buffer (Perkin Elmer); 0.1 _I (300 _M) of
primer TF2 (5'-CTCACTGTAGACACTGCCTCAGTTTC-3'(SEQ ID NO: 23); and
0.1 _I (300_M) of primer TR2 (540 CAGAGGCTGGCACTGGAACTCAAGATC-3(-
SEQ ID NO: 24) in a total volume of .about.25 _I. RT-PCR conditions
include a six minute treatment at 94 degrees C. to denature
protein-RNA complexes; a thirty minute treatment at 65 degrees C.
for the reverse transcription reaction; a 1.5 minute treatment at
94 degrees C. to denature DNA-RNA complexes; thirty cycles of PCR
amplification with each cycle comprising a 30 second treatment at
94 degrees C. and a 30 second treatment at 65 degrees C.; and a
final extension reaction by treatment for seven minutes at 60
degrees C. After PCR, the samples can be analyzed by gel
electrophoresis using 1.times.TBE polyacrylamide gels and staining
with SYBR-Green I. Tests showed that this primer set amplifies band
of correct size in both mortal and immortal cell lines and
demonstrate that the TPC3 mRNA is expressed more abundantly in
immortal cell lines.
[0190] D. RT-PCR Protocol for hTR
[0191] First strand cDNA synthesis is performed by mixing total RNA
(1 _g) with 40 to 80 ng random hexamer in 11 _I, heating to 95
degrees C. for 5 min. to denature the nucleic acids (the thermal
cycler may be used for this step), and then cooling on ice. The
reaction mixture (8 _I) containing 4 _I of 5.times.buffer (BRL,
provided with the RTase), 2 _I of 0.1 M DTT,1 _I of 10 mM dNTP
(each), and 1 _I of RNAse inhibitor (Pharmacia) is added to the
denatured RNA and hexamer mixture and placed in a water bath at 42
degrees C. After a 1-2 min. incubation, 1 _I of Superscript II.TM.
RTase (BRL) is added to the mixture and the incubation continued
for 60 min. at 42 degrees C. The reaction is stopped by heating the
tube containing the reaction mixture for 10 min. at 95 degrees C.
The first strand cDNA is collected by precipitation and brief
centrifugation and aliquoted to new tubes, in which it can be
quickly frozen on dry ice and stored at -80 degrees C., if
necessary, for later use.
[0192] PCR amplification of hTR cDNA with specific primer sets can
be generally accomplished as follows. About 1 _I of cDNA is used
for each primer set. For a 10 _I PCR with .sup.32P-dATP nucleotide,
1 _I of cDNA is mixed with 1 _I of 10.times.Taq buffer, 20 pmol of
each primer, 1 _I of 2.5 mM dNTP, 5 _Ci alpha-.sup.32P-dATP, 1 unit
of Taq polymerase (Boehringer Mannheim), 1 unit of Taq antibody
(Clontech), 0.2 _g of T4 gene 32 protein (Boehringer Mannheim), and
water to 10 _I. One drop of mineral oil is then added to the tube.
The conditions for PCR amplification for hTR are about 20 cycles of
amplification, with each cycle comprising a treatment at 94 degrees
C. for 45 sec., 60 degrees C. for 45 sec., and 72 degrees C. for
1.5 min. The primers used for the RT-PCR of hTR are shown
below.
[0193] Upstream primer: F3b, 5'-TCTAACCCTAACTGAGAAGGGCGTAG-3'(SEQ
ID NO: 25);
[0194] Downstream primer: R3c, 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3'(SEQ
ID NO: 26).
[0195] Amplification of hTR with the F3b and R3c primer pair
produces a 126 bp product. PCR products labeled with .sup.32p can
be conveniently detected by adding 5 _I of a formamide/dye mixture
to the products, heating the products to denature the nucleic
acids, separating the products by 6% urea polyacrylamide gel
electrophoresis, and then exposing a Phosphorlmager.TM. cassette or
X-ray film to the gel.
[0196] The invention has been described in terms of preferred
embodiments and illustrated by way of example and is claimed below.
Sequence CWU 1
1
* * * * *