Methods and reagents for regulating telomere length and telomerase activity

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.

FIELD OF THE INVENTION 
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 
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). 
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 31 Aug. 1995, and 08/330,123, filed 27 Oct. 
1994, each of which is incorporated herein by reference. 
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 15 Apr. 96, 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 
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 one embodiment, the invention provides recombinant mammalian host cells 
containing: 
(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 
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 
(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 
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; 
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. 
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). 
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. 
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: 
(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 
(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. 
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. 
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. 
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. 
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: 
(i) an .about.3.5 kb NotI-BstEII restriction fragment of plasmid pGRN109; 
or 
(ii) an .about.1.4 kb EcoRI-BamHI restriction fragment of plasmid pGRN92. 
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. 
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. 
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 20 Oct. 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. 
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: 
(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; 
(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; 
(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 
(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. 
These and other embodiments of the invention will be described in detail 
below.

These Figures are discussed in more detail below, where a variety of 
preferred embodiments of the invention are described. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
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. 
I. DEFINITIONS 
II. CLONING AND CHARACTERIZATION OF THE TPC2 AND TPC3 GENES 
III. RECOMBINANT HOST CELLS 
IV. OLIGONUCLEOTIDES AND NUCLEIC ACIDS 
V. PEPTIDES AND PROTEINS 
VI. ANTIBODIES 
VII. METHODS 
VIII. EXAMPLES 
I. DEFINITIONS 
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. 
"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. 
"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". 
"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. 
"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. 
"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. 
"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. 
"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.3 H, .sup.14 C., .sup.35 S, .sup.125 I, .sup.131 I) 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. 
"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. 
"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. 
"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. 
"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). 
"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 NaCl or KCl, pH 6.5-8.5, 20-45 degrees C., and 0.001-10 
mM divalent cation (e.g., Mg.sup.++, Ca.sup.++); preferably about 150 mM 
NaCl or KCl, 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 NaCl, 5-50 mM Tris HCl, 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. 
"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. 
"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. 
"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. 
"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. 
"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. 
"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. 
"Selected from" refers, in connection with sequences, to one sequence 
sharing identity with another sequence. 
"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. 
"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, New 
York. 
"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. 
"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. 
"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. 
"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 (&lt;500 Daltons), stabilizers (e.g., BSA), and elemental ion 
species are not considered macromolecular species for purposes of this 
definition. 
"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 MgCl.sub.2, 50 mM KCl, 10 mM Tris-HCl, pH 9.0, and 0.1% Triton 
X-100. 
"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. 
"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. 
II. CLONING AND CHARACTERIZATION OF THE TPC2 AND TPC3 GENES 
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." 
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. 
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). 
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. 
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: 
tpc-p1 5'-ATGGGGATTCCAGGGTGGAGCT-3', (SEQ ID NO: 6)and 
tpc-p4 5'-ACCTGCTCTCAGGGCCCACAAGT-3', (SEQ ID NO: 7); 
and the primers used for TPC3 were: 
tpc-p13 5'-TAAGACAAAGAACAGGTCACAACA-3' (SEQ ID NO: 8), and 
tpc-p14 5'-ATTTGTGCTTAGAGGTCGTGCCAG-3' (SEQ ID NO: 9). 
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. 
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. 
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 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. 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. 
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. 
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. 
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. 
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. 
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). 
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. 
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. 
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, 22 Feb. 1996, Nature 379:694. The homeobox domain 
in TPC3 is: LAMCTNLPEARVQVWFKNRRAKFR (SEQ ID NO: 10). 
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!-YA/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: WSYGVCRDGRVFFINDQLRCTTWLHP (SEQ ID NO: 
13); WFVLADYCLFYYKAEKKRSSXSIP (SEQ ID NO: 14); and 
WEEGFTEEGASYFIDHNQQTTAFRHP (SEQ ID NO: 15). 
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. 
III. RECOMBINANT HOST CELLS 
In one embodiment, the invention provides recombinant mammalian host cells 
containing: 
(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 
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 
(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 
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; 
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. 
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). 
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. 
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. 
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). 
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. 
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. 
IV. OLIGONUCLEOTIDES AND NUCLEIC ACIDS 
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: 
(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 
(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. 
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. 
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. 
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. 
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. 
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. 
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 .mu.g/ml) and puromycin (0.2 .mu.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. 
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. 
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. 
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. 
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. 
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.). 
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. 
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. 
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. 
V. PEPTIDES AND PROTEINS 
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: 
(i) an .about.3.5 kb NotI-BstEII restriction fragment of plasmid pGRN109; 
or 
(ii) an .about.1.4 kb EcoRI-BamHI restriction fragment of plasmid pGRN92. 
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. 
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. 
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. 
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). 
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. 
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 Ecl136II-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 SalI-PstI restriction 
fragment of plasmid pMALc2 with a SalI-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. 
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. 
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. 
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 20 Oct. 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. 
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. 
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. 
VI. ANTIBODIES 
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. 
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. 
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. 
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. 
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. 
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. 
VII. METHODS 
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. 
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). 
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. 
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. 
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. 
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. 
VIII. EXAMPLES 
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. 
A. Methods In Molecular Genetics 
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. 
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. 
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. 
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. 
B. Subtractive Hybridization Differential Display 
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. 
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. 
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. 
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. 
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 29 Apr. 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. 
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. 
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. 
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. 
Thus, double stranded cDNA is made from total RNA purified by CsCl gradient 
centrifugation. In general, mix 5 .mu.g of total RNA, 0.5 .mu.g oligo dT 
(12 to 18 bases), and water (deionized water is routinely used) in a total 
of 7 .mu.l, 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 .mu.l of 5.times.first strand synthesis buffer (BRL), 2 .mu.l of 0.1 M 
DTT (BRL), 1 .mu.l of dNTP (10 mM each), and 1 .mu.l of RNAsin 
(Pharmacia), and warmed for 2 minutes at 42 degrees C. About 5 .mu.l 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 .mu.l of water, 16 .mu.l of 10.times.E. coli DNA ligase 
buffer, 3 .mu.l of dNTP (10 mM each), 1.5 .mu.l of E. coli DNA ligase (15 
units, BRL), 7.7 .mu.l E. coli DNA polymerase (40 units, Pharmacia), and 
0.7 .mu.l of E. coli RNAse H (BRL). The reaction mixture is incubated for 
two hours at 16 degrees C., and then 1 .mu.l 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 
.mu.l of 0.5 M EDTA and phenol/chloroform extraction, usually performed 
twice. The double-stranded cDNA is precipitated with ethanol and 
resuspended in 12 .mu.l of TE buffer. 
The cDNA is then modified by the addition of linkers. Mix 10 .mu.l of cDNA 
prepared as above with 4 .mu.l of 10.times. buffer for RsaI, 21 .mu.l of 
water, and 5 .mu.l of RsaI (25-50 units), and incubate the mixture for two 
hours at 37 degrees C. Four .mu.l 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. 
The linkers are prepared as double stranded oligonucleotides by mixing 10 
.mu.g of each of: 
NotA (5'-pATAGCGGCCGCAAGAATTCA-NH.sub.2 -3'(SEQ ID NO: 19); and 
NotB (5'-TGAATTCTTGCGGCCGCTAT-3'(SEQ ID NO: 20); or 
Ancol (5'-pCAGAAGCTTGGTTGGATCCAGCAAG-NH.sub.2 -3'(SEQ ID NO: 21); and 
PCR02 (5'-CTTGCTGGATCCAACCAAGCTTCTG-3'(SEQ ID NO: 22), with 5.6 .mu.l 
10.times. buffer (One for all.TM., Pharmacia) and water to a final volume 
of 56 .mu.l. 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 
.mu.l 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 .mu.l of 
double stranded oligonucleotide Ancol-PCR02 to the tube containing the 
digested driver cDNA (HUVEC). To both tubes add 2 .mu.l of 100 mM ATP, 3.3 
.mu.l of 10.times. lipase buffer (Pharmacia), 1 .mu.l of T4 DNA ligase 
(Pharmacia), and water to 100 .mu.l. 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 .mu.l 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. 
PCR amplification of the tester and driver cDNA libraries is carried out by 
taking about 1 .mu.l of each gel slice isolated as above (melted at 65 
degrees C. before use) and mixing with 10 .mu.l of NotB (for testers--this 
oligonucleotide serves as both the 5' and 3' primers) or PCR02 (for the 
driver), 5 .mu.l of 10.times.PCR buffer, 6 .mu.l dNTP (2.5 mM each), 1 
unit of Taq polymerase (Boehringer Mannheim or Perkin Elmer), 1 unit of 
Pfu polymerase (Stratagene), 0.2 .mu.g of gene 32 protein (Boehringer 
Mannheim), and water to 50 .mu.l. 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. 
Photobiotinylation of the driver cDNA is conveniently accomplished as 
follows. About 100 .mu.g of driver cDNA in 1 mM EDTA is mixed with 100 
.mu.l 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 .mu.l of 1 M Tris-Cl (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 .mu.g/.mu.l. 
Subtraction hybridization is conveniently accomplished as follows. Mix 8 
.mu.g of biotinylated driver DNA with 0.4 .mu.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 .mu.l 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 
.mu.l of 2.times.hybridization solution containing 1.5 M NaCl, 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. 
A second subtraction is performed by mixing recovered tester DNA (about 80 
.mu.l) with 8 .mu.l (8 .mu.g) of biotinylated driver DNA and then 
precipitation with ethanol. The precipitated DNA pellet is resuspended in 
10 .mu.l 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 .mu.l) 
for 18 cycles in a reaction mixture containing 2 .mu.l of 10.times. Pfu 
polymerase buffer, 2.5 .mu.l of 2.5 mM dNTP, 0.2 .mu.l of Taq polymerase 
(1 unit), 0.4 .mu.l of Pfu polymerase (1 unit), 0.04 .mu.l of T4 gene 32 
protein, and water to 20 .mu.l. 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. 
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 .mu.l of one 5' 
primer (20 .mu.M stock) or two 5' primers (half of each) or 1.2 .mu.l of 
one 5' primer (1 .mu.l) and one 3' primer (0.2 .mu.l) to the tube. Add 1 
.mu.l of subtracted DNA to the same tube. To this mixture, add 8 .mu.l of 
cocktail mix containing 1 .mu.l of 10.times. PCR buffer for Pfu polymerase 
(commercially available), 1 .mu.l of dNTP (2.5 mM each), 0.3 mM 
alpha-.sup.32 P-dATP, 0.1 .mu.l of Taq polymerase, 0.2 .mu.l of Pfu 
polymerase (Stratagene), 0.02 .mu.l of T4 gene 32 protein (Boehringer 
Mannheim), and 5.38 .mu.l 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 .mu.l 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). 
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 .mu.l 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 .mu.l of TE buffer containing 100 mM NaCl 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. 
This DNA can be PCR-amplified by placing 1-3 .mu.l of recovered DNA in a 50 
.mu.l total reaction volume in a reaction mixture containing 6 .mu.l of 
total primer(s), 5 .mu.l of 10.times. PCR buffer for Pfu polymerase, 6 
.mu.l of dNTP (2.5 mM each), 0.25 .mu.l of Taq polymerase, 0.5 .mu.l of 
Pfu polymerase, 0.05 .mu.l 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. 
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. 
C. RT-PCR Protocol for TPC3 
Cell extracts are prepared using CHAPS, as described for the TRAP assay 
(TRAP-eze.TM. kit, Oncor). About 2 .mu.l 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 .mu.l of water; 2.5 
.mu.l of 25 .mu.M Mn(OAc).sub.2 ; 5.5 .mu.l of 5.times. EZ buffer (Perkin 
Elmer); 0.3 .mu.l of 25 .mu.M dNTPs; 1 .mu.l of rTth DNA polymerase buffer 
(Perkin Elmer); 0.1 .mu.l (300 .mu.M) of primer TF2 
(5'-CTCACTGTAGACACTGCCTCAGTTTC -3'(SEQ ID NO: 23); and 0.1 .mu.l 
(300.mu.M) of primer TR2 (540 CAGAGGCTGGCACTGGAACTCAAGATC-3(SEQ ID NO: 24) 
in a total volume of .about.25 .mu.l. 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. 
D. RT-PCR Protocol for hTR 
First strand cDNA synthesis is performed by mixing total RNA (1 .mu.g) with 
40 to 80 ng random hexamer in 11 .mu.l, 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 .mu.l) 
containing 4 .mu.l of 5.times. buffer (BRL, provided with the RTase), 2 
.mu.l of 0.1 M DTT,1 .mu.l of 10 mM dNTP (each), and 1 .mu.l 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 
.mu.l 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. 
PCR amplification of hTR cDNA with specific primer sets can be generally 
accomplished as follows. About 1 .mu.l of cDNA is used for each primer 
set. For a 10 .mu.l PCR with .sup.32 P-dATP nucleotide, 1 .mu.l of cDNA is 
mixed with 1 .mu.l of 10.times. Taq buffer, 20 pmol of each primer, 1 
.mu.l of 2.5 mM dNTP, 5 .mu.Ci alpha-.sup.32 P-dATP, 1 unit of Taq 
polymerase (Boehringer Mannheim), 1 unit of Taq antibody (Clontech), 0.2 
.mu.g of T4 gene 32 protein (Boehringer Mannheim), and water to 10 .mu.l. 
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. 
Upstream primer: F3b, 5'-TCTAACCCTAACTGAGAAGGGCGTAG-3'(SEQ ID NO: 25); 
Downstream primer: R3c, 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3'(SEQ ID NO: 26). 
Amplification of hTR with the F3b and R3c primer pair produces a 126 bp 
product. PCR products labeled with .sup.32 P can be conveniently detected 
by adding 5 .mu.l 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 PhosphorImager.TM. 
cassette or X-ray film to the gel. 
The invention has been described in terms of preferred embodiments and 
illustrated by way of example and is claimed below: 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 26 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4232 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3315 
(D) OTHER INFORMATION: /product="TPC2" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 651 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 660 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1142 
(D) OTHER INFORMATION: /note= "N = probably T" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1160 
(D) OTHER INFORMATION: /note= "N = maybe G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1188 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1211 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1225 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1230 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1238 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1247 
(D) OTHER INFORMATION: /note= "N = probably A" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1301 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1375 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1379 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1391 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1407 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1530 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1543 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1545 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1586 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1588 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1652 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1685 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1688 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1707 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1719 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1725 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1734 
(D) OTHER INFORMATION: /note= "N = probably C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1789 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1796 
(D) OTHER INFORMATION: /note= "N = probably A" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1816 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1824 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1929 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1935 
(D) OTHER INFORMATION: /note= "N = maybe C" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 1941 
(D) OTHER INFORMATION: /note= "N = probably G" 
(ix) FEATURE: 
(A) NAME/KEY: unsure 
(B) LOCATION: 3828 
(D) OTHER INFORMATION: /note= "N = maybe A" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCGCGCTCGGCGAACATGGCGGCGGCGACGGTCGGGCGGGACACTTTACCTGAGCATTGG60 
TCCTACGGGGTGTGCCGGGATGGCCGCGTCTTCTTCATCAATGACCAGCTCCGCTGCACG120 
ACCTGGCTGCACCCGCGCACCGGGGAGCCCGTCAACTCGGGCCACATGATCCGCTCAGAC180 
CTGCCCCGCGGCTGGGAGGAGGGCTTCACGGAGGAGGGCGCCAGCTACTTCATCGACCAT240 
AACCAGCAGACCACAGCATTCAGGCATCCTGTGACGGGACAGTTTTCTCCAGAAAATAGT300 
GAATTCATTCTTCAAGAAGAGCCGAATCCACATATGTCGAAGCAAGACAGAAACCAAAGA360 
CCGTCCAGCATGGTCAGTGAAACATCCACGGCTGGGACCGCCTCCACCCTGGAGGCCAAG420 
CCTGGACCCAAGATCATAAAGTCCAGCAGTAAAGTCCACAGCTTTGGGAAGAGAGACCAG480 
GCCATTCGGAGGAACCCCAATGTTCCCGTGGTGGTGAGGGGCTGGCTGCACAAGCAGGAC540 
AGTTYTGGGATGAGGCTGTGGAAAAGGAGGTGGTTTGTGCTTGCTGATTACTGCTTATTT600 
TACTATAAAGCCGAGAAGAAGCGGTCCTCGNGGAGCATCCCCTTGCCCAGNTACGTGATN660 
TCTCCTGTGGCCCCTGAGGATCGCATAAGCCGCAAATATTCCTTTAAGGCTGTGCACACG720 
GGGATGCGAGCGCTCATCTATAACAGCTCCACAGCGGGCTCTCAGGCCGAGCAGTCAGGC780 
ATGAGGACCTACTACTTCAGTGCCGACACCCAGGAGGACATGAACGCTTGGGTCAGGGCC840 
ATGAACCAGGCTGCACAGGTGCTGTCTCGATCGTCACTGAAGAGGGATATGGAGAAGGTG900 
GAGCGGCAGGCTGTCCCCCAGGCCAACCACACAGAGTCCTGTCACGAATGTGGCCGGGTG960 
GGACCCGGACATACGAGAGATTGTCCTCATCGTGGCCATGATGACATTGTCAACTTCGAG1020 
AGGCAGGAGCAGGAGGGAGAGCAGTACCGTTCCCAGAGGGACCCACTGGAGGGCAAGCGG1080 
GACCGGAGCAAGGCCAGGTCTCCGTACTCGCCAGCCGAGGAGGATGCCTTGTTTATGGAT1140 
TNACCCAYTGGCCCAAGAGNCCAGCAGGCACAGCCCCAACGGGCAGANAARAATGGAATG1200 
CTGCCTGCYTNATATGGCCCAGGANAACANAATGGGANTGGTGGGTNCCAGCGGGCYTTT1260 
CYTCCCAGGACCAACCYTGAAAAACACAGCCAAAGGAAGANCAATCTGGCCCAGGTGGAG1320 
CACTGGGCAAGGGCCCAGAAAGGGGATAGCAGGAGTCTTCCCTTGGACCAGACGNTTCNT1380 
CGCCAGGGTCNTGGCCAATCCCTGTCNTTCCCAGAAAACTACCAGAYTYTTCCCAAGAGC1440 
ACCCGACACCCCTCGGGGGGYTCYTCGCCACYTCCCCGAAACCTGCCAAGTGACTACAAG1500 
TATGCGCAGGACCGAGCCAGCCACCTGAANATGTCGAGTGAANANCGCCGNGGCGCACCG1560 
GGATGGCACCGTGTGGCAGYTCTACNANTGGCAGCAGCGCCAGCAGTTCCGGCACGGCAG1620 
CCCCACAGCGCCCATCTGCCTTGGCTCCCCANAGTTCACCGACCAGGGCCGGAGCAGGAG1680 
CATGNTANAGGTGCCCCGCTCCATYTNTGTGCCTCCATNTCCYTNGGACATCCNTCCCCC1740 
AGGACCCCCAAGGGTYTTCCCACCCCGGCGGCCACACACACCAGCAGANCGAGTCNCAGT1800 
GAAGCCACCGGACCANARGARGANTGTGGACATCTCCCTGGGGGATTCTCCATGGGTTAC1860 
ATGAMCCACACCGTCAGCGCTCCCAGTTTACATGGAAAATCGGCTGATGATACCTACCTC1920 
CAGCTGAANAAARANCTGGANTACCTGGATCTAAAGATGACAGGCCGGGACCTTCTCAAG1980 
GATCGAAGTCTGAAGCCTGTGAAGATCGCTGAGAGCGACACTGACGTCAAACTGAGCATC2040 
TTCTGTGAACAAGACAGGGTCCTCCAGGACTTGGAAGACAAGATACGAGCCCTTAAAGAG2100 
AACAAAGACCAGCTAGAATCTGTGCTGGAGGTGTTGCACAGACAGATGGAGCAGTACCGA2160 
GACCAGCCCCAGCACTTGGAGAAGATTGCCTACCAGCAGAAGTTGCTGCAGGAGGACCTT2220 
GTCCATATCCGAGCTGAGCTCTCCAGAGAGTCCACTGAGATGGAAAATGCTTGGAACGAA2280 
TACCTGAAGTTGGAGAATGATGTGGAACAGCTGAAGCAGACCCTGCAGGAGCAACACAGA2340 
AGAGCCTTTTTTTTCCAGGAGAAATCGCAGATACAGAAAGATCTATGGAGAATTGAAGAT2400 
GTCACTGCAGGCCTGAGTGCAAATAAAGAGAACTTCAGAATTCTAGTGGAGTCAGTAAAA2460 
AATCCGGAGAGAAAAACGGTGCCTTTGTTTCCTCACCCGCCTGTGCCTTCACTCTCAACT2520 
TCTGAGAGCAAGCCGCCCCCACAGCCCAGTCCTCCCACCAGCCCTGTGCGGACCCCTCTG2580 
GAGGTTCGACTCTTCCCCCAGCTGCAAACCTACGTGCCGTACCGACCTCACCCACCCCAG2640 
CTGAGGAAAGTGACATCCCCCCTTCAGTCACCAACTAAGGCGAAGCCCAAAGTTCAGGAA2700 
GATGAAGCACCTCCCAGGCCCCCACTCCCCGAACTCTACAGCCCAGAGGACCAGCCCCCG2760 
GCTGTGCCGCCTCTGCCAAGAGAGGCCACCATCATCCGGCACACATCTGTGCGGGGCCTC2820 
AAGCGGCAGTCAGACGAGAGGAAGCGAGACCGGGAGCTGGGGCAGTGTGTGAATGGGGAT2880 
TCCAGGGTGGAGCTGCGGTCGTATGTCAGTGAGCCTGAGCTGGCGACCCTCAGCGGGGAC2940 
ATGGCCCAGCCCTCCCTAGGACTTGTGGGCCCTGAGAGCAGGTACCAGACGCTGCCAGGC3000 
AGAGGGCTCTCAGGGTCCACGTCAAGGCTCCAGCAGTCGTCCACCATTGCTCCCTACGTC3060 
ACACTCCGGAGGGGTCTCAATGCCGAAAGCAGCAAGGCGACCTTCCCTAGACCTAAGAGT3120 
GCCTTGGAGCGCCTGTACTCAGGGGATCACCAGCGAGGCAAGATGAGTGCAGAGGAGCAG3180 
CTGGAGCGCATGAAGCGACACCAGAAGGCCCTGGTCCGAGAGCGCAAGAGGACACTGGGC3240 
CAAGGGGAGAGGACGGGCCTGCCCTCATCTCGCTACCTCAGCCGGCCGCTCCCTGGAGAT3300 
CTTGGCTCAGTATGTTAGGAGGGGCCAGGCAGCGGGGCAGGGACAGGGAGCCGAGTGCCC3360 
CTCAGAGTCCCCCAAACACAAGCACATCACACCTCCCAGTGAGAGAGCTGTCCATTGACC3420 
TACATGGTTCAGAGAACACCCCACGGGGCTGTTTGTCCACGACCCAGGCTGGACGAATGC3480 
CTGGTCAGAGGGTGACCTGAACCAGAGCTGGAGTGAGGATCAAACAGGCCCAGGAGCCTG3540 
AGGAAATACCCAGTCAGTCCTCCCAGCCGCGATGGAGAGGGGCCTTTGCAGGCGTTCGGA3600 
ATCTCGGCTGAATTCAGGACCTGGGAATACAGGGTTCAGAGAGGAGAGGAGGAAGATGGT3660 
GACATGATTTGGTTAGAAGCACAAGCAAACTGATCAGCCTCCCAGACCTGCCAGCAGATG3720 
CTGTGTGAGGGTGATGGAGCACGGGGTCACACCCCTGCCCCAAGGGCCACTGGTCTCCCT3780 
GGGCTTGCAGTGCAGAGGCCTCAGGGTGTCTGGGATTGCTGGGGAGGNCTGTGCTGCCCC3840 
CTGGTGGCGCTTCCTGGCGCTGCGCCCTGTCCACAGTCACCTTAGGACCCTTTGGAAACA3900 
TTCCATTTGACTTTTCCCTGTTGYTTGAAATCCCATGTTTCCCTAAACCTCTAGCCTGAT3960 
TGTTCTTTCCCTAATTCATTGCACAAGCTCCTTTGCTTTTAGTGTTACCGCTCATTGCCT4020 
CTCTAATCCTGCCTGATTGTGTTTACAGAAGCTTCTGATTTGCATTGAACATGCTCTAAC4080 
TGGCCTGTGCTACTTATTACCGGGCTTGTAATAGCGGTTCTTGTCTCCATAGCCTGTTGA4140 
GTGTTCCCAGATGTGACTCACCTTTCTGCTGCCCTCTTCATGCAGGCCTACTGACTCATA4200 
ATTCACTTGTCCGTCGACGCGGCCGCGAATTC4232 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1105 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..1105 
(D) OTHER INFORMATION: /note= "deduced amino acid sequence of 
TPC2 open reading frame" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ProArgSerAlaAsnMetAlaAlaAlaThrValGlyArgAspThrLeu 
151015 
ProGluHisTrpSerTyrGlyValCysArgAspGlyArgValPhePhe 
202530 
IleAsnAspGlnLeuArgCysThrThrTrpLeuHisProArgThrGly 
354045 
GluProValAsnSerGlyHisMetIleArgSerAspLeuProArgGly 
505560 
TrpGluGluGlyPheThrGluGluGlyAlaSerTyrPheIleAspHis 
65707580 
AsnGlnGlnThrThrAlaPheArgHisProValThrGlyGlnPheSer 
859095 
ProGluAsnSerGluPheIleLeuGlnGluGluProAsnProHisMet 
100105110 
SerLysGlnAspArgAsnGlnArgProSerSerMetValSerGluThr 
115120125 
SerThrAlaGlyThrAlaSerThrLeuGluAlaLysProGlyProLys 
130135140 
IleIleLysSerSerSerLysValHisSerPheGlyLysArgAspGln 
145150155160 
AlaIleArgArgAsnProAsnValProValValValArgGlyTrpLeu 
165170175 
HisLysGlnAspSerXaaGlyMetArgLeuTrpLysArgArgTrpPhe 
180185190 
ValLeuAlaAspTyrCysLeuPheTyrTyrLysAlaGluLysLysArg 
195200205 
SerSerXaaSerIleProLeuProXaaTyrValXaaSerProValAla 
210215220 
ProGluAspArgIleSerArgLysTyrSerPheLysAlaValHisThr 
225230235240 
GlyMetArgAlaLeuIleTyrAsnSerSerThrAlaGlySerGlnAla 
245250255 
GluGlnSerGlyMetArgThrTyrTyrPheSerAlaAspThrGlnGlu 
260265270 
AspMetAsnAlaTrpValArgAlaMetAsnGlnAlaAlaGlnValLeu 
275280285 
SerArgSerSerLeuLysArgAspMetGluLysValGluArgGlnAla 
290295300 
ValProGlnAlaAsnHisThrGluSerCysHisGluCysGlyArgVal 
305310315320 
GlyProGlyHisThrArgAspCysProHisArgGlyHisAspAspIle 
325330335 
ValAsnPheGluArgGlnGluGlnGluGlyGluGlnTyrArgSerGln 
340345350 
ArgAspProLeuGluGlyLysArgAspArgSerLysAlaArgSerPro 
355360365 
TyrSerProAlaGluGluAspAlaLeuPheMetAspXaaProXaaGly 
370375380 
ProArgXaaGlnGlnAlaGlnProGlnArgAlaXaaLysAsnGlyMet 
385390395400 
LeuProXaaXaaTyrGlyProGlyXaaXaaAsnGlyXaaGlyGlyXaa 
405410415 
GlnArgXaaPheXaaProArgThrAsnXaaGluLysHisSerGlnArg 
420425430 
LysXaaAsnLeuAlaGlnValGluHisTrpAlaArgAlaGlnLysGly 
435440445 
AspSerArgSerLeuProLeuAspGlnThrXaaXaaArgGlnGlyXaa 
450455460 
GlyGlnSerLeuXaaPheProGluAsnTyrGlnXaaXaaProLysSer 
465470475480 
ThrArgHisProSerGlyXaaXaaSerProXaaProArgAsnLeuPro 
485490495 
SerAspTyrLysTyrAlaGlnAspArgAlaSerHisLeuXaaMetSer 
500505510 
SerGluXaaArgXaaGlyAlaProGlyTrpHisArgValAlaXaaLeu 
515520525 
XaaXaaAlaAlaAlaProAlaValProAlaArgGlnProHisSerAla 
530535540 
HisLeuProTrpLeuProXaaValHisArgProGlyProGluGlnGlu 
545550555560 
HisXaaXaaGlyAlaProLeuHisXaaCysAlaSerXaaSerXaaGly 
565570575 
HisXaaSerProArgThrProLysGlyXaaProThrProAlaAlaThr 
580585590 
HisThrSerArgXaaSerXaaSerGluAlaThrGlyProXaaGluXaa 
595600605 
CysGlyHisLeuProGlyGlyPheSerMetGlyTyrMetXaaHisThr 
610615620 
ValSerAlaProSerLeuHisGlyLysSerAlaAspAspThrTyrLeu 
625630635640 
GlnLeuXaaLysXaaLeuXaaTyrLeuAspLeuLysMetThrGlyArg 
645650655 
AspLeuLeuLysAspArgSerLeuLysProValLysIleAlaGluSer 
660665670 
AspThrAspValLysLeuSerIlePheCysGluGlnAspArgValLeu 
675680685 
GlnAspLeuGluAspLysIleArgAlaLeuLysGluAsnLysAspGln 
690695700 
LeuGluSerValLeuGluValLeuHisArgGlnMetGluGlnTyrArg 
705710715720 
AspGlnProGlnHisLeuGluLysIleAlaTyrGlnGlnLysLeuLeu 
725730735 
GlnGluAspLeuValHisIleArgAlaGluLeuSerArgGluSerThr 
740745750 
GluMetGluAsnAlaTrpAsnGluTyrLeuLysLeuGluAsnAspVal 
755760765 
GluGlnLeuLysGlnThrLeuGlnGluGlnHisArgArgAlaPhePhe 
770775780 
PheGlnGluLysSerGlnIleGlnLysAspLeuTrpArgIleGluAsp 
785790795800 
ValThrAlaGlyLeuSerAlaAsnLysGluAsnPheArgIleLeuVal 
805810815 
GluSerValLysAsnProGluArgLysThrValProLeuPheProHis 
820825830 
ProProValProSerLeuSerThrSerGluSerLysProProProGln 
835840845 
ProSerProProThrSerProValArgThrProLeuGluValArgLeu 
850855860 
PheProGlnLeuGlnThrTyrValProTyrArgProHisProProGln 
865870875880 
LeuArgLysValThrSerProLeuGlnSerProThrLysAlaLysPro 
885890895 
LysValGlnGluAspGluAlaProProArgProProLeuProGluLeu 
900905910 
TyrSerProGluAspGlnProProAlaValProProLeuProArgGlu 
915920925 
AlaThrIleIleArgHisThrSerValArgGlyLeuLysArgGlnSer 
930935940 
AspGluArgLysArgAspArgGluLeuGlyGlnCysValAsnGlyAsp 
945950955960 
SerArgValGluLeuArgSerTyrValSerGluProGluLeuAlaThr 
965970975 
LeuSerGlyAspMetAlaGlnProSerLeuGlyLeuValGlyProGlu 
980985990 
SerArgTyrGlnThrLeuProGlyArgGlyLeuSerGlySerThrSer 
99510001005 
ArgLeuGlnGlnSerSerThrIleAlaProTyrValThrLeuArgArg 
101010151020 
GlyLeuAsnAlaGluSerSerLysAlaThrPheProArgProLysSer 
1025103010351040 
AlaLeuGluArgLeuTyrSerGlyAspHisGlnArgGlyLysMetSer 
104510501055 
AlaGluGluGlnLeuGluArgMetLysArgHisGlnLysAlaLeuVal 
106010651070 
ArgGluArgLysArgThrLeuGlyGlnGlyGluArgThrGlyLeuPro 
107510801085 
SerSerArgTyrLeuSerArgProLeuProGlyAspLeuGlySerVal 
109010951100 
Cys 
1105 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4080 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 79..1380 
(D) OTHER INFORMATION: /product="TPC3" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGAAACGCAGTTTAAAACTCCAGCCCAGGCCCCGTCGCGCGTAGATGGCAGCGGAGGCGG60 
CGGCGCGGGCCGGGGTGACCAGATCCCGTTCAAACTCAGCTGCCACCAAGT111 
ProAspProValGlnThrGlnLeuProProSer 
1510 
GCGCCTTTTCTCTCTGGATTGCGATTCTGCACGAATTTTCCAGTTGAG159 
AlaProPheLeuSerGlyLeuArgPheCysThrAsnPheProValGlu 
152025 
GGTGGTTCGGCGCTCAGCCAGCCTCTGCCCTCGAAGACGCGGCCTTGG207 
GlyGlySerAlaLeuSerGlnProLeuProSerLysThrArgProTrp 
303540 
TCTAGGAACCTTCAGGCGGATGCCGCCATGCAGCACTACGGGGTGAAC255 
SerArgAsnLeuGlnAlaAspAlaAlaMetGlnHisTyrGlyValAsn 
455055 
GGCTACTCACTGCACGCCATGAACTCACTCAGCGCCATGTACAACCTG303 
GlyTyrSerLeuHisAlaMetAsnSerLeuSerAlaMetTyrAsnLeu 
60657075 
CACCAGCAGGCAGCCCAGCAGGCCCAGCATGCCCCCGACTACCGGCCT351 
HisGlnGlnAlaAlaGlnGlnAlaGlnHisAlaProAspTyrArgPro 
808590 
TCAGTGCATGCGCTTACATTGGCTGAGCGCCTGGCTGGCTGTACATTT399 
SerValHisAlaLeuThrLeuAlaGluArgLeuAlaGlyCysThrPhe 
95100105 
CAAGACATCATCTTGGAGGCCCGTTATGGTTCCCAGCACCGCAAACAA447 
GlnAspIleIleLeuGluAlaArgTyrGlySerGlnHisArgLysGln 
110115120 
CGTCGCAGCCGCACAGCGTTCACGGCTCAGCAGCTCGAGGCCCTGGAA495 
ArgArgSerArgThrAlaPheThrAlaGlnGlnLeuGluAlaLeuGlu 
125130135 
AAGACCTTCCAGAAGACTCACTACCCAGATGTGGTGATGCGTGAGAGG543 
LysThrPheGlnLysThrHisTyrProAspValValMetArgGluArg 
140145150155 
CTGGCCATGTGCACCAACCTGCCTGAGGCCCGGGTGCAGGTGTGGTTC591 
LeuAlaMetCysThrAsnLeuProGluAlaArgValGlnValTrpPhe 
160165170 
AAGAACCGCCGGGCCAAGTTCCGGAAGAAGCAGCGTAGCCTGCAGAAG639 
LysAsnArgArgAlaLysPheArgLysLysGlnArgSerLeuGlnLys 
175180185 
GAACAGCTCCAGAAGCAGAAGGAGGCTGAGGGCTCCCATGGGGAAGGC687 
GluGlnLeuGlnLysGlnLysGluAlaGluGlySerHisGlyGluGly 
190195200 
AAGGCCGAGGCCCCCACTCCAGATACCCAGCTGGACACTGAGCAGCCC735 
LysAlaGluAlaProThrProAspThrGlnLeuAspThrGluGlnPro 
205210215 
CCACGTCTGCCTGGCAGCGACCCCCCTGCTGAGCTTCACCTGAGTCTG783 
ProArgLeuProGlySerAspProProAlaGluLeuHisLeuSerLeu 
220225230235 
TCTGAGCAGTCAGCCAGTGAGTCAGCCCCTGAGGATCAGCCGGACCGT831 
SerGluGlnSerAlaSerGluSerAlaProGluAspGlnProAspArg 
240245250 
GAGGAGGACCCCAGGGCAGGGGCTGAGGACCCCAAAGCTGAGAAGAGC879 
GluGluAspProArgAlaGlyAlaGluAspProLysAlaGluLysSer 
255260265 
CCTGGGGCTGACAGCAAGGGGCTGGGCTGCAAGAGGGGCAGCCCCAAG927 
ProGlyAlaAspSerLysGlyLeuGlyCysLysArgGlySerProLys 
270275280 
GCAGATTCCCCAGGCAGCCTGACCATCACTCCTGTGGCCCCAGGGGGT975 
AlaAspSerProGlySerLeuThrIleThrProValAlaProGlyGly 
285290295 
GGCCTCCTGGGCCCCTCCCACTCCTATTCCTCGTCCCCGCTGAGCCTC1023 
GlyLeuLeuGlyProSerHisSerTyrSerSerSerProLeuSerLeu 
300305310315 
TTCCGTCTGCAGGAGCAATTCCGCCAGCATATGGCGGCCACCAACAAC1071 
PheArgLeuGlnGluGlnPheArgGlnHisMetAlaAlaThrAsnAsn 
320325330 
CTGGTGCACTACTCGTCCTTCGAAGTAGGGGGTCCGGCCCCTGCTGCT1119 
LeuValHisTyrSerSerPheGluValGlyGlyProAlaProAlaAla 
335340345 
GCAGCGGCGGCTGCTGCTGTGCCCTACCTGGGCGTCAACATGGCCCCG1167 
AlaAlaAlaAlaAlaAlaValProTyrLeuGlyValAsnMetAlaPro 
350355360 
CTGGGCTCACTGCACTGCCAGTCCTACTACCAGTCCCTGTCAGCAGCC1215 
LeuGlySerLeuHisCysGlnSerTyrTyrGlnSerLeuSerAlaAla 
365370375 
GCTGCTGCCCACCAGGGTGTGTGGGGGTCTCCTCTGCTGCCTGCACCC1263 
AlaAlaAlaHisGlnGlyValTrpGlySerProLeuLeuProAlaPro 
380385390395 
CCAGCAGGCCTGGCTCCTGCATCAGCTACCCTGAACAGTAAAACCACA1311 
ProAlaGlyLeuAlaProAlaSerAlaThrLeuAsnSerLysThrThr 
400405410 
AGCATCGAGAACCTGCGGCTCCGGGCCAAGCAGCACGCGGCCTCCCTG1359 
SerIleGluAsnLeuArgLeuArgAlaLysGlnHisAlaAlaSerLeu 
415420425 
GGACTCGATACGCTGCCCAACTGACTGTCTGGCTTCCAACCCAGCCAGGGG1410 
GlyLeuAspThrLeuProAsn 
430 
TCTTAGGTGTCCCCTCCTAGCCCTGTGGTTATCCCTAGGTGGCTCTCGAGGAGTTAACTC1470 
CATGAGCCCAGGGATCCTAGGGCCTGGGGTCCTGTTCCCTGCTCCGCTTCCCCATACCCC1530 
AGCCCGAGGTGAAGCCCACACCTACACACCCTCTGCATGGCCCTGCCTGGACCCCATGGA1590 
GGCCGAATAGGGAGGAGGTGAGAGGCTGGGGTGCCCCAAGCTTCCCTCGGAGAAGTGAGA1650 
GGCTCTCCCTGGCTAGATCCTCATCTCAATAGCACCTCCTCCCTTTTCTCCCTATCCTTC1710 
TGCCCCCTAGTAAGTCTACGTGTGGAATGTGAGATATAAATATAAATATATAAAGCTATA1770 
TTTTCAGGCTCCTGCCTGCCCCAGGCCCCCTGCCCCACTCCCATCTCTTCTTCCCTGCCA1830 
CCCCTCCCTGCAGCCTCCGCGGCTCACTCCAGCCATCCCTTCTGTTTCTCCTTCTCTCTC1890 
CTTCCTTCTTCCCTTGATCTCCCTCTTCCTGTCTCTGTCCTGGTCCCTGCCCCCGTCTCG1950 
GCCCAGCCTCTGTATTCTCCACCCTTGATCTTTCTCCTTGTCTCTCCCGCTGCCCCTGGT2010 
TTCTTCCTTTGGTGTTGGCTGTGTTGGTATCATCAGTTCTTGAGCTATATTTTGTTTGGG2070 
GTTGTGGCTGGTTTTGGTTTTAGTAATTTTGCGACTTCCCGTTGCTCTCCTTCTATTCCC2130 
TTCCTTCTGCCCTGCCTGCCTCCCTGCACCTGCGGCCTCTCTAGGAAGCTGTTCCTTTCT2190 
ATGCCCAATAGAAGCAACAAGGCCCTAGCTGGAGACTCTGGGGATCTGGAGCTGCAGGCA2250 
GGAGGTGGCACTGGCTCCCACTCCCACCCCTGCCCAGGCTGGCATCTAGAAGGCGTCATG2310 
AATTACTTTCTCTTCTCTCTTCTCAATTTTGAGGTCCTCATTCCCAAGATTGAGGAGGCA2370 
GTAGTTAATCTGGGAAGGCAGTAGAATGGCCAGCATTCCTGCCTGTAAGTCTTCCCAAGA2430 
CAGAGGCCTGGTGACACAGTTCAGCCAGGACTGACCACAGGGCTCTAGAGCTCTCTTTGG2490 
TGAGACTTCCCTGGATGGAGAGCAGCAGCAGGGGAAGAGGTGCTCTCAGAGACAGCAGGG2550 
CTGGTGCTCTTCTCCCACAAGCTGAGCTCCACGTTCAGCAGATACTGTCCAAGGCAGGGG2610 
TACGGCTGACCAGGAATGAAGGTTGAACTCTGCTCCTGAGCACGGTGCGTGCAAAGCATA2670 
TAGCAGCACATAGGCTCAGGCTTCTGTAGGCTTCCTGTCCCAGAGCCAATTATGGAAGTA2730 
AGGGCTTCCCTCCAGCTAGTCACTGGAATGGAAAAGTGTGTGTCCTGTTCATAGCCAGGA2790 
AACCCAGCTCAGCAAACTCCCTTTCAAAGCTGTGTGACCGGCTGGGCATGGTGGCTCACA2850 
CCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCAATCACCTGAGGTCAGGAGTTCAA2910 
GACCAGCCTGGCTAACATGTGAAACTAATAATAATACAAAAATTAGCTGGGCGTGGTGGC2970 
ACATGCCTGTAATCCCAGCTACTTGGGAGGCTGAGTTGGGAGGATTGCTGCAATCTGGGA3030 
GGTGGAAGTTGCAGTGAGCCGAGATCATGCCACTGCACTCCAGCCTGGGCGACGGAGTGA3090 
GACTCCATCTCAAAAAAAAAAAAAATAAAAATAAAAGCTGTGTGACCTTGGGCAABCCTG3150 
TAGCCTCTCTGGGTCTGTTTCCCTGTCTGGGTTAAATGGCCTGTAAGGTCCTAGCCAGCT3210 
CTACATTCTGCATTTGCTCGCAACCTTGTAACACAGAAGTTTTTAGTTAAATTGACAACA3270 
GAAGGTTCTCAAAAGCACAATATATGAAGTAGGAAATTACTATTGCCTTTCTGTGGAGCA3330 
AGGGGTGTTGTACACACAAGCCTCACTGTAGACACTGCCTCAGTTTCCCCATAGGCATAA3390 
TGGGTCCCTTCTAGTTCAGGCAATCTGGATTTGATCTTGAGTTCCAGTGCCAGCCTCTGG3450 
AGTCACTCCATTTTCATACCTTTTCATGATCTCAGGGGCTCTGGGCAGTGGGAGGTGATG3510 
GCTTGGACAGATTCTTGGTCATGCTCCCCAACTCTTGGTGGCTCACCACTGAACACTCCA3570 
AACCCTGCTTAAAGAAGTTGATTTATTTGAAAGCCAGGGTAAAGATTGCTAAGGCTTGTC3630 
TCCTCTCCCAGTGGGAAGAGAGAGGTTCTGTTGGTGTCCTGGTTGAATTGCTTTGCAGAG3690 
AAGTCAATGCCCATCACCCTTGATGGGGGTCAGCCTAGGCTGGGGCAGATGGAGAAGGCT3750 
TTGGACAGGAAAAAAGTGAGCAGGATGGTAGTCTAGGCCAGGAGAAGTGTTTGAACAAAG3810 
CAGCAGAGATGAGACTCAGTAGACCATGGGAAGGGGGTGGCTGGCTTCACGAGAGGTGGG3870 
GGCTAAGGGGCCTGGAATCCAGGCTAAAGACCACACCTACATGTGGCAAGCACCAAGACA3930 
GGCATTTGAGGGTTTCCAAATCCTCAGGTCTCTTGCTGGGGTCTGGAATTTGGAAGGGGA3990 
ATCCACCAGCCATGGGGGCATCAGAGGAGAGACTTAGGCAGCGCTGTGGGAGGTTGGCAG4050 
ATTCCAGGAGTGACAGAGGAGGTTTTTGGT4080 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 434 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ProAspProValGlnThrGlnLeuProProSerAlaProPheLeuSer 
151015 
GlyLeuArgPheCysThrAsnPheProValGluGlyGlySerAlaLeu 
202530 
SerGlnProLeuProSerLysThrArgProTrpSerArgAsnLeuGln 
354045 
AlaAspAlaAlaMetGlnHisTyrGlyValAsnGlyTyrSerLeuHis 
505560 
AlaMetAsnSerLeuSerAlaMetTyrAsnLeuHisGlnGlnAlaAla 
65707580 
GlnGlnAlaGlnHisAlaProAspTyrArgProSerValHisAlaLeu 
859095 
ThrLeuAlaGluArgLeuAlaGlyCysThrPheGlnAspIleIleLeu 
100105110 
GluAlaArgTyrGlySerGlnHisArgLysGlnArgArgSerArgThr 
115120125 
AlaPheThrAlaGlnGlnLeuGluAlaLeuGluLysThrPheGlnLys 
130135140 
ThrHisTyrProAspValValMetArgGluArgLeuAlaMetCysThr 
145150155160 
AsnLeuProGluAlaArgValGlnValTrpPheLysAsnArgArgAla 
165170175 
LysPheArgLysLysGlnArgSerLeuGlnLysGluGlnLeuGlnLys 
180185190 
GlnLysGluAlaGluGlySerHisGlyGluGlyLysAlaGluAlaPro 
195200205 
ThrProAspThrGlnLeuAspThrGluGlnProProArgLeuProGly 
210215220 
SerAspProProAlaGluLeuHisLeuSerLeuSerGluGlnSerAla 
225230235240 
SerGluSerAlaProGluAspGlnProAspArgGluGluAspProArg 
245250255 
AlaGlyAlaGluAspProLysAlaGluLysSerProGlyAlaAspSer 
260265270 
LysGlyLeuGlyCysLysArgGlySerProLysAlaAspSerProGly 
275280285 
SerLeuThrIleThrProValAlaProGlyGlyGlyLeuLeuGlyPro 
290295300 
SerHisSerTyrSerSerSerProLeuSerLeuPheArgLeuGlnGlu 
305310315320 
GlnPheArgGlnHisMetAlaAlaThrAsnAsnLeuValHisTyrSer 
325330335 
SerPheGluValGlyGlyProAlaProAlaAlaAlaAlaAlaAlaAla 
340345350 
AlaValProTyrLeuGlyValAsnMetAlaProLeuGlySerLeuHis 
355360365 
CysGlnSerTyrTyrGlnSerLeuSerAlaAlaAlaAlaAlaHisGln 
370375380 
GlyValTrpGlySerProLeuLeuProAlaProProAlaGlyLeuAla 
385390395400 
ProAlaSerAlaThrLeuAsnSerLysThrThrSerIleGluAsnLeu 
405410415 
ArgLeuArgAlaLysGlnHisAlaAlaSerLeuGlyLeuAspThrLeu 
420425430 
ProAsn 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 981 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- RNA 
(B) LOCATION: 267..715 
(D) OTHER INFORMATION: /product="hTR" 
/note= "hTR transcript serves as 
template in the telomerase 
ribonucleoprotein" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTGCAGAGGATAGAAAAAAGNCCCTCTGATACCTCAAGTTAGTTTCACCTTTAAAGAAGG60 
TCGGAAGTAAAGACGCAAAGCCTTTCCCGGACGTGCGGAAGGGCAACGTCCTTCCTCATG120 
GCCGGAAATGGAACTTTAATTTCCCGTTCCCCCCAACCAGCCCGCCCGAGAGAGTGACTC180 
TCACGAGAGCCGCGAGAGTCAGCTTGGCCAATCCGTGCGGTCGGCGGCCGCTCCCTTTAT240 
AAGCCGACTCGCCCGGCAGCGCACCGGGTTGCGGAGGGTGGGCCTGGGAGGGGTGGTGGC300 
CATTTTTTGTCTAACCCTAACTGAGAAGGGCGTAGGCGCCGTGCTTTTGCTCCCCGCGCG360 
CTGTTTTTCTCGCTGACTTTCAGCGGGCGGAAAAGCCTCGGCCTGCCGCCTTCCACCGTT420 
CATTCTAGAGCAAACAAAAAATGTCAGCTGCTGGCCCGTTCGCCCCTCCCGGGGACCTGC480 
GGCGGGTCGCCTGCCCAGCCCCCGAACCCCGCCTGGAGGCCGCGGTCGGCCCGGGGCTTC540 
TCCGGAGGCACCCACTGCCACCGCGAAGAGTTGGGCTCTGTCAGCCGCGGGTCTCTCGGG600 
GGCGAGGGCGAGGTTCAGGCCTTTCAGGCCGCAGGAAGAGGAACGGAGCGAGTCCCCGCG660 
CGCGGCGCGATTCCCTGAGCTGTGGGACGTGCACCCAGGACTCGGCTCACACATGCAGTT720 
CGCTTTCCTGTTGGTGGGGGGAACGCCGATCGTGCGCATCCGTCACCCCTCGCCGGCAGT780 
GGGGGCTTGTGAACCCCCAAACCTGACTGACTGGGCCAGTGTGCTGCAAATTGGCAGGAG840 
ACGTGAAGGCACCTCCAAAGTCGGCCAAAATGAATGGGCAGTGAGCCGGGGTTGCCTGGA900 
GCCGTTCCTGCGTGGGTTCTCCCGTCTTCCGCTTTTTGTTGCCTTTTATGGTTGTATTAC960 
AACTTAGTTCCTGCTCTGCAG981 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ATGGGGATTCCAGGGTGGAGCT22 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ACCTGCTCTCAGGGCCCACAAGT23 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TAAGACAAAGAACAGGTCACAACA24 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ATTTGTGCTTAGAGGTCGTGCCAG24 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
LeuAlaMetCysThrAsnLeuProGluAlaArgValGlnValTrpPhe 
151015 
LysAsnArgArgAlaLysPheArg 
20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
SerSerSerLysValHisSerPheGlyLysArgAspGlnAlaIleArg 
151015 
ArgAsnProAsnValProValValVal 
2025 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /product="OTHER" 
/note= "Xaa = Ala or Pro" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /product="OTHER" 
/note= "Xaa = Ala or Pro" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
XaaProProXaaTyr 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
TrpSerTyrGlyValCysArgAspGlyArgValPhePheIleAsnAsp 
151015 
GlnLeuArgCysThrThrTrpLeuHisPro 
2025 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
TrpPheValLeuAlaAspTyrCysLeuPheTyrTyrLysAlaGluLys 
151015 
LysArgSerSerXaaSerIlePro 
20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
TrpGluGluGlyPheThrGluGluGlyAlaSerTyrPheIleAspHis 
151015 
AsnGlnGlnThrThrAlaPheArgHisPro 
2025 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 100 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ProAsnIleProGlnMetSerAlaPheTrpTyrAlaValArgThrAla 
151015 
ValIleAsnAlaAlaSerGlyArgGlnThrValAspGluAlaLeuLys 
202530 
AspAlaGlnThrAsnSerSerSerAsnAsnAsnAsnAsnAsnAsnAsn 
354045 
AsnAsnLeuGlyIleGluGlyArgIleSerGluPheAlaAlaAlaSer 
505560 
ThrLeuAspLeuLysMetThrGlyArgAspLeuLeuLysAspArgSer 
65707580 
LeuLysProValLysIleAlaGluSerAspThrAspValLysLeuSer 
859095 
IlePheCysGlu 
100 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
ArgGlyLeuLysArgGlnSerAspGluArgLysArgAspArgGlu 
151015 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
LysValThrSerProLeuGlnSerProThrLysAlaLysProLys 
151015 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /mod.sub.-- base=OTHER 
/note= "N = 5'-phosphorylated adenine 
(p-A)" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 20 
(D) OTHER INFORMATION: /mod.sub.-- base=OTHER 
/note= "N = adenine substituted at the 
3'position of deoxyribose with an amino 
group (A-NH- 2)" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
NTAGCGGCCGCAAGAATTCN20 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TGAATTCTTGCGGCCGCTAT20 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /mod.sub.-- base=OTHER 
/note= "N = 5'-phosphorylated cytosine 
(p-C)" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 25 
(D) OTHER INFORMATION: /mod.sub.-- base=OTHER 
/note= "N = guanine substituted at the 
3'position of deoxyribose with an amino 
group (G-NH- 2)" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
NAGAAGCTTGGTTGGATCCAGCAAN25 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CTTGCTGGATCCAACCAAGCTTCTG25 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CTCACTGTAGACACTGCCTCAGTTTC26 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CAGAGGCTGGCACTGGAACTCAAGATC27 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
TCTAACCCTAACTGAGAAGGGCGTAG26 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
GTTTGCTCTAGAATGAACGGTGGAAG26 
__________________________________________________________________________