EP0953042A1 - Verfahren und reagentien zur regulation der telomerenlänge und der telomerase-aktivität - Google Patents

Verfahren und reagentien zur regulation der telomerenlänge und der telomerase-aktivität

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Publication number
EP0953042A1
EP0953042A1 EP96933011A EP96933011A EP0953042A1 EP 0953042 A1 EP0953042 A1 EP 0953042A1 EP 96933011 A EP96933011 A EP 96933011A EP 96933011 A EP96933011 A EP 96933011A EP 0953042 A1 EP0953042 A1 EP 0953042A1
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EP
European Patent Office
Prior art keywords
tpc3
tpc2
protein
recombinant
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP96933011A
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English (en)
French (fr)
Inventor
Bryant Villeponteau
Junli Feng
William H. Andrews
Robert R. Adams
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Geron Corp
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Geron Corp
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Publication date
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Publication of EP0953042A1 publication Critical patent/EP0953042A1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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 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.
  • telomeres 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.
  • hTR human telomerase
  • hTR human telomerase
  • 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 and PCT patent publication No. 96/01835, each of which is incorporated herein by reference.
  • 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.
  • 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.
  • 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 invention provides recombinant mammalian host cells containing:
  • 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 ⁇ 3.5 kb Notl-Bst ⁇ l 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
  • 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 -1.4 kb EcoRI-B ⁇ mHI 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.
  • mammalian host cells 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 ⁇ 2.5 kb Hi ⁇ dlll- Sacl restriction fragment of pGRN33 (ATCC 75926).
  • the recombinant host cells of the invention have application in many useful methods also provided by the invention.
  • 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.
  • 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.
  • 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 --3.5 kb Notl-BstEll restriction fragment of plasmid pGRN109; or
  • 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., mRN A 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.
  • telomere positive cancer cells inhibition of telomerase activity can prevent telomere maintenance in those cells, inducing upon continued proliferation telomere loss, cell crisis, and death.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides a subtraction hybridization differential display method to identify, isolate, and clone expressed sequence tags (ESTs) of mRN A 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:
  • mRN A 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 cDN A preparations differing from one another with respect to presence or absence of cDN A molecules encoding said rare protein and a label incorporated into one of said first and second cDN A preparations;
  • Steps (iii) and (iv) of the above method can be repeated as often as desired, and the cDN A isolated after completion of step (iv) can be amplified by PCR, to provide cDNA preparations greatly enriched for the desired cDN A.
  • Figure 1 in parts A, B, and C, is a bar graph showing the results of RT-PCR analysis using primers specific for TPC2 ( Figure 1 A) or TPC3 ( Figure IB) cDNA.
  • Figure 1 A the number over each bar is the numerical result obtained; for RT-PCR results, this number was generated by scanning autoradiograms or PhosphorlmagerTM screens (Molecular Dynamics) of the RT- PCR products after gel electrophoresis.
  • 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).
  • Figure 1C shows TPC3 mRNA levels normalized to G APDH levels and illustrates the difference in TPC3 mRNA levels between mortal and immortal cells (the spaces marked "0.0" are provided merely as breaks in the graphed data).
  • GAPDH mRNA was used as a control; due to its greater abundance, the RT-PCR of the GAPDH samples was allowed to complete fewer cycles of PCR than used for the TPC2 or TPC3 samples.
  • Figure 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.
  • Figure 2 A 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.
  • Figure 2B shows how TPC3 mRNA levels correlate with telomerase activity (as measured using the TRAP assay) in a variety of cell lines.
  • FIG. 2C shows how hTR RNA levels correlate with telomerase activity levels in a variety of cell lines. Taken together, these results show that TPC2 and TPC3 mRNA levels correlate with hTR levels and with telomerase activity levels in a variety of mortal and immortal cells lines.
  • Figure 3 shows a restriction site and function map of the ⁇ 7.2 kb plasmid pGRN109, which contains an -3.5 kb Notl-BsiE restriction fragment that contains an -3.3 kb open reading frame encoding the TPC2 protein (labeled "ORF” and "TPC2").
  • Figure 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.
  • nucleotides and amino acids are represented using standard abbreviations and designations; however, ambiguous nucleotides are represented as shown in the key at the bottom of Figure 4.
  • the initiating methionine codon is believed to be at nucleotides 16-18 of the sequence; the termination codon is marked with " — ".
  • Figure 5 shows a restriction site and function map of the -8 kb plasmid pGRN92, which contains an -1.4 kb EcoRl-BamHl restriction fragment that contains an -1.1 kb open reading frame encoding the TPC3 protein (labeled "ORF” and "TPC3").
  • Figure 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 " — ".
  • Figure 7 shows the results of an analysis of telomerase activity levels in stable recombinant HeTe7 clones expressing the sense or antisense mRNA of gene TPC3 or a control vector.
  • the recombinant sense TPC3 mRNA reduced telomerase activity markedly in these cells.
  • Figure 8 shows the results of an analysis of telomere length in stable recombinant HeTe7 clones expressing the sense or antisense mRNA of gene TPC3 or a control vector.
  • the recombinant TPC3 sense mRNA decreased telomere length (mean TRF) in the cells.
  • Figure 9 lists the nucleotide sequence of the hTR gene and corresponding RNA transcript; the sequence shown is that of one strand of an -1 kb Psfl restriction fragment that can be isolated from plasmid pGRN33.
  • the sequence of the mature hTR transcript, which serves as the template in the telomerase ribonucleoprotein, is marked with asterisks — the 3' end of the transcript is marked with an ">" .
  • 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
  • Antibody refers to naturally occurring and recombinant poly peptides 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)'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.
  • 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 cDN A.
  • 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 opera tively 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 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., , ⁇ C, ⁇ S, * ⁇ I, " ⁇ 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).
  • enzymatic activity e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatas
  • 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).
  • 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), phosphor amidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like.
  • PNAs peptide- nucleic acids
  • oligonucleotide is 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).
  • enteral i.e., oral
  • 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 vitro physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-45 degrees C, and 0.001-10 mM divalent cation (e.g., Mg ++ , Ca ++ ); preferably about 150 mM NaCl or KC1, 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.
  • RNA transcripts 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".
  • Poly nucleotides 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, CA, which are incorporated herein by reference.
  • Poly peptide “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.
  • 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.
  • 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.
  • references 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.
  • 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, FAST A, and TF AST A in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI) 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.
  • 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.
  • non- complementary nucleotides may be attached to the 5'-end of the primer, with the remainder of the primer sequence being complementary to the template.
  • 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.
  • 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.
  • recombinant host cells can express genes that are not found within the native (non-recombinant) form of the cell.
  • 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.
  • 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.
  • 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.
  • Probe polynucleotide e.g., a polynucleotide of the invention which may include substitutions, deletions, and/ or additions
  • a specific target polynucleotide e.g., a polynucleotide having the-sequence of a TPC2 or TPC3 gene or gene product
  • 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 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 m ) for the specific sequence at a defined ionic strength and pH. The T 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.
  • 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.
  • 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.
  • suitable polymerase chain reaction (PCR) conditions include those conditions specified in U.S. Patents 4,683,202; 4,683,195; 4,800,159; and 4,965,188, each of which is incorporated herein by reference.
  • suitable reaction conditions can comprise: 0.2 mM each dNTP, 2.2 mM MgCl 2 , 50 mM KC1, 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 PhosphorlmagerTM 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 PhosphorlmagerTM screens. See also the commercially available TRAP-ezeTM telomerase assay kit (Oncor); and Morin, 1989, Cell 59:521-529.
  • 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 JRNAs 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.”
  • 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 cDN As is then separated into two different populations, one comprising the label and one that does not, thereby forming an isolated, unlabeled preparation of cDN A 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.
  • cDNAs corresponding to abundant transcripts are depleted more than 100-fold and low abundant transcripts are enriched in the subtracted cDNA libraries.
  • telomere length and telomerase regulatory proteins 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 cDN A libraries were subjected to subtraction hybridization against the telomerase negative HUVEC cDNA library.
  • differential display was performed by first replicating each of the six subtracted cDN A 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.
  • RT-PCR reverse-transcription and PCR
  • 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.
  • 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.
  • RT-PCR with primers specific for nucleotide sequences in GAPDH mRNA was performed as well.
  • the primers used for TPC2 were: tpc-pl S'-ATGGGGATTCCAGGGTGGAGCT-S 1 , and tpc-p4 5'-ACCTGCTCTCAGGGCCCACAAGT-3'; and the primers used for TPC3 were: tpc-pl3 5'-TAAGACAAAGAACAGGTCACAACA-3', and tpc-pl4 S'-ATTTGTGCTTAG AGGTCGTGCC AG-3' .
  • the RT-PCR was performed by making first strand cDN A 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.
  • Figure 1 in parts A, B, and C, shows the results of RT-PCR analysis using primers specific for the TPC2 ( Figure 1 A) or TPC3 ( Figure IB) cDNA.
  • 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).
  • TPC2 and TPC3 mRNA are 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.
  • Figure 1C shows TPC3 mRNA levels normalized to GAPDH levels and illustrates the clear difference in TPC3 mRNA levels between mortal and immortal cells.
  • TPC2 and TPC3 mRNA is present in very low abundance even in telomerase positive cells (TPC2 or TPC3 mRNA amplification products detected after -25 cycles; GAPDH or HPRT detected after -15 or -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 -1.4 million plaques was positive, indicating a very rare transcript.
  • Figure 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.
  • Figure 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.
  • Figure 2B shows how TPC3 mRNA levels correlate with telomerase activity levels in a variety of cell lines.
  • 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 Figure 9.
  • 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, such as telomere length, 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.
  • telomere negative mortal cells 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 mammalian 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.
  • 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.
  • TPC2 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 (-35.2 kb
  • TRF TRF
  • testes embryonic carcinoma Tera- 27.0 kb
  • lung carcinoma NCI-H23 17.5 kb
  • 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.
  • 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 cDN As obtained by subtraction hybridization differential display.
  • 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.
  • cDNA fragments were cloned into pBluescript Ilsk vector (Stratagene) to generate vectors for sequencing and analysis.
  • FIG 3 shows a restriction site and function map of the -7.2 kb plasmid pGRN109, which contains an -3.5 kb Notl-BstE ⁇ l restriction fragment that contains an -3.3 kb open reading frame encoding the TPC2 protein (labeled "ORF” and "TPC2").
  • Figure 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.
  • Figure 5 shows a restriction site and function map of the -8 kb plasmid pGRN92, which contains an -1.4 kb EcoRl-Bam l restriction fragment that contains an -1.1 kb open reading frame encoding the TPC3 protein (labeled "ORF” and "TPC3").
  • Figure 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 Figure 6.
  • 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.
  • TPC3 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.
  • 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, [APJ-P-P- [AP]-Y, 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.
  • Trp conserved aromatic positions
  • this domain is represented by three amino acid residue sequences: WSYGVCRDGRVFFINDQLRCTTWLHP;
  • TPC2 and TPC3 open reading frames provide 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.
  • the invention provides recombinant mammalian host cells containing:
  • 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 -3.5 kb Notl-BsfEU restriction fragment of plasmid pGRN109; or a synthetic or recombinant peptide or protein comprising at least about 6 to
  • 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 -1.4 kb EcoRl-BamHl 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.
  • mammalian host cells 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 -2.5 kb H dlll- Sacl restriction fragment of pGRN33 (ATCC 75926).
  • the recombinant host cells of the invention have application in many useful methods also provided by the invention.
  • 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.
  • genomic clones of a gene that regulates telomere length or telomerase activity 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.
  • 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).
  • a TPC2 or TPC3 cDNA or genomic clone may be used to construct transgenes for expressing poly peptides 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).
  • a constitutive promoter e.g., an HSV-tk or pgk (phosphoglycerate kinase) promoter
  • a cell-lineage specific transcriptional regulatory sequence e.g., an CD4 or CD8 gene promoter/ enhancer
  • 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.
  • 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 -10 kb in size.
  • 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 -3.5 kb Notl-BstEU restriction fragment of plasmid pGRN109; or
  • 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 -3.5 kb Notl-Bst Ell insert of ⁇ GRN109 or the -1.4 kb EcoRl-BamHl insert of pGRN92 and are useful as probes, primers, antisense therapeutics, and ribozyme therapeutics, for example.
  • 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.
  • 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.
  • the polynucleotides of the invention need not encode a functional polypeptide.
  • 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.
  • 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.
  • 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.
  • the antisense polynucleotides comprise methylphosphonate or other synthetic moieties.
  • the inhibitory nucleic acid also can be a so-called "sense" or other nucleic acid, i.e., a triplex-forming nucleic acid.
  • expression of recombinant TPC3 mRNA in a cancer cell line resulted in the inhibition of telomerase activity by over 90%.
  • the entire -1.1 kb coding sequence of the TPC3 gene was isolated as an EcoRI fragment (-2.1 kb) from vector pTATPC3.9 and inserted into the EcoRI site of mammalian expression vector pBBS212 to give rise to two vectors: pGRNlll, 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 (-2.1 kb) into pCRII vector (Invitrogen).
  • the sense and antisense vectors were used to transform HeTe7 cells by electroporation.
  • the medium was changed to selection medium containing hygromycin (300 ⁇ g/ml) and puromycin (0.2 ⁇ g/ml) for four weeks to obtain individual clones.
  • the individual clones were then isolated, expanded, and assayed for the expression of sense or antisense TPC3 gene product and vector transcription by RT-PCR.
  • the positive clones were then assayed for telomerase activity using the TRAP assay, and mean TRF values were measured at different time points.
  • Figure 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.
  • Figure 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.
  • 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.
  • 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.
  • 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.
  • the gene therapy vectors of the invention that express a nonfunctional 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.
  • 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).
  • 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 pcDNAl (Invitrogen, San Diego, CA), 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.
  • 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
  • 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, Wisconsin).
  • 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. (USA) 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).
  • yeast Gal4 transcription protein which consists of separable domains responsible for DNA-binding and transcriptional activation, serves as the transcriptional activator.
  • a reporter gene e.g., lacZ, HIS3
  • the two- hybrid 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., beta- galactosidase) 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.
  • a reporter gene e.g., beta- galactosidase
  • Yeast two-hybrid systems may be used to screen a mammalian (typically human) cDN A 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.)).
  • a mammalian (typically human) cDN A 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.)).
  • 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.
  • 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:
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • a label such as a polyhistidine polypeptide or a maltose binding protein
  • 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.
  • 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.
  • plasmids pGRN103, pGRN104, pGRN106, and pGRNHO 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).
  • MBP maltose binding protein
  • Plasmid pGRN103 encodes a fusion protein comprising the amino- terminal portion of TPC3 protein and MBP and was prepared by replacing the Xmnl-Pstl restriction fragment of plasmid pMALc2 with the Pvull-Pstl 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 £c/136II-Bfl HI restriction fragment of plasmid pM ALc2 with the BspEl (treated with Klenow in the presence of dCTP and dGTP only) - B ⁇ wHI 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 Sall-Pstl restriction fragment of plasmid pMALc2 with a S ⁇ /I-Sst?8387I restriction fragment that can be isolated from plasmid pGRN109.
  • Plasmid pGRNHO 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 pM ALc2 such that the fusion protein shown below results from expression of the plasmid in £. coli W3110 cells (only the ends of the MBP and TPC2 proteins at the junction region are shown):
  • 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.
  • 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 and KVTSPLQSPTKAKPK, 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.
  • 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.
  • 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.
  • 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.
  • TPC2 or TPC3 proteins, fragments thereof, or analogs thereof can be used to immunize an animal for the production of specific antibodies.
  • 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.
  • 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 x 10° " 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
  • 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 x 10' M"l.
  • 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 immunized and used for a source of antiserum and/ or for making monoclonal antibody secreting hybridomas.
  • 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, NY, 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.
  • 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.
  • the desired antiserum or monoclonal antibody (ies) is/ are not monospecific.
  • 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.
  • diseases of excessive or inappropriate expression e.g., neoplasia
  • Other methods of the invention are discussed in the following section.
  • 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.
  • disease states e.g., neoplasia or pre-neoplasia
  • 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.
  • 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).
  • telomere-related and telomere length regulatory components TPC2, TPC3, and hTR
  • recombinant telomerase is produced by expressing a TPC2 or TPC3 protein or active TPC2 or TPC3 analog and/or recombinant hTR in a cell.
  • 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 -2.5 kb Hindlll-Sacl insert of pGRN33 (ATCC 75926).
  • 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.
  • telomere-maintenance or telomerase activity administered 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.
  • pathological conditions e.g., cancer, inflammation, lymphoproliferative diseases, autoimmune disease, neurodegenerative diseases, and the like
  • 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.
  • 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. Patent No.4,458,066.
  • PCR Technology Principles and Applications for DNA Amplification, Ed. Erlich, Stockton Press, New York, NY (1989); PCR Protocols: A Guide to Methods and Applications, eds. Innis, Gelfland, Sninsky, and White, Academic Press, San Diego, CA (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.
  • 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.
  • an agent for polymerization 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.
  • 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.
  • 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.
  • Both the subtractive hybridization method and the differential display method have disadvantages for isolating rare mRN As 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 cDN A pool, making difficult the isolation of very rare cDN A 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 mRN As, such as those expressed by the TPC2 and TPC3 genes.
  • this method comprises the steps of: (1) one or more cycles of subtractive hybridization of two cDNA populations to generate a population of subtracted cDN A 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 cDN A 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.
  • 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 cDN A 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 cDN A hybridized thereto is selectively removed from the reaction mixture.
  • the remaining reaction mixture is enriched for tester cDN A 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 cDN A, which may be substantially identical to the initial pool of driver cDN A 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;
  • 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.
  • a separation system such as a polyacrylamide gel.
  • 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 liga table 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 cDN A 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 can be subcloned into a replicable vector with or without attachment of linkers, amplified further, and/ or sequenced directly.
  • EST(s) 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, CA) 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.
  • double stranded cDNA is made from total RNA purified by CsCl gradient centrifugation.
  • 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 ⁇ l of water, 16 ⁇ l of lOx E. coli DNA ligase buffer, 3 ⁇ l of dNTP (10 mM each), 1.5 ⁇ l of E. coli DNA ligase (15 units, BRL), 7.7 ⁇ l E. coli DNA polymerase (40 units, Pharmacia), and 0.7 ⁇ l of E. coli RNAse H (BRL).
  • the reaction mixture is incubated for two hours at 16 degrees C, and then 1 ⁇ 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 ⁇ 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 ⁇ l of TE buffer.
  • the cDNA is then modified by the addition of linkers.
  • the linkers are prepared as double stranded oligonucleotides by mixing
  • NotA (5 , -pATAGCGGCCGCAAGAATTCA-NH 2 -3');
  • PCR02 (5 , CTTGCTGGATCCAACCAAGOTCTG-3'), with 5.6 ⁇ l lOx buffer (One for allTM, Pharmacia) and water to a final volume of 56 ⁇ 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 ⁇ 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).
  • PCR amplification of the tester and driver cDN A libraries is carried out by taking about 1 ⁇ l of each gel slice isolated as above (melted at 65 degrees C before use) and mixing with 10 ⁇ l of NotB (for testers — this oligonucleotide serves as both the 5' and 3' primers) or PCR02 (for the driver), 5 ⁇ l of lOx PCR buffer, 6 ⁇ l dNTP (2.5 mM each), 1 unit of Taq polymerase (Boehringer Mannheim or Perkin Elmer), 1 unit of Pfu polymerase (Stratagene), 0.2 ⁇ g of gene 32 protein
  • 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 cDN A is conveniently accomplished as follows. About 100 ⁇ g of driver cDNA in 1 mM EDTA is mixed with 100 ⁇ 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.
  • Subtraction hybridization is conveniently accomplished as follows. Mix 8 ⁇ g of biotinylated driver DNA with 0.4 ⁇ g of tester DNA (concentrations estimated by OD measurement and ethidium bromide staining of the gel). The mixed DNA is precipitated with ethanol and resuspended in 10 ⁇ 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 ⁇ l of 2X 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.
  • a second subtraction is performed by mixing recovered tester DNA (about 80 ⁇ l) with 8 ⁇ l (8 ⁇ g) of biotinylated driver DNA and then precipitation with ethanol.
  • the precipitated DNA pellet is resuspended in 10 ⁇ 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.
  • 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 cDN A 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 ⁇ l of one 5' primer (20 ⁇ M stock) or two 5' primers (half of each) or 1.2 ⁇ l of one 5' primer (1 ⁇ l) and one 3' primer (0.2 ⁇ l) to the tube.
  • Add 1 ⁇ l of subtracted DNA to the same tube.
  • cocktail mix containing 1 ⁇ l of 10X PCR buffer for Pfu polymerase (commercially available), 1 ⁇ l of dNTP (2.5 mM each), 0.3 mM alpha- 32 P-dATP, 0.1 ⁇ l of Taq polymerase, 0.2 ⁇ l of Pfu polymerase (Stratagene), 0.02 ⁇ l of T4 gene 32 protein (Boehringer Mannheim), and 5.38 ⁇ l water.
  • the gel is dried under vacuum at 80 degrees C for 45 min. and exposed to PhosphorlmagerTM 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 cDN A 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 EppendorfTM tube.
  • This DNA can be PCR-amplified by placing 1-3 ⁇ l of recovered DNA in a 50 ⁇ l total reaction volume in a reaction mixture containing 6 ⁇ l of total primer(s), 5 ⁇ l of lOx PCR buffer for Pfu polymerase, 6 ⁇ l of dNTP (2.5 mM each), 0.25 ⁇ l of Taq polymerase, 0.5 ⁇ l of Pfu polymerase, 0.05 ⁇ 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.
  • 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.
  • Cell extracts are prepared using CHAPS, as described for the TRAP assay
  • RNA is prepared using the TRIzolTM RNA extraction method (Life Technologies) on cell pellets or CHAPS extracts.
  • PCR tube contains: 15 ⁇ l of water; 2.5 ⁇ l of 25 ⁇ M Mn(OAc) 2 ; 5.5 ⁇ l of 5X EZ buffer
  • primer TF2 5'-CTCACTGTAGACACTGCCTC-
  • 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.
  • the samples can be analyzed by gel electrophoresis using IX 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.
  • First strand cDNA synthesis is performed by mixing total RNA (1 ⁇ g) with 40 to 80 ng random hexamer in 11 ⁇ 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 ⁇ l) containing 4 ⁇ l of 5X buffer (BRL, provided with the RTase), 2 ⁇ l of 0.1 M DTT,1 ⁇ l of 10 mM dNTP (each), and 1 ⁇ 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.
  • PCR amplification of hTR cDNA with specific primer sets can be generally accomplished as follows. About 1 ⁇ l of cDN A is used for each primer set. For a 10 ⁇ l PCR with 32 P-dATP nucleotide, 1 ⁇ l of cDNA is mixed with 1 ⁇ l of 10X Taq buffer, 20 pmol of each primer, 1 ⁇ l of 2.5 mM dNTP, 5 ⁇ Ci alpha- 32 P-dATP, 1 unit of Taq polymerase (Boehringer Mannheim), 1 unit of Taq antibody (Clontech), 0.2 ⁇ g of T4 gene 32 protein (Boehringer Mannheim), and water to 10 ⁇ 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.
  • Downstream primer R3c, 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3*. Amplification of hTR with the F3b and R3c primer pair produces a 126 bp product.
  • PCR products labeled with 32 P can be conveniently detected by adding 5 ⁇ 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 PhosphorlmagerTM cassette or X-ray film to the gel.

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EP96933011A 1996-09-13 1996-09-13 Verfahren und reagentien zur regulation der telomerenlänge und der telomerase-aktivität Withdrawn EP0953042A1 (de)

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US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
JP2001231567A (ja) * 2000-02-18 2001-08-28 Inst Of Physical & Chemical Res テロメア長の調節方法
CA2401076A1 (en) * 2000-02-24 2001-08-30 Incyte Genomics, Inc. Molecules for disease detection and treatment

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