text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 29, 1999
|Filing date
|Jun 16, 1998
|Priority date
|Jul 8, 1996
|Fee status
|Also published as
|Publication number
|Kathleen Collins
|Original Assignee
|The Regents Of The University Of California
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|Patent Citations (1), Referenced by (47), Classifications (18), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a divisional application of U.S. Ser. No. 08/676,974, filed on Jul. 8, 1996, now U.S. Pat. No. 5,770,422, which is incorporated herein by reference.
The field of this invention is an enzyme involved in cell replication.
DNA at chromosome ends is maintained in a dynamic balance of loss and addition of telomeric simple sequence repeats. Sequence loss occurs during cell replication, in part from incomplete replication of chromosome termini by DNA-dependent DNA polymerase. Telomeric repeat addition is catalyzed by the enzyme telomerase: a ribonucleoprotein enzyme which uses a short region within the RNA as a template for the polymerase reaction. Although cells can maintain a constant number of telomeric repeats by balancing repeat loss and addition, not all cells do so. Human germline and cancer cells maintain a constant number of telomeric repeats, while normal human somatic cells lose telomeric repeats with each cycle of cell division. Cells which do not maintain stable telomere length demonstrate a limited proliferative capacity: these cells senesce after a number of population doublings correlated with the erosion of telomeres to a critical minimum length.
Because normal somatic cells do not appear to express or require telomerase and do not maintain chromosome ends, and because all or almost all cancer cells express high levels of telomerase activity and maintain chromosome ends, molecules that inhibit or alter telomerase activity could provide effective and non-toxic anti-cancer agents. Similarly, inhibition of telomerase in parasitic or infectious agents (e.g. trypanosomes, fungi, etc.) could provide a specific approach for reducing the viability or proliferation of these agents. Conversely, activation of telomerase in proliferation-restricted cells (such as normal somatic cells of the blood, vasculature, liver, skin, etc.) could provide a mechanism for promoting additional proliferative lifespan.
Purification of telomerase from the ciliate Tetrahymena and cloning of genes encoding two protein components of the enzyme is reported in Collins et al. (1995) Cell 81, 677-686 and copending U.S. Pat. application No. 08/359,125, filed Dec. 19, 1994. Literature relating to human telomerase include; Kim et al. (1994) Science 266, 2011-2014; and Feng et al. (1995) Science 269, 1236-1241. Literature relating to telomerase template modifications include Autexier et al. (1994) Genes and Devel 8, 563-575; Yu et al. (1991) Cell 67, 823-832; and Yu et al. (1990) Nature 344, 126-132. The Washington University-Merck EST Project contains an EST, reportedly deposited by Hillier et al. on Nov. 1, 1995, which has sequence similarity with the 3' end of SEQ ID NO:3, disclosed herein. For a general review, see Blackburn et al., Eds. (1995) Telomeres, Cold Spring Harbor Laboratory Press.
The invention provides methods and compositions relating to a human telomerase and related nucleic acids. Included are four distinct human telomerase subunit proteins, called p140, p105, p48 and p43 and telomerase protein domains thereof having telomerase-specific activity. The proteins may be produced recombinantly from transformed host cells from the subject telomerase encoding nucleic acids or purified from human cells. Also included are human telomerase RNA components, as well as specific, functional derivatives thereof.
The invention provides isolated telomerase hybridization probes and primers capable of specifically hybridizing with the disclosed telomerase gene, telomerase-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for telomerase transcripts), therapy (e.g. gene therapy to modulate telomerase gene expression) and in the biopharmaceutical industry (e.g. reagents for screening chemical libraries for lead pharmacological agents and nucleic acid polymerase reagents).
SEQ ID NO: 1: p105 protein (amino acid sequence)
SEQ ID NO: 2: p105 ambiguity maximized synthetic DNA
SEQ ID NO: 3: p105 natural cDNA (the coding region is bp 97-2370)
SEQ ID NO: 4: p105 E. coli optimized synthetic DNA
SEQ ID NO: 5: p105 mammalian optimized synthetic DNA
SEQ ID NO: 6: telomerase RNA
SEQ ID NO: 7: telomerase RNA template region modification 1
SEQ ID NO: 8: telomerase RNA template region modification 2
SEQ ID NO: 9: telomerase RNA template region modification 3
SEQ ID NO: 10 p43 peptide (XXXEAAT I/L!D I/L!PQQGANK, where the three X's are indeterminant residues)
The invention provides isolated human telomerase proteins including human telomerase proteins p140, p105, p48 and p43, having molecular weights of about 140 kD, about 105 kD, about 48 kD and about 43 kD, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions (Matsudaira and Burgess (1978) Anal Biochem 87, 386-396), and telomerase protein domains thereof. The telomerase proteins comprise assay-discernable functional domains including RNA recognition motifs and subunit binding domains and may be provided as fusion products, e.g with non-telomerase polypeptides. The human telomerase proteins of the invention, including the subject protein domains, all have telomerase-specific activity or function.
Telomerase-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of a telomerase protein with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a telomerase subunit (e.g. another protein subunit or RNA subunit), a substrate, agonist, antagonist, chaperone, or other regulator that directly modulates telomerase activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or a telomerase specific agent such as those identified in assays described below. Generally, telomerase-binding specificity is assayed by telomere polymerase activity (see, e.g. Collins et al. 1995, Cell 81, 677-686), by binding equilibrium constants (usually at least about 107 M-1, preferably at least about 108 M-1, more preferably at least about 109 M-1), by the ability of the subject protein to function as negative mutants in telomerase-expressing cells, to elicit telomerase specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the telomerase binding specificity of the subject telomerase proteins necessarily distinguishes ciliate telomerase, preferably distinguishes non-mammalian telomerases and more preferably distinguishes non-human telomerases. Exemplary telomerase proteins which are shown to have telomerase binding specificity include the telomerase RNA (e.g. SEQ ID NO:6) binding domains (e.g. RRM 1-4: SEQ ID NO:1, about residues 5-81, residues 115-192, residues 336-420, and residues 487-578, respectively), telomerase primer binding domains, nucleotide triphosphate binding domains and binding domains of regulators of telomerase such as nuclear localization proteins, etc. As used herein, a protein domain comprises at least 12, preferably at least about 20, more preferably at least about 40, most preferably at least about 80 residues of the disclosed respective SEQ ID NO.
The claimed human telomerase proteins are isolated or pure: an "isolated" protein is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total protein in a given sample and a pure protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample. The telomerase proteins and protein domains may be synthesized, produced by recombinant technology, or purified from human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, New York) or that are otherwise known in the art. An exemplary method for isolating each of human telomerase protein p140, p105, p48 and p43 from human cells is as follows:
Several thousand (two to twelve thousand) liters of HeLa cells are grown in spinner culture. The cells are lysed by dounce homogenization in low-salt buffer to produce crude cell lysates. The lysates are supplemented with 15% glycerol and centrifuged at 125,000�g for 50 minutes to obtain a first soluble fraction enriched for telomerase activity (S-100 fraction). The S-100 fraction is adjusted to 0.2 M ammonium sulfate, bound to SP Sepharose (Pharmacia), and developed with a gradient in sodium chloride, to obtain a second soluble fraction enriched for telomerase (SP fraction). The SP fraction is adjusted to about 0.3-0.4 M ionic strength and bound to Q Sepharose (Pharmacia), and developed with a gradient in sodium chloride, to obtain a third soluble fraction enriched for telomerase (Q fraction). The Q fraction is adjusted to about 0.3-0.4 M ionic strength, bound to phosphocellulose (Whatman), and developed with sodium chloride, to obtain a fourth soluble fraction enriched for telomerase (PC fraction). The PC fraction is adjusted to about 0.3-0.4 M ionic strength, bound to 2'Omethyl RNA oligonucleotide immobilized on streptavidin agarose (Sigma), and eluted with a electrophoresis sample medium comprising 5% β-mercaptoethanol and 2% Sodium Dodecyl Sulfate to obtain a fifth soluble fraction (2'Omethyl fraction). The 2'Omethyl fraction is separated by polyacrylamide gel electrophoresis under denaturing conditions (Matsudaira and Burgess (1978) Anal Biochem 87, 386-396) to obtain gel protein bands at a molecular weight of about 140 kD, 105 kD, 48 kD or 43 kD having telomerase activity. The gel bands are excised or blotted to obtain purified human telomerase proteins p140, p105, p48 and p43.
The subject telomerase proteins find a wide variety of uses including use in isolating, enriching for and concentrating telomerase RNA and telomerase proteins, as immunogens, in the methods and applications described below, as reagents in the biotechnology industries, and in therapy. Recombinant telomerases are used in many applications where nascent oligonucleotides of predetermined sequence are desired. For example, native nucleic acid molecules are labeled or extended at their 3' ends by addition of a precleterrnined repeat sequence (for double-stranded oligonucleotides, both ends of the molecule may be tagged). Oligonucleotides complementary to the repeat are then used to amplify, sequence, affinity purify, etc. the nucleic acid molecules. The use of a repeat sequence for 3' end tagging improves specificity and provides sequence alternatives compared with non-templated enzymes presently available for this purpose, e.g. terminal transferase. Repeats encoding restriction enzyme sites provide repeat tagging to facilitate cloning and the use of telomerase alleviates the restrictive conditions required for optimal ligation with available ligase enzymes. Telomerase also finds use in regulating cell growth or increasing cell density tolerance; for example, cells contacted with an effective amount of exogenous telomerase to overcome the growth control limitation otherwise imposed by short telomere length. Telomerase may be introduced, expressed, or repressed in specific populations of cells by any convenient way such as microinjection, promoter-specific expression of recombinant enzyme, targeted delivery of lipid vesicles, etc. Advantageously, only a brief period of telomerase activity is required to allow many generations of continued proliferation of the contacted cell, due to the ability of telomerase to extend telomeres in one cell cycle by more sequence than is lost with each cell division.
The invention provides natural and non-natural human telomerase-specific binding agents including substrates, agonist, antagonist, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, human telomerase-specific agents are useful in a variety of diagnostic and therapeutic applications. Novel human telomerase-specific binding agents include human telomerase-specific receptors, such as somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Agents of particular interest modulate human telomerase function, e.g. human telomerase antgonists and find use methods for modulating the binding of a human telomerase or telomerase protein to a human telomerase binding target.
For diagostic uses, the binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, brother easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent. Binding agents also find use in modulating the telomerase activity present in a cell. For example, isolated cells, whole tissues, or individuals may be treated with a telomerase binding agent to activate, inhibit, or alter the specificity of telomerase assembly, localization, substrate interaction, or synthesis activity. Effectively treated cells have increased or decreased replication potential, or suffer from loss of proper telomere structure (resulting in lethality). These binding agents also find therapeutic use to control cell proliferation; for example, the uncontrolled growth of transformed cells (e.g. cancer cells) is managed by administration to the cells or patient comprising such cells of a telomerase binding agent which reduces telomerase activity. In contrast to many current chemotherapies, the present invention provides enhanced specificity of lethality, with minimum toxicity to dividing yet normal somatic cells.
The amino acid sequences of the disclosed telomerase proteins are used to back-translate telomerase protein-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural telomerase encoding nucleic acid sequences ("GCG" software, Genetics Computer Group, Inc, Madison Wis.). As examples, SEQ ID NO:2 discloses an ambiguity-maximized p105 coding sequence encompassing all possible nucleic acids encoding the full-length p105 protein. SEQ ID NO:3 discloses a natural human cDNA sequence encoding p105, SEQ ID NO:4 is a p105 coding sequence codon-optimized for E. coli, SEQ ID NO:5 is a p105 coding sequence codon optimized for mammalian cell expression. Telomerase encoding nucleic acids may be part of human telomerase-expression vectors and may be incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with human telomerase-mediated signal transduction, etc. Expression systems are selected and/or tailored to effect human telomerase protein structural and functional variants through alternative post-translational processing.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a human telomerase cDNA specific sequence contained in SEQ ID NO:3, bases 1-2345, and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with SEQ ID NO:3, bases 1-2345 in the presence of natural ciliate telomerase cDNA, preferably in the presence of non-mammalian telomerase cDNA and more preferably, in the presence of murine telomerase cDNA). Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5�SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42� C. and remaining bound when subject to washing at 42� C. with 0.2�SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5�SSPE buffer at a temperature of 42� C. and remaining bound when subject to washing at 42� C. with 0.2�SSPE buffer at 42� C. Human telomerase cDNA homologs can also be distinguished from other protein using alignment algorithms, such as BLASTX (Altschul et al (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).
The invention also provides non-natural sequence, recombinant and isolated natural sequence human telomerase RNA. Natural human telomerase RNA sequences include the nucleic acid disclosed as SEQ ID NO:6, or a fragment thereof sufficient to specifically hybridize with a nucleic acid having the sequence defined by SEQ ID NO:6 in the presence of a nucleic acid having the sequence disclosed in Feng et al. 1995, Science 269, 1236-1241 (SEQ ID NO.11). Such fragments necessarily distinguish the previously described (Feng et al. 1995, Science 269, 1236-1241) human RNA species. Preferred such fragments comprise SEQ ID NO:6, bases 191-210, bases 245-259, bases 341-369 or bases 381-399. Non-natural sequences include derivatives and/or mutations of SEQ ID NO:6, where such derivatives/mutations provide alteration in template, protein binding, or other regions to effect altered telomerase substrate specificity or altered reaction product (e.g. any predetermined sequence), etc.; see, e.g. Autexier et al., 1994, Genes & Develop 8, 563-575; Collins et al. (1995) EMBO J. 14, 5422-5432; Greider et al. (1995) Structure and Biochemistry of Ciliate and Mammalian Telomerases, in DNA Replication, DePamphlis, Ed., Cold Spring Harbor Laboratory Press. Additional derivatives function as dominant negative fragments which effectively compete for telomerase assembly. For examples, SEQ ID NO:7, 8 and 9 are derivatives which provide for modified substrate specificity and polymerase reaction product to interfere with cellular function (see, e.g. Hanish et al. (1994) Proc Natl Acad Sci 91, 8861-8865).
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ ID NO:3 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc. The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of human telomerase genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional human telomerase homologs and structural analogs.
In diagnosis, human telomerase hybridization probes find use in identifying wild-type and mutant human telomerase alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic human telomerase nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active telomerase. A wide variety of indications may be treated, either prophylactically or therapeutically with the subject compositions. For example, where limitation of cell growth is desired, e.g. neoproliferative disease, a reduction in telomerase expression is effected by introducing into the targeted cell type human telomerase nucleic acids which reduce the functional expression of human telomerase gene products (e.g. nucleic acids capable of inhibiting translation of a functional telomerase transcript). Conditions for treatment include various cancers, where any of a wide variety of cell types may be involved, restenosis, where vascular smooth muscle cells are involved, inflammatory disease states, where endothelial cells, inflammatory cells and glomerular cells are involved, myocardial infarction, where heart muscle cells are involved, glomerular nephritis, where kidney cells are involved, transplant rejection where endothelial cells are involved, infectious diseases such as HIV infection where certain immune cells and other infected cells are involved, or the like.
Telomerase inhibitory nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed natural telomerase coding sequences. Antisense modulation of the expression of a given telomerase protein may employ telomerase antisense nucleic acids operably linked to gene regulatory sequences. Cell are transfected with a vector comprising a human telomerase sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous human telomerase protein encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given human telomerase protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein.
In other indications, e.g. certain hypersensitivities, atrophic diseases, etc., an increase in cell growth or proliferation is desired. In these applications, an enhancement in human telomerase expression is effected by introducing into the targeted cell type human telomerase nucleic acids which increase the functional expression of human telomerase gene products. Conditions for treatment include multiple sclerosis, where certain neuronal cells are involved, inflammatory disease states such as rheumatoid arthritis, where bystander cells are involved, transplant rejection where graft cells are involved, infectious diseases such as HIV infection where certain uninfected host cells are involved, or the like. Such nucleic acids may be human telomerase expression vectors, vectors which upregulate the functional expression of an endogenous human telomerase allele, or replacement vectors for targeted correction of human telomerase mutant alleles.
Various techniques may be employed for introducing of the nucleic acids into viable cells, e.g. transfection with a retrovirus, viral coat protein-liposome mediated transfection. The techniques vary depending upon whether one is using the subject compositions in culture or in vivo in a host. In some situations it is desirable to provide the nucleic acid source with an agent which targets the target cells, such as an antibody specific for a surface membrane protein on the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
The invention provides methods and compositions for enhancing the yield of many recombinantly produced proteins by increasing maximum cell densities and survival time of host production cells in culture. Specifically, cultured cells are transfected with nucleic acids which effect the up-regulation of endogenous telomerase or the expression of an exogenous telomerase. For example, nucleic acids encoding functional human telomerase operably linked to a transcriptional promoter are used to over-express the exogenous telomerase in the host cell. Telomerase-expressing cells demonstrate enhanced survival ability at elevated cell densities and over extended culture periods.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a human telomerase modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate human telomerase interaction with a natural human telomerase binding target. A wide variety of assays for binding agents are provided including labeled in vitro telomere polymerase assays, proteinprotein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Target indications may include infection, genetic disease, cell growth and regulatory disfunction, such as neoplasia, inflammation, hypersensitivity, etc. Target cells also include progenitor cells for repopulating blood or bone marrow, tissue grafts, and tissue subject to degredation/high turnover such as digestive and vascular endothelia and pulmunary and dermal epithelia.
In vitro binding assays employ a mixture of components including a human telomerase protein, which may be part of multi-subunit telomerase, a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular human telomerase binding target, e.g. a substrate. While native binding targets may be used, it is frequently preferred to use portions (e.g. peptides, nucleic acid fragments) thereof so long as the portion provides binding affinity and avidity to the subject human telomerase conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the human telomerase specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.
After incubation, the agent-biased binding between the human telomerase and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation (e.g. TCA precipitation, immunoprecipitation, etc.), immobilization (e.g on a solid substrate), etc., followed by washing by, for examples, membrane filtration (e.g. Whatman's P-81 ion exchange paper, Polyfiltronic's hydrophobic GFC membrane, etc.), gel chromatography (e.g. gel filtration, affinity, etc.). For telomere polymerase assays, binding is detected by a change in the polymerization by the telomerase of a nucleic acid or nucleic acid analog on the substrate.
Detection may be effected in any convenient way. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
A difference in the binding affinity of the human telomerase protein to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the human telomerase protein to the human telomerase binding target. Analogously, in the cell-based transcription assay also described below, a difference in the human telomerase transcriptional induction in the presence and absence of an agent indicates the agent modulates human telomerase-induced transcription. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The following examples are offered by way of illustration and not by way of limitation.
1. Protocol for high-throughput human telomere polymerization assay.
A. Reagents:
Neutralite Avidin: 20 μg/ml in PBS.
human telomerase: 10-8 -10-5 M human telomerase in PBS.
Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.
Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1 mM dATP, 1 mM dTTP, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.
32 P!α-dGTP 10�stock: 2�10-5 M "cold" dGTP with 100 μCi 32 P!α-dGTP. Place in the 4� C. microfridge during screening.
telomerase substrate: 10-7 -10-4 M biotinylated telomerase substrate (5'-biotin-d(TTAGGG)3 -3'! in PBS.
Protease inhibitor cocktail (1000�): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo3 (Sigma #S-6508) in 10 ml of PBS.
B. Preparation of assay plates:
Coat with 120 μl of stock N Avidin per well overnight at 4� C.
Wash 2 times with 200 μl PBS.
Block with 150 μl of blocking buffer.
Wash 2 times with 200 μl PBS.
C. Assay:
Add 40 μl assay buffer/well.
Add 40 μl human telomerase (1-1000 fmoles/40 μl in assay buffer)
Add 10 μl compound or extract.
Add 10 μl 32 P!α-dGTP 10� stock.
Add 40 μl biotinylated telomerase substrate (0.1-10 pmoles/40 ul in assay buffer)
Shake at 25� C. for 15 minutes.
Incubate additional 45 minutes at 25� C.
Stop the reaction by washing 4 times with 200 μl PBS.
Add 150 μl scintillation cocktail.
Count in Topcount.
D. Controls for all assays (located on each plate):
a. Non-specific binding
b. cold dGTP at 80% inhibition.
2. Protocol for high throughput human telomerase subunit-RNA complex formation assay.
A. Reagents:
Neutralite Avidin: 20 μg/ml in PBS.
Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.
Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1 % glycerol, 0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.
33 P human telomerase protein 10�stock: 10-8 -10-6 M "cold" human telomerase subunit (p105) supplemented with 200,000-250,000 cpm of labeled human telomerase (Beckman counter). Place in the 4� C. microfridge during screening.
Protease inhibitor cocktail (1000�): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo3 (Sigma #S-6508) in 10 ml of PBS.
telomerase RNA: 10-7 -10-4 M biotinylated RNA (SEQ ID NO:6) in PBS.
B. Preparation of assay plates:
Coat with 120 μl of stock N-Avidin per well overnight at 4� C.
Wash 2 times with 200 μl PBS.
Block with 150 μl of blocking buffer.
Wash 2 times with 200 μl PBS.
C. Assay:
Add 40 μl assay buffer/well.
Add 10 μl compound or extract.
Add 10 μl 33 P-human telomerase protein (20,000-25,000 cpm/0.1-10 pmoles/well=10-9 -10-7 M final concentration).
Shake at 25� C. for 15 minutes.
Incubate additional 45 minutes at 25� C.
Add 40 μl biotinylated RNA (0.1-10 pmoles/40 μl in assay buffer)
Incubate 1 hour at room temperature.
Stop the reaction by washing 4 times with 200 μl PBS.
Add 150 μl scintillation cocktail.
Count in Topcount.
D. Controls for all assays (located on each plate):
a. Non-specific binding
b. Soluble (non-biotinylated telomerase) at 80% inhibition.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 11- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 759 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: Not Relev - #ant- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Met Ala Gly Leu Thr Leu Phe Val - # Gly Arg Leu Pro Pro Ser AlaArg# 15- Ser Glu Gln Leu Glu Glu Leu Phe - # Ser Gln Val Gly Pro Val LysGln# 30- Cys Phe Val Val Thr Glu Lys Gly - # Ser Lys Ala Cys Arg Gly PheGly# 45- Tyr Val Thr Phe Ser Met Leu Glu - # Asp Val Gln Arg Ala Leu LysGlu# 60- Ile Thr Thr Phe Glu Gly Cys Lys - # Ile Asn Val Thr Val Ala LysLys# 80- Lys Leu Arg Asn Lys Thr Lys Glu - # Lys Gly Lys Asn Glu Asn SerGlu# 95- Cys Pro Lys Lys Glu Pro Lys Ala - # Lys Lys Ala Lys Val Ala AspLys# 110- Lys Ala Arg Leu Ile Ile Arg Asn - # Leu Ser Phe Lys Cys Ser GluAsp# 125- Asp Leu Lys Thr Val Phe Ala Gln - # Phe Gly Ala Val Leu Glu ValAsn# 140- Ile Pro Arg Lys Pro Asp Gly Lys - # Met Arg Gly Phe Gly Phe ValGln# 160- Phe Lys Asn Leu Leu Glu Ala Gly - # Lys Ala Leu Lys Gly Met AsnMet# 175- Lys Glu Ile Lys Gly Arg Thr Val - # Ala Val Asp Trp Ala Val AlaLys# 190- Asp Lys Tyr Lys Asp Thr Gln Ser - # Val Ser Ala Ile Gly Glu GluLys# 205- Ser His Glu Ser Lys His Gln Glu - # Ser Val Lys Lys Lys Gly ArgGlu# 220- Glu Glu Asp Met Glu Glu Glu Glu - # Asn Asp Asp Asp Asp Asp AspAsp# 240- Asp Glu Glu Asp Gly Val Phe Asp - # Asp Glu Asp Glu Glu Glu GluAsn# 255- Ile Glu Ser Lys Val Thr Lys Pro - # Val Gln Ile Gln Lys Arg AlaVal# 270- Lys Arg Pro Ala Pro Ala Lys Ser - # Ser Asp His Ser Glu Glu AspSer# 285- Asp Leu Glu Glu Ser Asp Ser Ile - # Asp Asp Gly Glu Glu Leu AlaGln# 300- Ser Asp Thr Ser Thr Glu Glu Gln - # Glu Asp Lys Ala Val Gln ValSer# 320- Asn Lys Lys Lys Arg Lys Leu Pro - # Ser Asp Val Asn Glu Gly LysThr# 335- Val Phe Ile Arg Asn Leu Ser Phe - # Asp Ser Glu Glu Glu Glu LeuGly# 350- Glu Leu Leu Gln Gln Phe Gly Glu - # Leu Lys Tyr Val Arg Ile ValLeu# 365- His Pro Asp Thr Glu His Ser Lys - # Gly Cys Ala Phe Ala Gln PheMet# 380- Thr Gln Glu Ala Ala Gln Lys Cys - # Leu Leu Ala Ala Ser Pro GluAsn# 400- Glu Ala Gly Gly Leu Lys Leu Asp - # Gly Arg Gln Leu Lys Val AspLeu# 415- Ala Val Thr Arg Asp Glu Ala Ala - # Lys Leu Gln Thr Thr Lys ValLys# 430- Lys Pro Thr Gly Thr Arg Asn Leu - # Tyr Leu Ala Arg Glu Gly LeuIle# 445- Arg Ala Gly Thr Lys Ala Ala Glu - # Gly Val Ser Ala Ala Asp MetAla# 460- Lys Arg Glu Arg Phe Glu Leu Leu - # Lys His Gln Lys Leu Lys AspGln# 480- Asn Ile Phe Val Ser Arg Thr Arg - # Leu Cys Leu His Asn Leu ProLys# 495- Ala Val Asp Asp Lys Gln Leu Arg - # Lys Leu Leu Leu Ser Ala ThrSer# 510- Gly Glu Lys Gly Val Arg Ile Lys - # Glu Cys Arg Val Met Arg AspLeu# 525- Lys Gly Val His Gly Asn Met Lys - # Gly Gln Ser Leu Gly Tyr AlaPhe# 540- Ala Glu Phe Gln Glu His Glu His - # Ala Leu Lys Ala Leu Arg LeuIle# 560- Asn Asn Asn Pro Glu Ile Phe Gly - # Pro Leu Lys Arg Pro Ile ValGlu# 575- Phe Ser Leu Glu Asp Arg Arg Lys - # Leu Lys Met Lys Glu Leu ArgIle# 590- Gln Arg Ser Leu Gln Lys Met Arg - # Ser Lys Pro Ala Thr Gly GluPro# 605- Gln Lys Gly Gln Pro Glu Pro Ala - # Lys Asp Gln Gln Gln Lys AlaAla# 620- Gln His His Thr Glu Glu Gln Ser - # Lys Val Pro Pro Glu Gln LysArg# 640- Lys Ala Gly Ser Thr Ser Trp Thr - # Gly Phe Gln Thr Lys Ala GluVal# 655- Glu Gln Val Glu Leu Pro Asp Gly - # Lys Lys Arg Arg Lys Val LeuAla# 670- Leu Pro Ser His Arg Gly Pro Lys - # Ile Arg Leu Arg Asp Lys GlyLys# 685- Val Lys Pro Val His Pro Lys Lys - # Pro Lys Pro Gln Ile Asn GlnTrp# 700- Lys Gln Glu Lys Gln Gln Leu Ser - # Ser Glu Gln Val Ser Arg LysLys# 720- Ala Lys Gly Asn Lys Thr Glu Thr - # Arg Phe Asn Gln Leu Val GluGln# 735- Tyr Lys Gln Lys Leu Leu Gly Pro - # Ser Lys Gly Ala Pro Leu AlaLys# 750- Arg Ser Lys Trp Phe Asp Ser 755- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2277 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- ATGGCNGGNY TNACNYTNTT YGTNGGNMGN YTNCCNCCNW SNGCNMGNWS NG - #ARCARYTN 60- GARGARYTNT TYWSNCARGT NGGNCCNGTN AARCARTGYT TYGTNGTNAC NG - #ARAARGGN 120- WSNAARGCNT GYMGNGGNTT YGGNTAYGTN ACNTTYWSNA TGYTNGARGA YG - #TNCARMGN 180- GCNYTNAARG ARATHACNAC NTTYGARGGN TGYAARATHA AYGTNACNGT NG - #CNAARAAR 240- AARYTNMGNA AYAARACNAA RGARAARGGN AARAAYGARA AYWSNGARTG YC - #CNAARAAR 300- GARCCNAARG CNAARAARGC NAARGTNGCN GAYAARAARG CNMGNYTNAT HA - #THMGNAAY 360- YTNWSNTTYA ARTGYWSNGA RGAYGAYYTN AARACNGTNT TYGCNCARTT YG - #GNGCNGTN 420- YTNGARGTNA AYATHCCNMG NAARCCNGAY GGNAARATGM GNGGNTTYGG NT - #TYGTNCAR 480- TTYAARAAYY TNYTNGARGC NGGNAARGCN YTNAARGGNA TGAAYATGAA RG - #ARATHAAR 540- GGNMGNACNG TNGCNGTNGA YTGGGCNGTN GCNAARGAYA ARTAYAARGA YA - #CNCARWSN 600- GTNWSNGCNA THGGNGARGA RAARWSNCAY GARWSNAARC AYCARGARWS NG - #TNAARAAR 660- AARGGNMGNG ARGARGARGA YATGGARGAR GARGARAAYG AYGAYGAYGA YG - #AYGAYGAY 720- GAYGARGARG AYGGNGTNTT YGAYGAYGAR GAYGARGARG ARGARAAYAT HG - #ARWSNAAR 780- GTNACNAARC CNGTNCARAT HCARAARMGN GCNGTNAARM GNCCNGCNCC NG - #CNAARWSN 840- WSNGAYCAYW SNGARGARGA YWSNGAYYTN GARGARWSNG AYWSNATHGA YG - #AYGGNGAR 900- GARYTNGCNC ARWSNGAYAC NWSNACNGAR GARCARGARG AYAARGCNGT NC - #ARGTNWSN 960- AAYAARAARA ARMGNAARYT NCCNWSNGAY GTNAAYGARG GNAARACNGT NT - #TYATHMGN1020- AAYYTNWSNT TYGAYWSNGA RGARGARGAR YTNGGNGARY TNYTNCARCA RT - #TYGGNGAR1080- YTNAARTAYG TNMGNATHGT NYTNCAYCCN GAYACNGARC AYWSNAARGG NT - #GYGCNTTY1140- GCNCARTTYA TGACNCARGA RGCNGCNCAR AARTGYYTNY TNGCNGCNWS NC - #CNGARAAY1200- GARGCNGGNG GNYTNAARYT NGAYGGNMGN CARYTNAARG TNGAYYTNGC NG - #TNACNMGN1260- GAYGARGCNG CNAARYTNCA RACNACNAAR GTNAARAARC CNACNGGNAC NM - #GNAAYYTN1320- TAYYTNGCNM GNGARGGNYT NATHMGNGCN GGNACNAARG CNGCNGARGG NG - #TNWSNGCN1380- GCNGAYATGG CNAARMGNGA RMGNTTYGAR YTNYTNAARC AYCARAARYT NA - #ARGAYCAR1440- AAYATHTTYG TNWSNMGNAC NMGNYTNTGY YTNCAYAAYY TNCCNAARGC NG - #TNGAYGAY1500- AARCARYTNM GNAARYTNYT NYTNWSNGCN ACNWSNGGNG ARAARGGNGT NM - #GNATHAAR1560- GARTGYMGNG TNATGMGNGA YYTNAARGGN GTNCAYGGNA AYATGAARGG NC - #ARWSNYTN1620- GGNTAYGCNT TYGCNGARTT YCARGARCAY GARCAYGCNY TNAARGCNYT NM - #GNYTNATH1680- AAYAAYAAYC CNGARATHTT YGGNCCNYTN AARMGNCCNA THGTNGARTT YW - #SNYTNGAR1740- GAYMGNMGNA ARYTNAARAT GAARGARYTN MGNATHCARM GNWSNYTNCA RA - #ARATGMGN1800- WSNAARCCNG CNACNGGNGA RCCNCARAAR GGNCARCCNG ARCCNGCNAA RG - #AYCARCAR1860- CARAARGCNG CNCARCAYCA YACNGARGAR CARWSNAARG TNCCNCCNGA RC - #ARAARMGN1920- AARGCNGGNW SNACNWSNTG GACNGGNTTY CARACNAARG CNGARGTNGA RC - #ARGTNGAR1980- YTNCCNGAYG GNAARAARMG NMGNAARGTN YTNGCNYTNC CNWSNCAYMG NG - #GNCCNAAR2040- ATHMGNYTNM GNGAYAARGG NAARGTNAAR CCNGTNCAYC CNAARAARCC NA - #ARCCNCAR2100- ATHAAYCART GGAARCARGA RAARCARCAR YTNWSNWSNG ARCARGTNWS NM - #GNAARAAR2160- GCNAARGGNA AYAARACNGA RACNMGNTTY AAYCARYTNG TNGARCARTA YA - #ARCARAAR2220- YTNYTNGGNC CNWSNAARGG NGCNCCNYTN GCNAARMGNW SNAARTGGTT YG - #AYWSN2277- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2733 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- TGAGCTTGGT TGTCCTACCA AAGCCAGCGT TTCGGCTCGC GTGCGCCGGC CT - #AGTTTGCT 60- CGCGTCCTCA CGCGCTTTGG GTTTCCCGGT CTCATGGCCG GCCTGACCTT AT - #TTGTGGGC 120- CGCCTCCCGC CCTCGGCCCG CAGTGAGCAG CTGGAGGAAC TGTTCAGTCA GG - #TGGGGCCG 180- GTGAAGCAGT GCTTCGTGGT GACTGAAAAA GGGAGTAAGG CATGTCGAGG CT - #TTGGCTAT 240- GTCACTTTTT CAATGCTGGA AGATGTTCAG AGGGCCCTCA AGGAGATTAC CA - #CCTTTGAA 300- GGTTGCAAGA TCAACGTGAC TGTTGCCAAG AAAAAACTGA GGAACAAGAC AA - #AGGAAAAG 360- GGGAAAAATG AAAACTCAGA GTGCCCAAAG AAGGAGCCGA AGGCTAAAAA AG - #CCAAAGTG 420- GCAGATAAGA AAGCCAGATT AATTATTCGG AACCTGAGCT TTAAGTGTTC AG - #AAGATGAC 480- TTGAAGACAG TATTTGCTCA ATTTGGAGCT GTCCTGGAAG TAAATATCCC TA - #GGAAACCA 540- GATGGGAAGA TGCGCGGTTT TGGTTTTGTT CAGTTCAAAA ACCTCCTAGA AG - #CAGGTAAA 600- GCTCTCAAAG GCATGAACAT GAAAGAGATA AAAGGCCGGA CAGTGGCTGT GG - #ATTGGGCC 660- GTGGCAAAGG ATAAATATAA AGATACACAG TCTGTTTCTG CTATAGGTGA GG - #AAAAGAGC 720- CATGAATCTA AACATCAGGA ATCAGTTAAA AAGAAGGGCA GAGAGGAAGA GG - #ATATGGAA 780- GAGGAAGAAA ACGATGATGA TGACGATGAT GATGATGAAG AAGATGGGGT TT - #TTGATGAT 840- GAAGATGAAG AGGAAGAGAA TATAGAATCA AAGGTGACCA AGCCTGTGCA AA - #TTCAGAAG 900- AGAGCAGTCA AGAGACCAGC CCCTGCAAAA AGCAGTGATC ATTCTGAGGA GG - #ACAGTGAC 960- CTAGAGGAAA GCGATAGTAT TGATGATGGA GAGGAACTGG CTCAGAGTGA TA - #CCAGCACT1020- GAGGAGCAAG AGGATAAAGC TGTGCAAGTC TCAAACAAAA AGAAGAGGAA AT - #TACCCTCT1080- GATGTGAATG AAGGGAAAAC TGTTTTTATC AGAAATCTGT CCTTTGACTC AG - #AAGAAGAA1140- GAACTTGGGG AGCTTCTCCA ACAGTTTGGA GAACTCAAAT ATGTCCGCAT TG - #TCTTGCAT1200- CCAGACACAG AGCATTCTAA AGGTTGTGCA TTTGCCCAGT TCATGACTCA AG - #AAGCAGCT1260- CAGAAATGCC TTCTAGCTGC TTCTCCAGAG AATGAGGCTG GTGGGCTTAA AC - #TGGATGGC1320- CGGCAGCTCA AGGTTGACTT GGCGGTGACC CGTGATGAGG CTGCAAAGCT TC - #AGACGACG1380- AAGGTGAAGA AGCCGACTGG CACCCGGAAT CTCTATCTGG CCCGAGAAGG CT - #TGATTCGT1440- GCTGGGACGA AGGCTGCAGA GGGTGTGAGT GCTGCTGATA TGGCCAAAAG AG - #AACGGTTT1500- GAGCTGCTGA AGCATCAGAA ACTCAAGGAC CAGAATATCT TTGTCTCCCG AA - #CCAGGCTC1560- TGCCTGCACA ATCTCCCAAA GGCTGTAGAT GACAAACAGC TCAGAAAGCT GC - #TGCTGAGT1620- GCTACTAGTG GAGAGAAAGG GGTGCGCATC AAGGAGTGTA GAGTGATGCG AG - #ACCTCAAA1680- GGAGTTCATG GGAACATGAA GGGTCAGTCC CTGGGCTACG CCTTTGCGGA GT - #TCCAAGAG1740- CACGAGCATG CCCTGAAAGC CCTCCGCCTC ATCAACAACA ATCCAGAAAT CT - #TTGGGCCT1800- CTGAAGAGAC CAATAGTGGA GTTCTCTTTA GAAGATCGAA GAAAACTTAA AA - #TGAAGGAA1860- TTAAGGATCC AGCGCAGCTT GCAAAAAATG AGATCCAAGC CTGCAACTGG TG - #AGCCTCAG1920- AAGGGGCAAC CAGAGCCTGC AAAAGACCAG CAACAGAAGG CAGCTCAACA CC - #ACACAGAG1980- GAACAAAGCA AGGTGCCCCC AGAGCAGAAG AGAAAGGCGG GCTCTACCTC AT - #GGACCGGG2040- TTCCAGACCA AGGCTGAAGT GGAGCAGGTG GAGCTGCCTG ATGGAAAGAA GA - #GAAGAAAG2100- GTCCTGGCGC TCCCCTCACA CCGAGGCCCC AAAATCAGGT TGCGGGACAA AG - #GCAAAGTG2160- AAGCCCGTCC ATCCCAAAAA GCCAAAGCCA CAGATAAACC AGTGGAAGCA GG - #AGAAGCAG2220- CAATTATCGT CCGAGCAGGT ATCTAGGAAA AAAGCTAAGG GAAATAAGAC GG - #AAACCCGC2280- TTCAACCAGC TGGTCGAACA ATATAAGCAG AAATTATTGG GACCTTCTAA AG - #GAGCACCT2340- CTTGCAAAGA GGAGCAAATG GTTTGATAGT TGATGATGGC AGCAGGCTGG GT - #AAGAAGCT2400- GGGTTGTATA CTTTCTGGTG ACACTCCTGG GCTCCTCCCC ATCCCCCGTG TC - #TCTCACTG2460- AGGGAAAGAA AATCCCCAAG GGCACTGCCA CTGTGCTCGG AGGTGCCCTG GA - #CTGTGTAC2520- ATCTGAACTT TGGTCCATCC TTTGATGTGT GGTTCGTTAG CCACAAAGAG AA - #ATATCTGA2580- AAGTCAACAT GATGCTTCTT GCATATTATC CAGATTATTG TATGAAGTTG TG - #TCTATAAT2640- TATTACCAAT TTTTATTCTT TATTTCTCAA ATGGAAACAC CTGAAAAAGC AA - #AAAAAAAA2700# 2733 GGGG GCCCGTACCC AAT- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2277 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- AUGGCUGGUC UGACCCUGUU CGUUGGUCGU CUGCCGCCGU CCGCUCGUUC CG - #AACAGCUG 60- GAAGAACUGU UCUCCCAGGU UGGUCCGGUU AAACAGUGCU UCGUUGUUAC CG - #AAAAAGGU 120- UCCAAAGCUU GCCGUGGUUU CGGUUACGUU ACCUUCUCCA UGCUGGAAGA CG - #UUCAGCGU 180- GCUCUGAAAG AAAUCACCAC CUUCGAAGGU UGCAAAAUCA ACGUUACCGU UG - #CUAAAAAA 240- AAACUGCGUA ACAAAACCAA AGAAAAAGGU AAAAACGAAA ACUCCGAAUG CC - #CGAAAAAA 300- GAACCGAAAG CUAAAAAAGC UAAAGUUGCU GACAAAAAAG CUCGUCUGAU CA - #UCCGUAAC 360- CUGUCCUUCA AAUGCUCCGA AGACGACCUG AAAACCGUUU UCGCUCAGUU CG - #GUGCUGUU 420- CUGGAAGUUA ACAUCCCGCG UAAACCGGAC GGUAAAAUGC GUGGUUUCGG UU - #UCGUUCAG 480- UUCAAAAACC UGCUGGAAGC UGGUAAAGCU CUGAAAGGUA UGAACAUGAA AG - #AAAUCAAA 540- GGUCGUACCG UUGCUGUUGA CUGGGCUGUU GCUAAAGACA AAUACAAAGA CA - #CCCAGUCC 600- GUUUCCGCUA UCGGUGAAGA AAAAUCCCAC GAAUCCAAAC ACCAGGAAUC CG - #UUAAAAAA 660- AAAGGUCGUG AAGAAGAAGA CAUGGAAGAA GAAGAAAACG ACGACGACGA CG - #ACGACGAC 720- GACGAAGAAG ACGGUGUUUU CGACGACGAA GACGAAGAAG AAGAAAACAU CG - #AAUCCAAA 780- GUUACCAAAC CGGUUCAGAU CCAGAAACGU GCUGUUAAAC GUCCGGCUCC GG - #CUAAAUCC 840- UCCGACCACU CCGAAGAAGA CUCCGACCUG GAAGAAUCCG ACUCCAUCGA CG - #ACGGUGAA 900- GAACUGGCUC AGUCCGACAC CUCCACCGAA GAACAGGAAG ACAAAGCUGU UC - #AGGUUUCC 960- AACAAAAAAA AACGUAAACU GCCGUCCGAC GUUAACGAAG GUAAAACCGU UU - #UCAUCCGU1020- AACCUGUCCU UCGACUCCGA AGAAGAAGAA CUGGGUGAAC UGCUGCAGCA GU - #UCGGUGAA1080- CUGAAAUACG UUCGUAUCGU UCUGCACCCG GACACCGAAC ACUCCAAAGG UU - #GCGCUUUC1140- GCUCAGUUCA UGACCCAGGA AGCUGCUCAG AAAUGCCUGC UGGCUGCUUC CC - #CGGAAAAC1200- GAAGCUGGUG GUCUGAAACU GGACGGUCGU CAGCUGAAAG UUGACCUGGC UG - #UUACCCGU1260- GACGAAGCUG CUAAACUGCA GACCACCAAA GUUAAAAAAC CGACCGGUAC CC - #GUAACCUG1320- UACCUGGCUC GUGAAGGUCU GAUCCGUGCU GGUACCAAAG CUGCUGAAGG UG - #UUUCCGCU1380- GCUGACAUGG CUAAACGUGA ACGUUUCGAA CUGCUGAAAC ACCAGAAACU GA - #AAGACCAG1440- AACAUCUUCG UUUCCCGUAC CCGUCUGUGC CUGCACAACC UGCCGAAAGC UG - #UUGACGAC1500- AAACAGCUGC GUAAACUGCU GCUGUCCGCU ACCUCCGGUG AAAAAGGUGU UC - #GUAUCAAA1560- GAAUGCCGUG UUAUGCGUGA CCUGAAAGGU GUUCACGGUA ACAUGAAAGG UC - #AGUCCCUG1620- GGUUACGCUU UCGCUGAAUU CCAGGAACAC GAACACGCUC UGAAAGCUCU GC - #GUCUGAUC1680- AACAACAACC CGGAAAUCUU CGGUCCGCUG AAACGUCCGA UCGUUGAAUU CU - #CCCUGGAA1740- GACCGUCGUA AACUGAAAAU GAAAGAACUG CGUAUCCAGC GUUCCCUGCA GA - #AAAUGCGU1800- UCCAAACCGG CUACCGGUGA ACCGCAGAAA GGUCAGCCGG AACCGGCUAA AG - #ACCAGCAG1860- CAGAAAGCUG CUCAGCACCA CACCGAAGAA CAGUCCAAAG UUCCGCCGGA AC - #AGAAACGU1920- AAAGCUGGUU CCACCUCCUG GACCGGUUUC CAGACCAAAG CUGAAGUUGA AC - #AGGUUGAA1980- CUGCCGGACG GUAAAAAACG UCGUAAAGUU CUGGCUCUGC CGUCCCACCG UG - #GUCCGAAA2040- AUCCGUCUGC GUGACAAAGG UAAAGUUAAA CCGGUUCACC CGAAAAAACC GA - #AACCGCAG2100- AUCAACCAGU GGAAACAGGA AAAACAGCAG CUGUCCUCCG AACAGGUUUC CC - #GUAAAAAA2160- GCUAAAGGUA ACAAAACCGA AACCCGUUUC AACCAGCUGG UUGAACAGUA CA - #AACAGAAA2220- CUGCUGGGUC CGUCCAAAGG UGCUCCGCUG GCUAAACGUU CCAAAUGGUU CG - #ACUCC2277- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2277 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- ATGGCCGGCC TGACCCTGTT CGTGGGCCGC CTGCCCCCCA GCGCCCGCAG CG - #AGCAGCTG 60- GAGGAGCTGT TCAGCCAGGT GGGCCCCGTG AAGCAGTGCT TCGTGGTGAC CG - #AGAAGGGC 120- AGCAAGGCCT GCCGCGGCTT CGGCTACGTG ACCTTCAGCA TGCTGGAGGA CG - #TGCAGCGC 180- GCCCTGAAGG AGATCACCAC CTTCGAGGGC TGCAAGATCA ACGTGACCGT GG - #CCAAGAAG 240- AAGCTGCGCA ACAAGACCAA GGAGAAGGGC AAGAACGAGA ACAGCGAGTG CC - #CCAAGAAG 300- GAGCCCAAGG CCAAGAAGGC CAAGGTGGCC GACAAGAAGG CCCGCCTGAT CA - #TCCGCAAC 360- CTGAGCTTCA AGTGCAGCGA GGACGACCTG AAGACCGTGT TCGCCCAGTT CG - #GCGCCGTG 420- CTGGAGGTGA ACATCCCCCG CAAGCCCGAC GGCAAGATGC GCGGCTTCGG CT - #TCGTGCAG 480- TTCAAGAACC TGCTGGAGGC CGGCAAGGCC CTGAAGGGCA TGAACATGAA GG - #AGATCAAG 540- GGCCGCACCG TGGCCGTGGA CTGGGCCGTG GCCAAGGACA AGTACAAGGA CA - #CCCAGAGC 600- GTGAGCGCCA TCGGCGAGGA GAAGAGCCAC GAGAGCAAGC ACCAGGAGAG CG - #TGAAGAAG 660- AAGGGCCGCG AGGAGGAGGA CATGGAGGAG GAGGAGAACG ACGACGACGA CG - #ACGACGAC 720- GACGAGGAGG ACGGCGTGTT CGACGACGAG GACGAGGAGG AGGAGAACAT CG - #AGAGCAAG 780- GTGACCAAGC CCGTGCAGAT CCAGAAGCGC GCCGTGAAGC GCCCCGCCCC CG - #CCAAGAGC 840- AGCGACCACA GCGAGGAGGA CAGCGACCTG GAGGAGAGCG ACAGCATCGA CG - #ACGGCGAG 900- GAGCTGGCCC AGAGCGACAC CAGCACCGAG GAGCAGGAGG ACAAGGCCGT GC - #AGGTGAGC 960- AACAAGAAGA AGCGCAAGCT GCCCAGCGAC GTGAACGAGG GCAAGACCGT GT - #TCATCCGC1020- AACCTGAGCT TCGACAGCGA GGAGGAGGAG CTGGGCGAGC TGCTGCAGCA GT - #TCGGCGAG1080- CTGAAGTACG TGCGCATCGT GCTGCACCCC GACACCGAGC ACAGCAAGGG CT - #GCGCCTTC1140- GCCCAGTTCA TGACCCAGGA GGCCGCCCAG AAGTGCCTGC TGGCCGCCAG CC - #CCGAGAAC1200- GAGGCCGGCG GCCTGAAGCT GGACGGCCGC CAGCTGAAGG TGGACCTGGC CG - #TGACCCGC1260- GACGAGGCCG CCAAGCTGCA GACCACCAAG GTGAAGAAGC CCACCGGCAC CC - #GCAACCTG1320- TACCTGGCCC GCGAGGGCCT GATCCGCGCC GGCACCAAGG CCGCCGAGGG CG - #TGAGCGCC1380- GCCGACATGG CCAAGCGCGA GCGCTTCGAG CTGCTGAAGC ACCAGAAGCT GA - #AGGACCAG1440- AACATCTTCG TGAGCCGCAC CCGCCTGTGC CTGCACAACC TGCCCAAGGC CG - #TGGACGAC1500- AAGCAGCTGC GCAAGCTGCT GCTGAGCGCC ACCAGCGGCG AGAAGGGCGT GC - #GCATCAAG1560- GAGTGCCGCG TGATGCGCGA CCTGAAGGGC GTGCACGGCA ACATGAAGGG CC - #AGAGCCTG1620- GGCTACGCCT TCGCCGAGTT CCAGGAGCAC GAGCACGCCC TGAAGGCCCT GC - #GCCTGATC1680- AACAACAACC CCGAGATCTT CGGCCCCCTG AAGCGCCCCA TCGTGGAGTT CA - #GCCTGGAG1740- GACCGCCGCA AGCTGAAGAT GAAGGAGCTG CGCATCCAGC GCAGCCTGCA GA - #AGATGCGC1800- AGCAAGCCCG CCACCGGCGA GCCCCAGAAG GGCCAGCCCG AGCCCGCCAA GG - #ACCAGCAG1860- CAGAAGGCCG CCCAGCACCA CACCGAGGAG CAGAGCAAGG TGCCCCCCGA GC - #AGAAGCGC1920- AAGGCCGGCA GCACCAGCTG GACCGGCTTC CAGACCAAGG CCGAGGTGGA GC - #AGGTGGAG1980- CTGCCCGACG GCAAGAAGCG CCGCAAGGTG CTGGCCCTGC CCAGCCACCG CG - #GCCCCAAG2040- ATCCGCCTGC GCGACAAGGG CAAGGTGAAG CCCGTGCACC CCAAGAAGCC CA - #AGCCCCAG2100- ATCAACCAGT GGAAGCAGGA GAAGCAGCAG CTGAGCAGCG AGCAGGTGAG CC - #GCAAGAAG2160- GCCAAGGGCA ACAAGACCGA GACCCGCTTC AACCAGCTGG TGGAGCAGTA CA - #AGCAGAAG2220- CTGCTGGGCC CCAGCAAGGG CGCCCCCCTG GCCAAGCGCA GCAAGTGGTT CG - #ACAGC2277- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 540 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- GGGTTGCGGA GGGTGGGCCT GGGAGGGGTG GTGGCCATTT TTTGTCTAAC CC - #TAACTGAG 60- AAGGGCGTAG GCGCCGTGCT TTTGCTCCCC GCGCGCTGTT TTTCTCGCTG AC - #TTTCAGCG 120- GGCGGAAAAG CCTCGGCCTG CCGCCTTCCA CCGTTCATTC TAGAGCAAAC AA - #AAAATGTC 180- AGCTGCTGGC CCGTTCGCCC CTCCCGGGGA CCTGCGGCGG GTCGCCTGCC CA - #GCCCCCGA 240- ACCCCGCCTG GAGGCCGCGG TCGGCCCGGG GCTTCTCCGG AGGCACCCAC TG - #CCACCGCG 300- AAGAGTTGGG CTCTGTCAGC CGCGGGTCTC TCGGGGGCGA GGGCGAGGTT CA - #GGCCTTTC 360- AGGCCGCAGG AAGAGGAACG GAGCGAGTCC CCGCGCGCGG CGCGATTCCC TG - #AGCTGTGG 420- GACGTGCACC CAGGACTCGG CTCACACATG CAGTTCGCTT TCCTGTTGGT GG - #GGGGAACG 480- CCGATCGTGC GCATCCGTCA CCCCTCGCCG GCAGTGGGGG CTTGTGAACC CC - #CAAACCTG 540- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 540 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- GGGTTGCGGA GGGTGGGCCT GGGAGGGGTG GTGGCCATTT TTTGTCCAAC CC - #CAACTGAG 60- AAGGGCGTAG GCGCCGTGCT TTTGCTCCCC GCGCGCTGTT TTTCTCGCTG AC - #TTTCAGCG 120- GGCGGAAAAG CCTCGGCCTG CCGCCTTCCA CCGTTCATTC TAGAGCAAAC AA - #AAAATGTC 180- AGCTGCTGGC CCGTTCGCCC CTCCCGGGGA CCTGCGGCGG GTCGCCTGCC CA - #GCCCCCGA 240- ACCCCGCCTG GAGGCCGCGG TCGGCCCGGG GCTTCTCCGG AGGCACCCAC TG - #CCACCGCG 300- AAGAGTTGGG CTCTGTCAGC CGCGGGTCTC TCGGGGGCGA GGGCGAGGTT CA - #GGCCTTTC 360- AGGCCGCAGG AAGAGGAACG GAGCGAGTCC CCGCGCGCGG CGCGATTCCC TG - #AGCTGTGG 420- GACGTGCACC CAGGACTCGG CTCACACATG CAGTTCGCTT TCCTGTTGGT GG - #GGGGAACG 480- CCGATCGTGC GCATCCGTCA CCCCTCGCCG GCAGTGGGGG CTTGTGAACC CC - #CAAACCTG 540- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 540 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- GGGTTGCGGA GGGTGGGCCT GGGAGGGGTG GTGGCCATTT TTTGTCTAAG CC - #TAAGTGAG 60- AAGGGCGTAG GCGCCGTGCT TTTGCTCCCC GCGCGCTGTT TTTCTCGCTG AC - #TTTCAGCG 120- GGCGGAAAAG CCTCGGCCTG CCGCCTTCCA CCGTTCATTC TAGAGCAAAC AA - #AAAATGTC 180- AGCTGCTGGC CCGTTCGCCC CTCCCGGGGA CCTGCGGCGG GTCGCCTGCC CA - #GCCCCCGA 240- ACCCCGCCTG GAGGCCGCGG TCGGCCCGGG GCTTCTCCGG AGGCACCCAC TG - #CCACCGCG 300- AAGAGTTGGG CTCTGTCAGC CGCGGGTCTC TCGGGGGCGA GGGCGAGGTT CA - #GGCCTTTC 360- AGGCCGCAGG AAGAGGAACG GAGCGAGTCC CCGCGCGCGG CGCGATTCCC TG - #AGCTGTGG 420- GACGTGCACC CAGGACTCGG CTCACACATG CAGTTCGCTT TCCTGTTGGT GG - #GGGGAACG 480- CCGATCGTGC GCATCCGTCA CCCCTCGCCG GCAGTGGGGG CTTGTGAACC CC - #CAAACCTG 540- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 538 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- GGGTTGCGGA GGGTGGGCCT GGGAGGGGTG GTGGCCATTT TTTGTCTACC CT - #ACTGAGAA 60- GGGCGTAGGC GCCGTGCTTT TGCTCCCCGC GCGCTGTTTT TCTCGCTGAC TT - #TCAGCGGG 120- CGGAAAAGCC TCGGCCTGCC GCCTTCCACC GTTCATTCTA GAGCAAACAA AA - #AATGTCAG 180- CTGCTGGCCC GTTCGCCCCT CCCGGGGACC TGCGGCGGGT CGCCTGCCCA GC - #CCCCGAAC 240- CCCGCCTGGA GGCCGCGGTC GGCCCGGGGC TTCTCCGGAG GCACCCACTG CC - #ACCGCGAA 300- GAGTTGGGCT CTGTCAGCCG CGGGTCTCTC GGGGGCGAGG GCGAGGTTCA GG - #CCTTTCAG 360- GCCGCAGGAA GAGGAACGGA GCGAGTCCCC GCGCGCGGCG CGATTCCCTG AG - #CTGTGGGA 420- CGTGCACCCA GGACTCGGCT CACACATGCA GTTCGCTTTC CTGTTGGTGG GG - #GGAACGCC 480- GATCGTGCGC ATCCGTCACC CCTCGCCGGC AGTGGGGGCT TGTGAACCCC CA - #AACCTG 538- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 14 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: Not Relev - #ant- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 5..13#/note= "Xaa represents isoleucine or leucin - #e"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Glu Ala Ala Thr Xaa Asp Xaa Pro - # Gln Gln Gly Ala Asn Lys# 10- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 534 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- GGGTTGCGGA GGGTGGGCCT GGGAGGGGTG GTGGCCATTT TTTGTCTAAC CC - #TAACTGAG 60- AAGGGCGTAG GCGCCGTGCT TTTGCTCCCC GCGCGCTGTT TTTCTCGCTG AC - #TTTCAGCG 120- GGCGGAAAAG CCTCGGCCTG CCGCCTTCCA CCGTTCATTC TAGAGCAAAC AA - #AAAATGTC 180- AGCTGCTGGC CCGTTCGCCT CCCGGGGACC TGCGGCGGGT CGCCTGCCCA GC - #CCCCGAAC 240- CCCGCCTGGA GCCGCGGTCG GCCCGGGGCT TCTCCGGAGG CACCCACTGC CA - #CCGCGAAG 300- AGTTGGGCTC TGTCAGCCGC GGGTCTCTCG GGGGCGAGGG CGAGGTTCAC CG - #TTTCAGGC 360- CGCAGGAAGA GGAACGGAGC GAGTCCCGCC GCGGCGCGAT TCCCTGAGCT GT - #GGGACGTG 420- CACCCAGGAC TCGGCTCACA CATGCAGTTC GCTTTCCTGT TGGTGGGGGG AA - #CGCCGATC 480- GTGCGCATCC GTCACCCCTC GCCGGCAGTG GGGGCTTGTG AACCCCCAAA CC - #TG 534__________________________________________________________________________
|Cited Patent
|Filing date
|Publication date
|Oct 27, 1994
|Dec 10, 1996
|Geron Corporation
|Mammalian telomerase
|Citing Patent
|Filing date
|Publication date
|Oct 18, 1999
|Jul 17, 2001
|Geron Corporation
|Purified telomerose
|Sep 29, 2000
|Aug 27, 2002
|Geron Corporation
|Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
|Nov 20, 2000
|Feb 11, 2003
|Geron Corporation
|Purified telomerase
|Nov 20, 2000
|Apr 8, 2003
|Geron Corporation
|Methods for purifying telomerase
|Dec 24, 2002
|Sep 7, 2004
|Geron Corporation
|Using purified telomerase to identify telomerase activators and inhibitors
|Jan 19, 2001
|Oct 26, 2004
|Geron Corporation
|Method for detecting polynucleotides encoding telomerase
|Jan 18, 2002
|Jul 26, 2005
|Regents Of The University Of Colorado
|Nov 12, 1999
|Aug 9, 2005
|Geron Corporation
|Genes for human telomerase reverse transcriptase and telomerase variants
|Jan 18, 2002
|Feb 28, 2006
|Geron Corporation
|Methods for detecting nucleic acids encoding human telomerase reverse transcriptase
|Apr 26, 2001
|Jun 6, 2006
|Geron Corporation
|Mar 26, 2004
|Jun 27, 2006
|Geron Corporation
|Producing telomerase activators and inhibitors by screening with purified telomerase
|Aug 9, 2002
|Dec 19, 2006
|Cornell Research Foundation, Inc.
|Telomerase immortalized neural progenitor cells
|Jan 11, 2002
|Mar 27, 2007
|Geron Corporation
|Mammalian cells that have increased proliferative capacity
|Nov 22, 2000
|Aug 28, 2007
|Geron Corporation
|Nucleic acids encoding human telomerase reverse transcriptase and related homologs
|Jan 18, 2002
|Oct 23, 2007
|Geron Corporation
|Antibody to telomerase reverse transcriptase
|Jul 30, 2002
|Jul 22, 2008
|Geron Corporation
|Method for identifying and killing cancer cells
|Jun 24, 2004
|Aug 19, 2008
|Geron Corporation
|Treating cancer using a telomerase vaccine
|Nov 22, 2000
|Apr 14, 2009
|Geron Corporation
|Muteins of human telomerase reverse transcriptase lacking telomerase catalytic activity
|Jan 11, 2002
|Jul 14, 2009
|Geron Corporation
|Nucleic acid compositions for eliciting an immune response against telomerase reverse transcriptase
|Nov 2, 1999
|Sep 8, 2009
|Geron Corporation
|Increasing the proliferative capacity of cells using telomerase reverse transcriptase
|Jun 24, 2004
|Nov 24, 2009
|Geron Corporation
|Human telomerase reverse transcriptase polypeptides
|Aug 17, 2005
|Jul 6, 2010
|Geron Corporation
|Antibody to telomerase reverse transcriptive
|Apr 27, 2006
|Nov 2, 2010
|Geron Corporation
|Cellular telomerase vaccine and its use for treating cancer
|Aug 20, 2007
|Jul 17, 2012
|Geron Corporation
|Kit for detection of telomerase reverse transcriptase nucleic acids
|Aug 21, 2009
|Aug 7, 2012
|Geron Corporation
|Human telomerase catalytic subunit
|Aug 20, 2007
|Apr 29, 2014
|Geron Corporation
|Method for eliciting an immune response to human telomerase reverse transcriptase
|Apr 30, 2004
|Jul 8, 2014
|Duke University
|Chimeric molecule and nucleic acid encoding same
|Aug 14, 2006
|Aug 5, 2014
|Geron Corporation
|Nucleic acids encoding inactive variants of human telomerase
|Apr 26, 2001
|Nov 7, 2002
|Cech Thomas R.
|Novel telomerase
|Jan 19, 2001
|Dec 12, 2002
|Cech Thomas R.
|Novel telomerase
|Jan 18, 2002
|Feb 13, 2003
|Cech Thomas R.
|Novel telomerase
|Jan 18, 2002
|Mar 6, 2003
|Cech Thomas R.
|Novel telomerase
|Jan 18, 2002
|Mar 27, 2003
|Cech Thomas R.
|Novel telomerase
|Aug 9, 2002
|May 1, 2003
|Goldman Steven A.
|Telomerase immortalized human neural stem cells and phenotypically-restricted progenitor cells
|Jan 11, 2002
|May 22, 2003
|Cech Thomas R.
|Human telomerase catalytic subunit: diagnostic and therapeutic methods
|Jan 11, 2002
|May 29, 2003
|Cech Thomas R.
|Human telomerase catalytic subunit: diagnostic and therapeutic methods
|Jun 24, 2004
|Dec 2, 2004
|Geron Corporation
|Vector encoding inactivated telomerase for treating cancer
|Jun 24, 2004
|Dec 9, 2004
|Geron Corporation
|Treating cancer using a telomerase vaccine
|Jun 24, 2004
|Jan 20, 2005
|Geron Corporation
|Vaccine containing the catalytic subunit of telomerase for treating cancer
|Mar 26, 2004
|Apr 28, 2005
|Weinrich Scott L.
|Producing telomerase activators and inhibitors by screening with purified telomerase
|Aug 17, 2005
|Feb 23, 2006
|Geron Corporation
|Human telomerase catalytic subunit
|Mar 13, 2006
|Jul 27, 2006
|Weinrich Scott L
|Protocol for optimizing purification of enzymatically active human telomerase
|Apr 27, 2006
|Sep 14, 2006
|Gaeta Federico C
|Cellular telomerase vaccine and its use for treating cancer
|Aug 14, 2006
|Dec 7, 2006
|Morin Gregg B
|Nucleic acids encoding inactive variants of human telomerase
|Apr 30, 2004
|Jan 25, 2007
|Counter Christopher M
|Telomere elongation
|Aug 20, 2007
|Nov 13, 2008
|Geron Corporation
|Immunogenic composition
|Aug 20, 2007
|Oct 29, 2009
|Geron Corporation
|Kit for detection of telomerase reverse transcriptase nucleic acids
|U.S. Classification
|536/23.2, 435/252.3, 530/350, 435/194, 536/24.31, 435/91.31, 536/23.1, 435/320.1
|International Classification
|Cooperative Classification
|European Classification
|Sep 21, 1999
Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, CALI
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:COLLINS, KATHLEEN;REEL/FRAME:010247/0335
Effective date: 19960702
|Dec 6, 2002
|Fee payment
Year of fee payment: 4
|Dec 29, 2006
|Fee payment
Year of fee payment: 8
|Jan 31, 2011
|Maintenance fee reminder mailed
|Jun 29, 2011
|Lapse for failure to pay maintenance fees
|Aug 16, 2011
|Expired due to failure to pay maintenance fee
Effective date: 20110629


text:
"Eight drunk immortals bray / endorse the drunken way / and what will be will be" - Let's get drunk and everything will more or less work out.
LordRetardon January 16, 2006 Link
Lyrics submitted by LordRetard
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subset: commoncrawl


text:
Sources informed that many have been arrested from the city for thefts. Citing the unrest and possibility of more accidents following the quake, the government issued a strict night curfew in Christchurch.
"All entry to a 'restricted access zone' would be blocked from 6:30 pm, to prevent crime and with safety fears for the collapse of further buildings," sources reported quoting the government spokesperson. Police said that they would arrest people who are found in the central city after 6:30 pm.
Emergency services cordoned off central Christchurch. Prime Minister, John Key, declared a national emergency. The latest tremor toppled many buildings and left central Christchurch strewn with debris. The city's landmark cathedral lost its spire. Dozens of aftershocks rocked the city, much of which was without power and water.
Rescue teams are still hopeful to save many out of the debris. Local media reported, "There is incredible carnage right throughout the city. There are bodies littering the streets, they are trapped in cars and crushed under rubble."
"We are getting texts and tapping sounds from some of these buildings and that's where the focus is at the moment," rescue operators stated.
Specialist teams from Australia, Britain, Japan, Singapore, South Korea, Taiwan and the United States were due to join the New Zealand rescuers as an international effort swung into action.
New Zealand had experienced another massive temblor with 7.0 magnitude in Sep 2010 but miraculously the quake caused no deaths. This time, the earthquake was of 6.3 magnitude which is believed to be New Zealand's worst natural disaster in the last 80 years.
OneIndia News
(With inputs from PTI)


text:
The purpose of this website is to share an experience, one that has originated within the mind of a single person, myself, its creator. Only you know your reason for visiting it and how that came about though. Therefore immediately I offer you a choice. You may visit The Fiction, The Facts or The Speculation but don't be misled by these titles. There may be as many facts within the fiction as there could be fiction within the facts. As to the speculation, who knows? In truth everything on this website may reside in the grey area between fact and fiction. Only you can decide where to draw the boundaries as you see fit. As this is a place for the telling of stories and little else let me try to explain with one now.
When I was young I owned a wooden jigsaw puzzle. It was double-sided with a picture of Mr. Toad from The Wind In The Willows on one side and Ichabod and Katrina from The Legend Of Sleepy Hollow on the other. I imagine that it was merchandise connected with the film containing both these stories released in 1949 by Walt Disney Productions although I never saw that. In fact, being English, I only knew about The Wind In The Willows and hadn't heard of the other story then. Therefore I would put the puzzle together with the picture of Mr. Toad, the one that I recognised, uppermost. When working on a double-sided jigsaw puzzle there is the additional problem that one must determine which side of each piece is relevant. The fragments of picture on it are clues but the shape of the piece regardless of the pictures may also be of assistance in placing it. When I had completed the puzzle I would carefully place it between sheets of cardboard or such so that I could turn it over to see the other picture, the one that I had assembled without seeing it. Once assembled it was impossible to determine which picture I had actually used to guide me during the assembly. That is the nature of a double-sided jigsaw puzzle, that it is extremely difficult to see both sides at the same time while assembling it. Perhaps it could just be done with the assistance of a mirror.
The two stories depicted by the puzzle only became connected historically through events within the Disney studios. Equally the two pictures were unrelated except by being two sides of the same physical medium. Their coexistence was just a coincidence which I could not understand at the time. What I present here on this website are equally two possibly unrelated stories which happen to coexist within my mind. Now that I know both of them I am faced with the same problem as with that jigsaw puzzle, that I can no longer be sure which was my guide while I was assembling this joint experience.
You now have to decide which story you will read first, but please bear in mind that it isn't just the information that one acquires but the order in which one acquires it that determines the nature of one's experiences. I recommend that you start with The Fiction. I cannot say exactly what you will find there as I am writing this at the end of 2015 and I don't know what has happened here since then. You will at least find the first part of a novel that I started writing at the beginning of 2011. It is a reasonably light-hearted fictional story which some might class as being in the science fiction or fantasy genre, but don't let that put you off. Regard it as being as wispily playful as The Wind In The Willows. After that you may like to read The Facts, which attempt to explain how I came to write the novel and the thoughts behind it. That is perhaps a darker tale with a hint of the paranormal, a fitting parallel with The Legend of Sleepy Hollow. Obviously reading that first would spoil the experience of reading the fictional story itself, but it really depends how much time you have to waste here. The complete fictional story that I conceived was very long and how much has been committed to text so far depends more than anything on how interested anyone has been in reading it. You may wonder in that case how I came to write any of it at all and so do I. Believe me, so do I. Maybe the answer lies in the third section, my speculations. If you are the sort who likes to read how a story ends to judge the rest, then by all means read that section first, but doing so would hardly be sharing in the experience.
All that I ask is that, as Samuel Taylor Coleridge once explained, you suspend your disbelief for the time being while you read. At exactly what point you choose to resume disbelief is entirely up to you. I happened to attend the same school as Coleridge, but not at the same time of course. To have done so I would have to be either incredibly old or a time traveller. In fact physically I have travelled through the seventy or so years of my life to this point at the conventional pace and in the normal direction. What my mind may have done is possibly a different matter, but all our minds wander from time to time, don't they?
My name? It's Rob, just Rob for now.


text:
A nurse has appeared in court accused of the murder of three people.
Victorino Chua, 48, spoke only to confirm his name, age and address for the three minute hearing at Manchester Magistrates' Court.
He was charged following a lengthy investigation by police at Stepping Hill Hospital in Stockport.
Chua also faces 22 counts of attempting to cause grievous bodily harm with intent, seven charges of attempting to administer poison and one count of causing poison to be administered with intent, under the Offences Against the Person Act.
Chua, was held last year on suspicion of tampering with medical records at Stepping Hill Hospital in Stockport.
He was questioned on suspicion of three counts of murder and various counts of causing grievous bodily harm.
Police are carrying out an investigation into the contamination of various medical products between 1 June and 15 July 15, 2011 at the hospital.
Eight patients who were poisoned by contaminated products later died.
They were Tracey Arden, 44, Arnold Lancaster, 71, Derek Weaver, 83, William Dickson, 82, Linda McDonagh, 60, John Beeley, 73, Beryl Hope, 70, and Mary Cartwright, 89.
Chua is charged with murder over the deaths of Ms Arden, Mr Lancaster and Mr Weaver.
He was remanded into custody to appear at Manchester Crown Court next Tuesday.
Additional reporting by PAReuse content


text:
Periarticular Bone Changes in Osteoarthritis
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s11420-011-9257-5
- Cite this article as:
- Weinans, H. HSS Jrnl (2012) 8: 10. doi:10.1007/s11420-011-9257-5
- Citations
- 590 Downloads
Keywordsosteoarthritis subchondral bone osteoclast micro-SPECT micro-CT
Osteoarthritis (OA) can be considered an organ failure with pathological aspects in cartilage, bone, ligaments, and synovium. Altogether, these tissue changes can result in pain and immobilization—a failure of the joint. It is well regarded that OA is a complex multifactorial disease with many risk factors and different etiological pathways that all lead to an apparently similar end stage. Bony changes are clearly observed in advanced OA. However, little understanding exists on the role of these changes, whether they are a consequence of cartilage damage or precede this damage and maybe play an important role in the etiological process. Even more important is the issue of pain. Radiological scores of OA do not match well with pain and mobility scores, which questions the value of these scoring systems. It seems that we do not assess the most relevant parameters. Evaluation of conventional and new radiographic parameters is still an extensive part of the OA research field. We may have overlooked certain (subtle) parameters that can be extracted from x-rays, but other imaging modalities such as MRI, CT, or SPECT might better represent OA in a clinically relevant manner.
Subchondral Bone Adaptation in Osteoarthritis
Another interesting hypothesis came from the work of Westacott et al. [13], who found that bone cells from OA patients can adversely influence cartilage metabolism. This provided a hint that the increased subchondral bone activity in OA joints can lead to cartilage degeneration. Of course, this hypothesis requires diffusion of harmful agents between the subchondral bone compartment and the cartilage, which might be difficult if the mineralized cartilage and subchondral plate are relatively dense. To further investigate the role of subchondral bone in OA, we decided to first investigate the cascade of events in subchondral bone during the progression of OA in animal models using micro-CT for in vivo evaluations at various time points after induction of OA.
Observations from a Murine Model of Osteoarthritis
Surprisingly, higher bone turnover is an extremely fast response in the MIA model, contrary to the idea that MIA directly targets the cartilage, thereby inducing OA. This finding could be interpreted as the bone alteration occurring first. However, care must be taken with this interpretation. What we call "first" also relates to how much we can see with our imaging and/or histological methods. The bone SPECT scan is an extremely sensitive tool for measuring a bone response, and the cartilage might have been responsive to the MIA trigger at the 2-day time point as well, but maybe the histology was not sensitive enough to show it.
The animal models and the imaging tools provide a powerful combination for studying OA. What did we learn from the current approaches? It has been known for some years that subchondral bone is a highly adaptive tissue, and changes in subchondral bone can be accurately visualized and quantified by CT [6] and scintigraphy [5] even in a clinical setting. Animal CT and SPECT have been improved in the last decade to higher resolutions [3, 12], enabling refined in vivo evaluation in small animal research. Besides evaluations of subchondral bone and cartilage with the microimaging modalities, SPECT/CT can image macrophage activation as well [8]. With repetitive imaging of animals, we will learn the precise sequence of events that occur in the various tissues of the diseased joint. The bone scan analyses, with its fast (2 days) response in bone turnover in the MIA OA model, substantiate the often suggested crosstalk between the bone and cartilage compartments. In the different animal models, this sequence of events might be different; in one model, the problems might start in the cartilage, whereas in another model the bone might be triggered first. Each model might expose a different etiological pathway, similar to the differences in OA etiology in humans. Hopefully, these new tools will elucidate concepts and targets that help us in the design of new therapeutic interventions for OA.
The author certifies that he has no commercial associations (e.g., consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.
The author certifies that his institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


text:
freedom trip
Sail Date: February 01, 2012
Reviewed: 5 years ago
Traveled As: Large Group
Room Type: Balcony
Recommends Freedom of the Seas? Yes!
Category Ratings
Click table then swipe to view all columns.
Click table then swipe to view all columns.
|Service and Staff
|Room attendant and some bartenders were great. Store staff not so much.
|Ship Quality
|Food and Dining
|Windjammer cafe most of the time more dirty tables than clean.
|Cabin / Stateroom


text:
B2 good or good enough for a particular need or purpose:
The outcome of the discussion was highly satisfactory (= very pleasing).
Más ejemplos
- I still haven't received a satisfactory explanation.
- The meeting failed to produce a satisfactory outcome.
- He was at the time making satisfactory progress at school.
- I am convinced that in time we will find a satisfactory solution to the problem.
- Can we now be sure that schools are providing education of a satisfactory standard?
Thesaurus: sinónimos y palabras relacionadas


text:
Sunrise Kids School Home
Caring, Loving & Experienced Staff
Fun play & learning
Age appropriate handling
FunGames to Teach Basics
Sensory activities
Motor Skills
Drawing & Colouring
Early Development is fundamental to the Growth of your Child
Quality pre-school education lays a strong foundation for life-long learning and has short and long term benefits for children.To nurture children as life-long learners, we need to build their self-Confidence, encourage them to explore the world around them and teach them how to regulate their behavior and emotions express themselves and get along with others
Our Aspiration
We at Sunrise kids School Home aspires to develop in every child:
• A Confident person who has a strong sense of right and wrong, is adaptable and resilient, knows himself, is discerning in judgment, thinks independently and critically, and communicates effectively.
• A Self-directed learner who takes responsibility for his own learning, who questions, reflects and perseveres in the pursuit of learning.
• An Active contributor who is able to work effectively in teams, Exercises initiative, takes calculated risks, is innovative and strives for excellence.
• A Responsible citizen of India, has a strong civic consciousness, is informed, and takes an active role in bettering the lives of others around him.
At the Sunrise kid's school home, the holistic development of children is most important. The key stage outcomes of pre-school education emphasize the need for children to build up confidence and social skills and be equipped with the necessary knowledge, skills and dispositions for life-long learning.
Our Outcome
At the end of their pre-school education in Sunrise kids School home, children would:
• know what is right and what is wrong
• be willing to share and take turns with others
• be able to relate to others
• be curious and able to explore
• be able to listen and speak with understanding
• be comfortable and happy with themselves
• have developed physical co-ordination, healthy habits, participate in and enjoy a variety of arts experiences
• love their families, friends, teachers and school
Our Learning Areas
Sunrise kids school home Learning Areas
At Sunrise kids school home the following are the six learning areas, through which children acquire knowledge, skills and learning dispositions
• Aesthetics and Creative Expression
• Discovery of the World
• Language and Literacy
• Motor Skills Development
• Numeracy
• Social and Emotional Development
Aesthetics and Creative Expression
Children enjoy creative expression and should be given opportunities to explore their environment and indulge in creative play and self-expression through art and music and movement.
Discovery of the World
Children are naturally curious and their sense of wonder should be nurtured and sustained. They should be encouraged to observe, ask questions and make sense of the world around them.
Language and Literacy
Language and Literacy is important in children's overall thought and learning development. Exposure to language-related activities such as role-playing, singing, rhyming and reading will develop their ability to listen with understanding and to communicate with others.
Motor Skills Development
Children's gross and fine motor skills develop over time and influence their physical fitness, agility and coordination. Children should be given ample space and time for physical play to develop their sense of balance, physical co-ordination and spatial awareness.
Being numerate enables children to understand and apply mathematical concepts, skills and processes effectively. Thus, children will be able to make sense of their encounters with mathematics in their daily lives.
Social and Emotional Development
Effective social and emotional development instills healthy self-esteem in children. Children are taught to manage their feelings, thoughts and behavior which will allow them to form strong and meaningful relationships later in life.
PRAISE learning @ Sunrise kid's school home
There are six learning dispositions (PRAISE) that Sunrise Kids School Home will seek to develop in every child:
Learning dispositions are positive behaviors and attitudes towards learning. They are important for children in their journey as life-long learners and they support children's learning and development in the various learning areas.
Children with perseverance keep working at a task to complete it. They do not give up even though they find it challenging.
Children with the ability to reflect can think about what they have done, seen, heard or felt in their daily experiences. They generate alternative ideas and gain new knowledge.
Children with a sense of appreciation recognize that everyone has different and unique qualities. They listen to the views of others and show respect for them.
Children who are inventive are flexible in their thinking and can do things in different ways. They explore various ideas and possibilities and generate different solutions to challenges.
Sense of wonder and curiosity
Children with a sense of wonder and curiosity are interested in the world around them. They confidently ask questions on why things happen and how things work. They like to explore and discover new things.
Children who are engaged are immersed in learning and enjoy what they do. They proactively seek new opportunities for learning.


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Married driver Brian Twohey, 57, of Witham, Essex, was taken to St Mary's Hospital by ambulance where he was pronounced dead on arrival.
Following the incident, on 21 February, a post mortem established the cause of Mr Twohey's death as a heart attack.
Today Sharon Roberts, 24, of Thessaly Road, Battersea, and 21-year-old Liane McPherson of West Road, West Ham, were charged with manslaughter.
They are also charged with affray, common assault and making off from a taxi cab without payment.
- More about:
- Battersea
- Essex, England
- Healthcare
- Transport


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London, October 16(ANI): A new study has found that the human tongue can taste the "flavour" of carbon dioxide when bubbles of fizzy drinks like Champagne break on it, a new research has revealed.
The study published in the journal 'Science,' revealed that similar to the way tongue detects the flavour of sour food, it also responds to the gases of carbonated or fermented drinks.
Neuroscientist at Columbia University in New York, Charles Zuker, is the man who found that a class of taste-receptor cells in the tongue responds to carbon dioxide.
He also specifically identified an enzyme called carbonic anhydrase 4 in the detection of the gas, reports the Independent.
The research was conducted on laboratory mice genetically modified to lack sour-sensing taste cells.
The scientists apparently found that the mice also lacked the ability to detect carbon dioxide in carbonated water.
However, those that did retain the sour-sensing cells showed a neurological response when drinking carbonated water.
The study also explains the the reason why people do not get the taste of champagne, beer and other fizzy drinks when on an acetazolamide, a prophylactic taken to avert altitude sickness. (ANI)


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Leisure & Tourism
Web design, viral marketing, SEO, CMS
Wickedweb has completed some exciting projects in this sector. We have designed the new website for South Africa Tourism, which also includes viral activity, video streaming, message boards and competitions. We were also appointed as the SEO specialists for Beachcomber Tours who are a specialist operator for holidays to Mauritius. Our expertise in this sector also extends to work for the Destinations Show, where we implemented a full CMS and SEO strategy for this popular event.
Visit Wickedweb case studies for the Leisure and Tourism sector
Fact Sheet
Download our fact sheet
Enquiry Form


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Use of the Perclose ProGlide device with the 9 French Merci retrieval system
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s00234-006-0049-4
- Cite this article as:
- Layton, K.F., White, J.B., Cloft, H.J. et al. Neuroradiology (2006) 48: 324. doi:10.1007/s00234-006-0049-4
- Citations
- 155 Downloads
Closure devices are commonly used in neurointerventional procedures to achieve groin hemostasis. These devices are particularly useful in procedures requiring anticoagulation and larger catheters. The suture-mediated Perclose ProGlide device is intended for use with 5F to 8F sheaths. We describe the use of the ProGlide device with 9F sheaths in acute stroke treatment using the Merci retrieval device.
The ProGlide device is advanced over a wire until the wire exit port is at the skin surface. The wire is removed and the device is advanced until pulsatile blood flow is encountered. The footplates are opened and the stitch is deployed. The footplates are then closed and the device is removed. After advancing the suture to the vessel, it is locked and trimmed. Firm pressure is necessary during deployment to prevent oozing around the device. If continued bleeding is encountered, direct manual pressure is used to achieve hemostasis.
We have successfully used the Perclose ProGlide device in four patients following the Merci retriever without groin or extremity complication. The Perclose ProGlide device can be successfully used after placement of a 9F system in patients who have undergone mechanical thrombectomy.
This technique may be of interest to endovascular surgical neuroradiologists because it affords fast hemostasis in large access sites following mechanical thrombectomy with the Merci retrieval device. Compared to the "Preclose" technique, it saves valuable time at the beginning of the case.


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Definition of ribbon
1a : a flat or tubular narrow closely woven fabric (as of silk or rayon) used for trimmings or knittingb : a narrow fabric used for tying packagesc : a piece of usually multicolored ribbon worn as a military decoration or in place of a medald : a strip of colored satin given for winning a place in a competition
2 : a strip of inked fabric (as in a typewriter)
3 ribbons plural : reins for controlling an animal
4 : tatter, shred —usually used in plural a sheet cut to ribbons
5 : ribband
ribbonlikeplay \-ˌlīk\ adjective
Examples of ribbon in a Sentence
She wore pink ribbons in her hair.
She tied a ribbon around the present.
The present was tied with ribbon.
The soldier proudly wore his ribbons and medals.
Her pie won a ribbon at the county fair.
Origin and Etymology of ribbon
Middle English riban, from Anglo-French ribane, rubane
First Known Use: 14th century
Definition of ribbon
transitive verb
1a : to adorn with ribbonsb : to divide into ribbonsc : to cover with or as if with ribbons
2 : to rip to shreds
Examples of ribbon in a Sentence
over the years the historic flag had become badly ribboned by the wind
First Known Use of ribbon
RIBBON Defined for English Language Learners
Definition of ribbon for English Language Learners
: a narrow piece of cloth (such as silk) that is used to tie things or for decoration
: a short piece of cloth that is given as a military award
: a piece of colored cloth that is given as an award in a competition
RIBBON Defined for Kids
Learn More about ribbon
See words that rhyme with ribbon Thesaurus: All synonyms and antonyms for ribbon Spanish Central: Translation of ribbon Nglish: Translation of ribbon for Spanish speakers Britannica English: Translation of ribbon for Arabic speakers
Seen and Heard
What made you want to look up ribbon? Please tell us where you read or heard it (including the quote, if possible).


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Super Mario RPG 3: Search For The Silver Statues is a game for Nintendo Throwback. It is the unexpected sequel to Super Mario RPG 2: Quest For The Crystal Keys. It will be released in late 2013.
Prologue: The Dark Order
A long time ago an evil queen named Darknasmea lived. One day she fused with a Dark Star Spirit. She took over the Mushroom World and made everyone her slaves. After years of grueling work 4 great heroes rose. They sealed her sprit in a golden stone. But Darknasmea put a spell on them. If they ever put 4 special statues together they will summon the golden stone and if ever sung to with pure and dark voices the witch would rise again. When the heroes died the story stayed alive but is now dismissed as a fairy tale. But there still is a special day for the 4 heroes.
Chapter 1: The Day Of The Heroes
It was the Day Of The Heroes and Peach invites Mario to a big party at the castle. Mario arrives and meets many fans. The Mayor Of Toad Town has a surprise, 4 silver statues honoring the heroes. Before he can put all 4 on their stands a mysterious ship comes overhead. A giant mechanical claw grabs Peach and the statues. It knocks people down in the process. You see Bowser on the ship too but it doesn't look like he is behind this. Mario faints. When he wakes up a man introduces himself as Disgustavoni. He tells him those were the Z-X-Team and they are determined to pull the Silver Statues together and awaken th evil queen. He tells him to collect the 4 Silver Staues and save the worl from the Z-X-Team. So Mario sets out on a journey to save the world starting with saving the first Silver Satues in Forest Of Lakes.
Chapter 2: The Forest Of Lakes
Mario arrives in the Forest Of Lakes. He travels through the big forest and battles a big blob of slime named Boblenette. He sees Yoshi at the end. Mario asks Yoshi if he will join him. Yoshi is busy but has his nephew Joycki joins Mario. In a big lake Mario and Joycki find the 1st Silver Statue. Back in Toad Town Mario places the Silver Statue on one of the stands. Mario learns from Disgustavoni that the next Silver Statue is at Platform Beach.
Chapter 3: Platform Beach Panic
Mario and co. travel to Platform Beach. The beach is very confusing and is like a big maze. At the end it reaches te clouds. Mario meets an old enemy from the last game Mallouitoucous. After beating him Mario realizes something he is Mallow. The monster changes back into Mallow and it is revealed Mallow was brainwashed and tuned evil. Mallow joins Mario. Dangling from a cloud ahead the next Silver Statue. Mario Joycki and Mallow return to Toad Town and puts the next statue on the stand. Disgustavoni tells Mario the next statue is at Volcano Vacation Paradise
Chapter 4: Vacation Under The Volcano
Mario and co. travel to VVP an exclusive resort. At the resort Mario and co. head towards the volcano. At the foot of the volcano they meet another enemy from last game Genoaaini. After defeating Genoasini he changes into Geno Mario's old friend. Geno joins Mario's team. At the top of the volcano hanging from a rope is the 3rd Silver Statue. Back in Toad Town they put the statue in its stand. Disgustavoni tells Mario and co. the last statue is in Epicotmo Desert Ruins.
Chapter 5: Epic Adventure In Epicotmo Desert
Mario and co. arrive in Epicotmo Desertc. They roam a little bit before getting to a big sand dune. At the foot of the dune Bowser Jr joins your group wanting to save his father. At the foot of the dune a Dark Magician named Damockato battles them. They win and get the last Silver Statue. They go back to Toad Town. They put all 4 statues together. Suddenly the eyes glow and create a stone. A golden stone!
Chapter 6: Disgustavoni's True Intentions
Disgustavoni suddenly comes with Peach and Bowser and makes them both sing. The voices make the stone glow and release the evil queen Darknasmea. The Z-X-Team leader Cleanartris tries to defeat them but fails. Mario and his partners suceed in defeating them and win the game.
The game did excellent.


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Jobber Drill, 6.53mm, 140, Carbide
OSG| Zoro #: G1274113| Mfr #: 551025711
Zoro #: G1274113Mfr. Part #: 551025711
$108.95 /ea
This item ships free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G1274113 Mfr #: 551025711
|List Number: 5510
|Item: Jobber Drill Bit
|Drill Bit Material: Solid Carbide
|Drill Bit Grouping: Drill Bit
|Drill Bit Point Angle: 140 Degrees
|Drill Bit Finish: WD1
|Point Type: R Thinning
|Coolant Through: Yes
|Cutting Direction: Right Hand
|Overall Length: 109.00mm
|Decimal Equivalent: 0.2570
|Drill Bit Dimension Type: Letter
|Flute Length: 53.00mm
|Drill Bit Size: F
|Country of Origin (subject to change): Japan
Product Description
Jobber Drill Bit, Letter, Drill Bit Size F, Decimal Equivalent 0.2570, Drill Bit Point Angle 140 Degrees , Drill Bit Material Solid Carbide, Drill Bit Finish WD1, Coolant Through Yes, Grade High Performance, Overall Length 109.00mm, Cutting Direction Right Hand, Shank Dia. 0.2656 In., Flute Length 53mm, Point Type R Thinning, List Number 5510, For Deeper Hole Drilling And High Penetration Rates In Cast Aluminum, Cast Iron, Carbon Steel, Alloy Die Steel, Stainless Steel And High Heat Alloys
Restrictions and Compliance
- *2 : California Proposition 65 : This product contains a chemical known to the State of California to cause cancer.


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|Reviews for Katnip and Ginger Tea
| uss71832 chapter 1 . 2/19/2013
He actually TRUSTED something that Chance made...mistake!
| CultTickler chapter 1 . 6/24/2012
I'd take a horrible stomach flu if Chance would make me tea...
| Max Evelyn chapter 1 . 1/16/2011
oh yeah I liked that hehe :)
| Tseu Tsumi chapter 1 . 9/8/2010
Stomach viruses do indeed suck.
And I love how this little one shot went. Poor Jake. XD He is so fun to torture.
| shadewatcher chapter 1 . 9/3/2010
Heh, poor Jake. lol Funny as heck though. lol Hmm, wonder if there's a sequel where Jake finds out about all this naughtiness. lol Could be interesting. XD
And actually catnip for humans is about as calming as chamomile. _ So really not so bad.
| smounged1989 chapter 1 . 1/30/2010
Ah this was great, not much of these two that I have found.
Good work.
| Unchecked account chapter 1 . 9/30/2009
I keep reminding myself that one day I just HAVE to review a Swat Kats story, after all, they were my first, favourite darlings. And, I keep forgetting.
Back to the subject.
I read all your work (Gender Bender as well, though my inner yaoi fangirl protested. Jake into a girl was hilarious in it's own way, but...) and I had FUN. I mean, seriously. (Though Jake is almost always sick, or troubled with something.) I specifically read your stuff because...you torture Jake. I'm slightly sadistic about him, even though I like him, but for some ridiculous reason, Jake getting hurt is so much more pleasing. (Is that the right word?)
In retrospective, there are rather less Swat Kats fanfictions on the web, and even less yaoi ones. It's kinda sad, because this was an awesome cartoon, and deserves a LOT of love. It's rather depressing, because had Hanna- Barbara publicised them more, they would have been so much more famous. But it's been nearly 14 years to their production, and people have forgotten who they are.
I've asked many people about them. Not many know. And as something I watched and loved as a child, it's so depressing to find out that nobody even knows what they are. So, I'm grateful to you for writing for this fandom (I was supposed to write for it one day, but stupid Naruto sidetracked me) But still, I'm glad you do. Your woork is detailed, comprehensive and with good english, which makes it an overall AWESOME read.
| ulyferal chapter 1 . 9/25/2009
LOL! Let's hear it for home remedies! We use them in our home too. Peppermint in warm water is great for heartburn! Don't think its right though for Chance not to tell his friend about their sudden intimacy...that is going to cause discomfort in Chance's conscious over time. Great piece...loved it.


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Great Cruise!
Sail Date: June 03, 2016
Reviewed: 11 months ago
Traveled As: Couple
Room Type: Suite
Very good itinerary. At sea days were timed well. Best buffet food of all the cruises we have been on. Enjoyed all ports, especially St. Petersburg (beautiful city).
Category Ratings
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|Cabin / Stateroom
|Our cabin steward, Ruen, did a great job! We tipped him over and above the standard gratuities.
|Embarkation and Disembarkation
|Ship Quality
|Destinations and Excursions
|Onboard Activities
|Children's Programs
|Service and Staff
|Food and Dining
Port Ratings
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|Copenhagen, Denmark
|Oslo, Norway
|Warnemunde (Berlin), Germany
|Tallinn, Estonia
|St. Petersburg, Russian Federation
|Helsinki, Finland
|Nynashamn (Stockholm), Sweden
We figured out after a day or two, when the peak times were at the restaurants and the buffet, and we avoided those times. I have read many reviews that stated that crowds and waiting were a problem. With a little observation, we did not have a problem.


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Ripon Racecourse
|Official website
|Ripon, North Yorkshire
|Owned by
|The Ripon Race Company Ltd.
|Screened on
|At The Races
|Course type
|Notable Races
|Great St. Wilfrid Stakes
Ripon Racecourse is a thoroughbred horse racing venue located in Ripon, North Yorkshire, England. It has been used for horse racing for more than 300 years and is nicknamed the Garden Racecourse. The course is a right handed oval of approximately 1m5f with a 5f finishing straight and a 6f chute. The racecourse competes with three other North Yorkshire racecourses, being Thirsk, Catterick and York. The West Yorkshire racecourses, Wetherby and Pontefract are also nearby.
Notable races
- Ripon Champion Two Yrs Old Trophy
External links
|This article about a horse racing venue in the United Kingdom is a stub. You can help Wikipedia by expanding it.
Coordinates: Template:Coord/input/dms
|The above article was imported from Wikipedia.org on 08/24/2010 and may be a modified version of the Original Wikipedia article. See this article's history page for a list of revisions which may have occurred after import. This article is displayed here courtesy of the Wikimedia Foundation, Wikipedia, and its authors/contributors in compliance with the Wikimedia Terms of Use Agreement. This article was imported and is licensed under the terms of the Creative Commons Attribution/Share-Alike License 3.0 (CC-BY-SA). Clicking on photos, videos, or drawings in this article will display copyright and license information for that non-text media where available.


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Brazil posts primary surplus of 19.921 bln in January
BRASILIA Feb 28 (Reuters) - Brazil posted a primary budget surplus of 19.921 billion reais ($8.53 billion) in January, the central bank said on Friday.
The primary budget balance, which represents the public sector's excess revenue over expenditures before debt payments, was expected to be a surplus of 21.2 billion reais, according to a median forecast of 13 analysts surveyed by Reuters.
The country had a surplus of 10.4 billion reais in December.
© Thomson Reuters 2017 All rights reserved.