Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1997-08-14
2002-11-05
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S320100, C435S069100, C536S023200, C424S094100
Reexamination Certificate
active
06475789
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to novel nucleic acids and polypeptides encoding the catalytic subunit of telomerase. In particular, the present invention is directed to the catalytic subunit of telomerases from
Euplotes aediculatus, Schizosaccharomyces pombe, Tetrahymena thermophila
, and humans. The invention provides methods and compositions relating to medicine, molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology.
BACKGROUND OF THE INVENTION
The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section is not to be construed as an admission of prior invention.
It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972
, Nature NewBiol
., 239:197; Olovnikov, 1973
, J. Theor. Biol
., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5′ to 3′. When the RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence (cell aging) of normal human somatic cells in vitro (see, e.g., Goldstein, 1990
, Science
249:1129) and in vivo (see, e.g., Martin et al., 1979
, Lab. Invest
. 23:86; Goldstein et al., 1969
, Proc. Natl. Acad. Sci. USA
64:155; and Schneider and Mitsui, 1976
, Proc. Natl. Acad. Sci. USA
, 73:3584).
The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity). Moreover, the ability of a cell to maintain (or increase) telomere length may allow a cell to escape senescence, i.e., to become immortal.
The structure of telomeres and telomeric DNA has been investigated in numerous systems (see, e.g, Harley and Villeponteau, 1995
, Curr. Opin. Genet. Dev
. 5:249). In most organisms, telomeric DNA consists of a tandem array of very simple sequences; in humans and other vertebrates telomeric DNA consists of hundreds to thousands of tandem repeats of the sequence TTAGGG. Methods for determining and modulating telomere length in cells are described in PCT Publications WO 95/13382 and WO 96/41016.
The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomere repeat DNA synthesis (Morin, 1997
, Eur. J. Cancer
33:750; Yu et al., 1990
, Nature
344:126; Singer and Gottschling, 1994
, Science
266:404; Autexier and Greider, 1994
, Genes Develop
., 8:563; Gilley et al., 1995
, Genes Develop
., 9:2214; McEachern and Blackburn, 1995
, Nature
367:403; Blackburn, 1992
, Ann. Rev. Biochem
., 61:113;. Greider, 1996
, Ann. Rev. Biochem
., 65:337). The RNA components of human and other telomerases have been cloned and characterized (see, PCT Publication WO 96/01835 and Feng et al., 1995
, Science
269:1236). However, the characterization of the protein components of telomerase has been difficult. In part, this is because it has proved difficult to purify the telomerase RNP, which is present in extremely low levels in cells in which it is expressed. For example, it has been estimated that human cells known to express high levels of telomerase activity may have only about one hundred molecules of the enzyme per cell.
Consistent with the relationship of telomeres and telomerase with the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989
, Cell
59: 521; Shay and Bacchetti 1997
, Eur. J. Cancer
33:787; Kim et al., 1994
, Science
266:2011; Counter et al., 1992
, EMBO J
. 11:1921; Counter et al., 1994
, Proc. Natl. Acad Sci. U.S.A
. 91, 2900; Counter et al., 1994
, J. Virol
. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997
, Hum. Pathol
. 28:416). Telomerase activity has also been detected in human germ cells, proliferating stem or progenitor cells, and activated lymphocytes. In somatic stem or progenitor cells, and in activated lymphocytes, telomerase activity is typically either very low or only transiently expressed (see, Chiu et al., 1996
, Stem Cells
14:239; Bodnar et al., 1996
, Exp. Cell Res
. 228:58; Taylor et al., 1996
, J. Invest. Dermatology
106: 759).
Human telomerase is an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer. Methods for diagnosing and treating cancer and other telomerase-related diseases in humans are described in U.S. Pat. Nos. 5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumor progression by monitoring telomerase are described in U.S. Pat. No. 5,639,613. The discovery and characterization of the catalytic protein subunit of human telomerase would provide additional useful assays for telomerase and for disease diagnosis and therapy. Moreover, cloning and determination of the primary sequence of the catalytic protein subunit would allow more effective therapies for human cancers and other diseases related to cell proliferative capacity and senescence.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an isolated, substantially pure, or recombinant protein preparation of a telomerase reverse transcriptase protein. In one embodiment the protein has an amino acid sequence (SEQ ID NOS: 11-12):
Trp-R
1
-X
7
-R
1
-R
1
-R
2
-X-Phe-Phe-Tyr-X-Thr-Glu-X
8-9
-R
3
-R
3
-Arg-R
4
-X
2
-Trp
where X is any amino acid and a subscript refers to the number of consecutive residues, R
1
is leucine or isoleucine, R
2
is glutamine or arginine, R
3
is phenylalanine or tyrosine, and R
4
is lysine or histidine. In one embodiment the protein has a sequence of human TRT. In another embodiment, the invention relates to peptides and polypeptides sharing substantial sequence identity with a subsequence of such proteins.
In a related embodiment the invention provides an isolated, substantially pure or recombinant nucleic acid that encodes a telomerase reverse transcriptase protein. In one embodiment the protein has an amino acid sequence (SEQ ID NO: 11-12):
Trp-R
1
-X
7
-R
1
-R
1
-R
2
-X-Phe-Phe-Tyr-X-Thr-Glu-X
8-9
-R
3
-R
3
-Arg-R
4
-X
2
-Trp. In one embodiment the nucleic acid has a sequence of human TRT. In other embodiment, the invention relates to oligonucleotides and polynucleotides sharing substantial sequence identity with a subsequence of such nucleic acids.
In one aspect the invention provides isolated human telomerase comprising human telomerase reverse transcriptase (hTRT). In one embodiment the hTRT is associated with human telomerase RNA (hTR).
In one aspect the invention provides a method of detecting a human telomerase reverse transcriptase (hTRT) gene product in a biological sample by contacting the biological sample with a probe that specifically binds the gene product, wherein the probe and the gene product form a complex, and detecting the complex where the presence of the complex is correlated with the presence of the hTRT gene product in the biological sample. The gene product may be RNA, DNA or a polypeptide. Examples of probes in that may be used for detection include, but are not limited to, nucleic acids and antibodies.
In one embodiment the gene product is a nucleic acid which is detected by amplifying the gene
Andrews William H.
Cech Thomas R.
Chapman Karen B.
Harley Calvin B.
Lingner Joachim
Andres Janet L.
Ausenhus Scott L.
Earp David J.
Eyler Yvonne
Schiff J. Michael
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