Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-24
2003-09-09
Prouty, Rebecca E. (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S194000, C435S069200, C435S325000, C435S029000, C536S023200
Reexamination Certificate
active
06617110
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to novel nucleic acids encoding the catalytic subunit of telomerase and related polypeptides. In particular, the present invention is directed to the catalytic subunit of human telomerase. 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 New Biol.,
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 or aging (see, e.g., Goldstein, 1990,
Science
249:1129; 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) of normal human somatic cells in vitro and in vivo.
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 93/23572 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 to 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, or a variant thereof, or a fragment thereof. In one embodiment the protein is characterized as having a defined motif that has an amino acid sequence:
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 (SEQ ID NOS:11 and 12)
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 other embodiments, 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 nucleic acid encodes a protein comprising an amino acid sequence (SEQ ID NOS:11 and 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 another embodiment, the nucleic acid has a sequence that encodes the human TRT protein. In other embodiments, the invention relates to oligonucleotides and polynucleotides sharing substantial sequence identity or complementarity with a subsequence of such nucleic acids.
In one embodiment, the invention relates to human telomerase reverse transcriptase (hTRT) protein. Thus, in one embodiment, the invention provides an isolated, substantially pure, or recombinant protein preparation of an hTRT protein, or a variant thereof, or a fragment thereof. In one embodiment, the protein is characterized by having an amino acid sequence with at least about 75% or at least about 80% sequence identity to the hTRT protein of
FIG. 17
(SEQ ID NO:2), or a variant thereof, or a fragment thereof. In a related aspect, the hTRT protein has the sequence of SEQ ID NO:2. In some embodiments, the protein has one or more telomerase activities, such as catalytic activity. In one embodiment, the hTRT protein fragment has at least 6 amino acid residues. In other embodiments, the hTRT protei
Andrews William H.
Cech Thomas R.
Chapman Karen B.
Harley Calvin B.
Lingner Joachim
Ausenhus Scott L.
Earp David J.
Geron Corporation
Prouty Rebecca E.
Schiff J. Michael
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