Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-03-16
2004-07-27
Fredman, Jeffrey (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S455000, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06767719
ABSTRACT:
FIELD OF THE INVENTION
The subject matter of this application provides novel recombinant telomerase enzyme genes and proteins and relates to the cloning and characterization of the catalytic protein component of mouse telomerase enzyme, referred to as
m
ouse
t
elomerase
r
everse
t
ranscriptase (“mTERT”).
This invention pertains generally to cell proliferation and aging, including the fields of age-related diseases, such as cancer and cell biology. In particular, this invention pertains to the discovery of a novel mTERT enzyme proteins and nucleic acids, and methods for isolating and expressing by recombinant means these nucleic acids and proteins. The invention provides antibodies specifically reactive with mTERT. The invention also pertains to methods of screening for novel mTERT activity modulators. The invention also includes means of mortalizing cells, creating indefinitely proliferating cells and immortalizing cells, including normal, diploid cells, using the novel reagents, proteins, nucleic acids, enzymes and methods of the invention.
BACKGROUND OF THE INVENTION
The following discussion is intended to provide general information regarding the field of the present invention. The citation of various references is not to be construed as an admission of prior invention.
Telomeres, the protein-DNA structures physically located on the ends of chromosomes in eukaryotic organisms, are required for chromosome stability and are involved in chromosomal organization within the nucleus (Zakian (1995)
Science
270:1601, Blackburn (1978)
J. Mol. Biol
., 120:33, Oka (1980)
Gene
10:301, Klobutcher (1981)
Proc. Natl. Acad. Sci. USA
78:3015). Telomeres are believed to be essential in most eukaryotes, as they allow cells to distinguish intact from broken chromosomes, protect chromosomes from degradation, and act as substrates for replication. Telomere loss, i.e., inability to maintain telomere structure, is associated with normal human cellular development, including cell aging and cellular senescence. Telomere gain, i.e., the ability to maintain telomere structure in cells, is associated with chromosomal changes and cancer.
Telomeres are generally replicated in a complex, cell cycle and developmentally regulated manner by a “ribonucleoprotein telomerase enzyme complex.” The telomerase reverse transcriptase enzyme is a telomere-specific RNA-dependent DNA polymerase comprising a telomerase reverse transcriptase (TERT) protein and an RNA component. Telomerase enzyme uses its RNA component to specify the addition of telomeric DNA repeat sequences to chromosomal ends (U.S. Pat. No. 5,583,016; Villeponteau (1996)
Cell and Develop. Biol
. 7:15-21). In addition to the template RNA component, other proteins have been found to be associated with TRT. For example, telomerase-associated proteins called p80 and p95 were found in
Tetrahymena
(Collins (1995)
Cell
81:677). Homologs of the p80 protein have been found in humans, rats and mice. Neither enzymatic activity nor amino acid motifs typically associated with RNA-dependent DNA polymerases have been found to be associated with these proteins (Harrington (1997)
Science
275:973-977). In contrast, mutational analysis and reconstitution in vitro have shown the TERT proteins contain the catalytic moieties of telomerase (Lingner (1997)
Science
276:561-567; Weinrich (1997)
Nature Genetics
17:498-502). Various structural proteins that interact with telomeric DNA that are distinct from the protein components of TRT have also been described. In mammals, most of the simple repeated telomeric DNA is packaged in closely spaced nucleosomes (Makarov (1993)
Cell
73:775, Tommerup (1994)
Mol. Cell. Biol
. 14:5777). However, the telomeric repeats located at the very ends of the human chromosomes appear to be in a non-nucleosomal structure that has been termed the telosome.
Telomeric DNA can consist of a variety of different structures. Typically, telomeres are tandem arrays of very simple sequences, such as simple repetitive sequences rich in G residues, in the strand that runs 5′ to 3′ toward the chromosomal end. In humans, the telomere repeat sequence is 5′-TTAGGG-3′ (SEQ ID NO:7). In contrast, telomeric DNA in Tetrahymena is comprised of repeats of the sequence T
2
G
4
, while in Oxytricha, the repeat sequence is T
4
G
4
(Zakian (1995)
Science
270:1601; Lingner (1994)
Genes Develop
. 8:1984). Heterogenous telomeric sequences have been reported in some organisms, such as the repeat sequence TG
1-3
in Saccharomyces. The repeated telomeric sequence in other organisms is much longer, such as the 25 base pair repeat sequence of
Kluyveromyces lactis
. Furthermore, telomeric structure can be completely different in other organisms. For example, the telomeres of Drosophila are comprised of a transposable element (Biessman (1990)
Cell
61:663, Sheen (1994)
Proc. Natl. Acad. Sci. USA
91:12510).
In most organisms, the size of the telomere fluctuates. For example, the amount of telomeric DNA at individual yeast telomeres in a wild-type strain may range from approximately 200 to 400 bp, with this amount of DNA increasing and decreasing stochastically (Shampay (1988)
Proc. Natl. Acad. Sci. USA
85:534). Heterogeneity and spontaneous changes in telomere length may reflect a complex balance between the processes involved in degradation and lengthening of telomeric tracts. In addition, genetic, nutritional and other factors may cause increases or decreases in telomeric length (Lustig (1986)
Proc. Natl. Acad. Sci. USA
83:1398, Sandell (1994)
Cell
91:12061).
Telomeres are not maintained via conventional replicative processes. Complete replication of the ends of linear eukaryotic chromosomes presents special problems for conventional methods of DNA replication. Conventional DNA polymerases cannot begin DNA synthesis de novo; rather, they require RNA primers that are later removed during replication. In the case of telomeres, removal of the RNA primer from the lagging-strand end would necessarily leave a 5′-terminal gap, resulting in the loss of sequence from the leading strand if the daughter telomere was subsequently blunt-ended (Watson, (1972)
Nature New Biol
. 239:197, Olovnikov (1973)
J. Theor. Biol
., 41:181).
While conventional DNA polymerases cannot accurately reproduce chromosomal DNA ends, specialized factors exist to ensure their complete replication. The telomerase enzyme is a key component in this process. In vivo, telomerase enzyme is assembled as a ribonucleoprotein (RNP) enzyme complex. It is an RNA-dependent DNA polymerase that uses a portion of its internal RNA moiety as a template for telomere repeat DNA synthesis (Yu (1990)
Nature
344:126; Singer (1994)
Science
266:404; Autexier (1994)
Genes Develop
. 8:563; Gilley (1995)
Genes Develop
. 9:2214; McEachern (1995)
Nature
367:403; Blackburn (1992)
Ann. Rev. Biochem
. 61:113; Greider (1996)
Ann. Rev. Became
. 65:337). A combination of factors, including telomerase processivity, frequency of action at individual telomeres, and the rate of degradation of telomeric DNA, contribute to the size of the telomeres (i.e., whether they are lengthened, shortened, or maintained at a certain size). In vitro, telomerases may be extremely processive; for example, Tetrahymena telomerase can add an average of approximately 500 bases to the G strand primer before dissociation of the enzyme (Greider (1991)
Mol. Cell. Biol
., 11:4572).
Telomere replication is regulated both by developmental and cell cycle factors. Telomere replication may play a signaling role in the cell cycle. For example, certain DNA structures or DNA-protein complex formations may act as a checkpoint to indicate that chromosomal replication has been completed (Wellinger (1993)
Mol. Cell. Biol
. 13:4057). Telomere length is also believed to serve as a mitotic clock, which serves to limit the replication potential of cells in vivo and in vitro.
In humans, telomerase activity is not detectable in most somatic tissues. Cell that express either no or only low amounts of telomerase, such as so
Allsopp Richard
DePinho Ronald A.
Greenberg Roger A.
Morin Gregg B.
Earp David J.
Fredman Jeffrey
Geron Corporation
Kaushal Sumesh
Schiff J. Michael
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