Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-30
2002-05-14
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S041000, C536S023100, C536S024300
Reexamination Certificate
active
06387619
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates to telomerase compositions and methods connected therewith. Particularly disclosed are genes encoding the template RNA of telomerase in
Saccharomyces cerevisiae
and various telomerase-associated proteins. Methods of using such genes and other related biological components are also provided.
B. Description of the Related Art
DNA polymerases synthesize DNA in a 5′ to 3′ direction and require a primer to initiate synthesis. These restrictions pose a problem for the complete replication of linear chromosomes (Watson, 1972; Olovnikov, 1973). In the absence of a specialized mechanism to maintain terminal sequences, multiple replication cycles would cause chromosomes to progressively shorten from their ends.
Telomeres are specialized nucleoprotein complexes that constitute the ends of eukaryotic chromosomes and protect them from degradation and end-to-end fusion (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson & Larson, 1991; Wright et al., 1992; Blackburn, 1994). When telomeres are absent, the instability of non-telomeric chromosomal ends leads to chromosome loss (Sandell & Zakian, 1993). In addition, telomeres are required for the complete replication of chromosomes (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson & Larson, 1991; Wright et al., 1992; Blackburn, 1993; 1994).
In many eukaryotes, telomeres are composed of simple tandem repeats, with the 3′-terminal strand composed of G-rich sequences (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson & Larson, 1991; Wright et al., 1992; Blackburn, 1994). Certain insights into the mechanism by which telomeric DNA is maintained has come from the identification of telomerase activity in several species of ciliates, as well as in extracts of Xenopus, mouse, and human cells (Greider & Blackburn, 1985; 1987; 1989; Zahler & Prescott, 1988; Morin, 1989; Prowse et al., 1993; Shippen-Lentz & Blackburn, 1989; Mantell & Greider, 1994).
Telomerase is a ribonucleoprotein enzyme that elongates the G-rich strand of chromosomal termini by adding telomeric repeats (Blackburn, 1993). This elongation occurs by reverse transcription of a part of the telomerase RNA component, which contains a sequence complementary to the telomere repeat. Following telomerase-catalyzed extension of the G-rich strand, the complementary DNA strand of the telomere is presumably replicated by more conventional means.
Germline cells, whose chromosomal ends must be maintained through repeated rounds of DNA replication, do not decrease their telomere length with time, presumably due to the activity of telomerase (Allsopp et al., 1992). In contrast, somatic cells appear to lack telomerase, and their telomeres shorten with multiple cell divisions (Allsopp et al., 1992; Harley et al., 1990; Hastie et al., 1990; Lindsey et al., 1991; Vaziri et al., 1993; Counter et al., 1992; Shay et al., 1993; Klingelhutz et al., 1994; Counter et al., 1994a;b).
Telomerase is believed to have a role in the process of cell senescence (de Lange, 1994; Greider, 1994; Harley et al., 1992). The repression of telomerase activity in somatic cells is likely to be important in controlling the number of times they divide. Indeed, the length of telomeres in primary fibroblasts correlates well with the number of divisions these cells can undergo before they senescence (Allsopp et al., 1992). The loss of telomeric DNA may signal to the cell the end of its replicative potential, as part of an overall mechanism by which multicellular organisms limit the proliferation of their cells.
Due to its role in controlling replication, telomerase has also recently been implicated in oncogenesis (de Lange, 1994; Greider, 1994; Harley et al., 1992). It is thought that late stage tumors probably require the reactivation of telomerase in order to avoid total loss of their telomeres and massive destabilization of their chromosomes. Immortalized cell lines produced from virally transformed cultures have active telomerase and stable telomere lengths (Counter et al., 1992; Shay et al., 1993; Klingelhutz et al., 1994; Counter et al., 1994b). Recently, telomerase activity has also been detected in human ovarian carcinoma cells (Counter et al., 1994a).
Telomerase is thus an important component of eukaryotic cells, the dysfunction of which can have significant consequences. Although present knowledge concerning telomerase is increasing, there is a marked need for individual telomerase components to be isolated and for further analytical methods to be developed. The creation of a system for manipulating telomerase in a genetically tractable eukaryotic organism would be particularly valuable.
SUMMARY OF THE INVENTION
The present invention overcomes these and other drawbacks inherent in the prior art by providing purified telomerase components and systems for isolating further components and for developing agents with the capacity to modify telomerase actions. Particular aspects of-this invention concern the isolation and uses of several telomerase-associated genes from
Saccharomyces cerevisiae
, including the telomerase RNA template gene.
In certain aspects, this invention concerns nucleic acid segments that hybridize to, or that have sequences in accordance with, SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. SEQ ID NO:1 represents a telomerase RNA template-encoding sequence, also termed TLC1; and each of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 and SEQ ID NO:23 represent sequences that encode telomerase-associated polypeptides, also termed STR sequences (STR1, STR3, STR4, STR5 and STR6, respectively).
Both the gene TLC1 (SEQ ID NO:1 and the complementary sequence, SEQ ID NO:4), and the template RNA, include a CA-rich region. The CA-rich region is represented by SEQ ID NO:3. In the RNA template, the CA-rich region is reversed transcribed to synthesize the GT-rich telomeric repeats. An example of the GT-rich telomeric sequence is represented by SEQ ID NO:2.
The present invention generally concerns non-ciliate eukaryotic telomerase components. These are represented by telomerase components from mammalian cells, including human cells, and telomerase components from other non-ciliate species. One significant contribution of this invention is the development of methods of utilizing telomerase components, which methods are functional in useful eukaryotic cells. “Useful eukaryotic cells” particularly include human cells, as these are directly relevant to the development of diagnostics and therapeutics for human use, and cells of genetically tractable eukaryotic organisms, as these are recognized to have significant value in scientific terms and, ultimately, in drug development. The preferred non-ciliate telomerase components of the invention are thus mammalian, drosophila and yeast telomerase components.
A. DNA Segments and Vectors
The invention thus provides nucleic acid segments that are characterized as nucleic acid segments that include a sequence region that consists of at least 17 contiguous nucleotides that have the same sequence as, or are complementary to, 17 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23.
The nucleic acid segments of the invention are further characterized as being of from 17 to about 10,000 nucleotides in length, which nucleic acid segments hybridize to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or the complement thereof, under standard hybridization conditions.
“Complementary” or “complement”, in terms of nucleic acid segments that are complementary to those listed above, or that hybridize to a complement of such nucleic acid segments, means that the nucleic acid sequences are capable of base-pairing to a given sequence, such as the sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, according to the standard Watson-Crick complementarity rules. That is, the larger purin
Gottschling Daniel E.
Singer Miriam S.
Arch Development
Fredman Jeffrey
Fulbright & Jaworski LLP
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