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C W Greider

Publications and source records attributed to C W Greider.

At least 55 records · Page 3Linked to original sources
PubMed1995

Developmental and tissue-specific regulation of mouse telomerase and telomere length.

Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.

Animals↗
PubMed1994

Telomerase activity in germline and embryonic cells of Xenopus.

Telomerase is a ribonucleoprotein which synthesizes telomere repeats onto chromosome ends. Telomerase activity is involved in telomere length maintenance. We used Xenopus laevis as a model system to study the expression of telomerase activity in germline cells and during early development. We identified a non-processive telomerase activity in manually dissected nuclei of Xenopus stage VI oocytes. Telomerase activity was detected throughout oogenesis and embryogenesis. Telomerase was active in both S and M phase cell cycle extracts, suggesting that telomerase activity is not regulated with chromosomal DNA replication.

Animals↗
PubMed1994

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

Animals↗
PubMed1994

Mammalian telomere dynamics: healing, fragmentation shortening and stabilization.

Telomeres are essential for stable chromosome maintenance. The simple G-rich sequence motif d(TTAGGG)n is all that is required in cis for telomere function in mammalian cells, as in other eukaryotes. Using this fact, telomeres have been used to specifically fragment mammalian chromosomes to dissect their structure and function. Telomere length maintenance is altered in cancer cells. Trans-acting factors, such as telomerase and telomere-binding proteins, may determine telomere function in both normal and cancer cells. Current experiments are aimed at understanding the role of telomerase and telomere-binding proteins in cellular senescence and immortalization.

Animals↗
PubMed1993

Identification of a nonprocessive telomerase activity from mouse cells.

Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.

Animals↗
PubMed1993

Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation.

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosomes, maintaining telomere length and stabilizing chromosome ends. In vitro, telomerase from the ciliate Tetrahymena elongates single-stranded, guanosine-rich DNA primers by adding repeats of the Tetrahymena telomeric sequence, dT2G4. We have identified two activities of Tetrahymena telomerase in addition to the previously described processive elongation reaction: a 3'-5' nucleolytic cleavage of primer or product DNA and a nonprocessive mode of elongation. The nucleolytic cleavage activity removed residues not conforming to the telomeric repeat sequence from a primer 3' end, eliminating mismatch between DNA primer and RNA template sequences. Template-matched residues were also cleaved from primer or product DNA. Specific primer lengths, sequences, and concentrations stimulated cleavage and processive or nonprocessive elongation differentially. These newly identified activities suggest that telomerase may catalyze a range of telomere synthesis and repair functions and suggest mechanistic similarities between telomerase and RNA polymerase enzymes. On the basis of our results, we propose a model for telomerase primer binding, cleavage, and elongation.

Animals↗
PubMed1992

Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.

Base Sequence↗
PubMed1992

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.

Adult↗
PubMed1992

Tetrahymena telomerase RNA levels increase during macronuclear development.

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2- to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells.

Animals↗
PubMed1992

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Cell Transformation, Neoplastic↗
PubMed1991

Telomerase primer specificity and chromosome healing.

Chromosome healing by de novo telomere addition at nontelomeric sites has been well characterized in several organisms. The Tetrahymena telomerase ribonucleoprotein uses an internal RNA template to catalyse d(TTGGGG)n telomere addition to the 3' end of telomeric sequence in vitro and in vivo. Studies of telomerase RNA indicated that hybridization of the RNA template region, 5'-CAACCCCAA-3', to the 3' end of single-stranded telomeric oligonucleotides might be important for primer recognition and utilization. The apparent requirement of telomerase for pre-existing telomeric sequence has raised questions regarding its role in chromosome healing. We report here that Tetrahymena telomerase can specifically elongate single-stranded DNA oligonucleotides whose termini are not complementary to the RNA template sequence 5'-CAACCCCAA-3'. These data suggest that telomerase may be able to heal chromosomes directly in vivo.

Animals↗
PubMed1991

Telomeres.

Telomeres are essential for chromosome stability and replication. Maintaining a balance between telomere shortening and lengthening is essential for cell viability. Recent work on telomeres from yeast, Drosophila and mammals, and on telomerase has provided insight into the mechanisms of both the shortening and lengthening processes.

Aging↗
PubMed1991

Telomerase is processive.

Telomerase synthesizes tandem repeats of the sequence d(TTGGGG) onto input d(TTGGGG)n primer oligonucleotides (C. W. Greider and E. H. Blackburn, Cell 43:405-413). An intrinsic RNA component of the enzyme provides the template for d(TTGGGG)n repeat synthesis [C. W. Greider and E. H. Blackburn, Nature (London) 337:331-337, 1989; G.-L. Lu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature (London) 344:126-132, 1990]. In a typical reaction, products greater than 2,000 nucleotides were synthesized in 60 min. Dilution and primer challenge experiments showed that these long products were synthesized processively. The apparent processivity was not due to a higher affinity of the enzyme for long d(TTGGGG) products over the shorter competitors. The degree of processivity was quantitated; telomerase synthesized approximately 520 nucleotides before half of the enzyme had dissociated. After dissociating, telomerase reinitiated d(TTGGGG)n synthesis on new primer oligonucleotides. The products from a telomerase reaction have a characteristic 6-nucleotide banding pattern (C. W. Greider and E. H. Blackburn, Cell 51:887-898, 1987). A strong pause in the reaction occurs after the addition of the first G in the sequence d(TTGGGG). Both the processivity and the banding pattern analysis imply that in the elongation mechanism there must be a translocation step after the 9 nucleotides of internal template RNA have been copied to the extreme 5' end.

Animals↗
PubMed1990

Telomeres shorten during ageing of human fibroblasts.

The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.

Aging↗