Telomeres: structure of a chromosome's aglet.
(9/4834)
Telomeres impart stability on linear eukaryotic chromosomes by acting as caps, preventing chromosomes from fusing together or being degraded. The structure of a telomere end binding protein in a complex with DNA provides the first molecular view of chromosome capping. (+info)
Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase.
(10/4834)
Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase. (+info)
A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors.
(11/4834)
Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4. Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background. (+info)
Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein.
(12/4834)
The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomaska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance. (+info)
Longevity, stress response, and cancer in aging telomerase-deficient mice.
(13/4834)
Telomere maintenance is thought to play a role in signaling cellular senescence; however, a link with organismal aging processes has not been established. The telomerase null mouse provides an opportunity to understand the effects associated with critical telomere shortening at the organismal level. We studied a variety of physiological processes in an aging cohort of mTR-/- mice. Loss of telomere function did not elicit a full spectrum of classical pathophysiological symptoms of aging. However, age-dependent telomere shortening and accompanying genetic instability were associated with shortened life span as well as a reduced capacity to respond to stresses such as wound healing and hematopoietic ablation. In addition, we found an increased incidence of spontaneous malignancies. These findings demonstrate a critical role for telomere length in the overall fitness, reserve, and well being of the aging organism. (+info)
The boundaries of the silenced HMR domain in Saccharomyces cerevisiae.
(14/4834)
The chromosomes of eukaryotes are organized into structurally and functionally discrete domains that provide a mechanism to compact the DNA as well as delineate independent units of gene activity. It is believed that insulator/boundary elements separate these domains. Here we report the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae. Deletion of these boundary elements led to the spread of silenced chromatin, whereas the ectopic insertion of these elements between a silencer and a promoter blocked the repressive effects of the silencer on that promoter at HMR and at telomeres. Sequence analysis indicated that the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function. (+info)
Postthymic development of CD28-CD8+ T cell subset: age-associated expansion and shift from memory to naive phenotype.
(15/4834)
During human aging, one of the major changes in the T cell repertoire is a dramatic expansion of T cells with the atypical CD28-CD8+ phenotype. In this study, we show that this increase is a consequence not only of an expansion in the CD28-CD8+ population but also of a decrease in the number of CD28+CD8+ T cells. The decrease in circulating CD28+CD8+ T cells is dramatically accelerated after the age of 50 and is not accompanied by an equivalent reduction in the CD28+CD8+ subset. Our findings confirm that aging leads to an accumulation of CD45RO+ T cells within the CD28+CD8+ subset as previously observed. Surprisingly, we found an increase in CD45RA+ expression with age in the CD28-CD8+ subset. Immune-phenotyping for activation markers, measurement of telomere DNA content, and cytokine production analysis indicate that the large majority of CD28-CD8+ T cells are Ag-experienced, despite their CD45RA+ phenotype. Our study further demonstrates that the poor proliferative response displayed by CD28-CD8+ T cells is not a consequence of telomere shortening. Also, analysis of cytokine production at the single cell level revealed that the proportions of IFN-gamma +, IL-4+, and IL-10+ T cells are considerably higher among the CD28-CD8+ than the CD28+CD8+ subset. In summary, these data explain the presence of CD45RA+ T cells in the elderly, shed light on the phylogenetic origin of CD28-CD8+ T cells, and suggest a role for these cells in the immune senescence process. (+info)
Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening.
(16/4834)
Human fibroblasts whose lifespan in culture has been extended by expression of a viral oncogene eventually undergo a growth crisis marked by failure to proliferate. It has been proposed that telomere shortening in these cells is the property that limits their proliferation. Here we report that ectopic expression of the wild-type reverse transcriptase protein (hTERT) of human telomerase averts crisis, at the same time reducing the frequency of dicentric and abnormal chromosomes. Surprisingly, as the resulting immortalized cells containing active telomerase continue to proliferate, their telomeres continue to shorten to mean lengths below those in control cells that enter crisis. These results provide evidence for a protective function of human telomerase that allows cell proliferation without requiring net lengthening of telomeres. (+info)