Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explae 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 stiking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublngs per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent slglcantiy fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker ofsomatic 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 ofthe donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.The cellular senescence model of aging was founded by landmark experiments of Hayflick and Moorhead (1), who firmly established that normal human fibroblasts have a finite life-span in vitro. Although much evidence supports this model (2-8), the mechanism accounting for the finite division capacity of normal somatic cells remains a mystery. Olovnikov (9, 10) suggested that the cause of cellular senescence is the gradual loss of telomeres due to the end-replication problem-i.e., the inability of DNA polymerase to completely replicate the 3' end of linear duplex DNA (11) (for review, see refs. 12 and 13).Telomeres play a critical role in chromosome structure and function. They prevent aberrant recombination (14-16) and apparently function in the attachment ofchromosome ends to the nuclear envelope (17 (31)(32)(33). These observations have led to the telomere hypothesis of cellular aging (13), in which loss of telomeres due to incomplete DNA replication and absence of telomerase provides a mitotic clock that ultimately signals cell cycle exit, limiting the replicative capacity of somatic cells. To further explore this hypothesis, we have examined the relationship between telomere length, in vivo age, and replicative capacity of fibroblasts from normal donors and subjects with the Hutchinson-Gilford syndrome of premature aging (34,35). We have also determined the relationship between telomere length in sperm DNA and donor age.MATERIALS AND MET...

The proliferative life-span of the stem cells that sustain hematopolesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD3810 phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilica cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These fiings strongly suggest that the proliferative potential of most, if not all, hematopoletic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopolesis as well as gene therapy.The requirement for primers and the undirectional 5' -3 3' nature of DNA synthesis by DNA polymerases results in incomplete replication of the terminal 3' strands of linear chromosomes (1-3). Eukaryotic chromosomes end in specialized nucleoprotein structures called telomeres (4) and in vertebrates, including humans, telomeres terminate in tan-