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.

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...

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WHI1‐1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose‐dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over‐expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C‐terminal third of the wild‐type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1‐1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.

cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.