S. Michal Jazwinski

Replication control and cellular life span

Cell proliferation involves both control of progress through the current cell cycle and coordination of successive cell cycles. We have focused our attention on the events that trigger traversal of the G1/S boundary of the cell cycle. A protein kinase activity was found in preparations of the DNA-replicative complex from the budding yeast Saccharomyces cerevisiae. The activity phosphorylated only a few of the proteins present in the replicative fraction, and it displayed a marked preference for a 48-kDa polypeptide. Most importantly, the protein kinase activity was heat-sensitive in replicative fractions from cdc7 cells, a mutant that arrests at the G1/S boundary at restrictive temperature. The results suggest that phosphorylation of components of the replication machinery may play a role in control of initiation of DNA replication during the cell cycle. We have also begun an analysis of cellular aging in yeast, as a means of addressing the problem of coordination of successive cell cycles. Yeast cells have a finite life span defined by reproductive capacity. With age, the generation time of yeast cells lengthened. The cell cycle of the daughter cell was under the control of the mother. This control was transient, and the daughter cell began dividing at the rate characteristic of its own age within three divisions of its birth. This suggests that the senescent phenotype, as manifested by lengthened generation time, is a dominant feature in yeast cells, and that it is determined by a diffusible cytoplasmic molecule(s) that undergoes turnover in young cells. In a search for this putative senescence factor(s), we are cloning genes that differentially expressed during the yeast life span. Several such genes have been isolated and partially characterized. Our goals are to determine whether the expression of one or more of these genes is casually associated with cell longevity. We propose the Cell Spiral model to describe the relationship between the cell cycle and cellular aging.

Epigenetic stratification: the role of individual change in the biological aging process

Aging is a complex process. It consists of a diverse assortment of seemingly random manifestations that occur in the individual, the mutual relationship and impact on mortality of which is frequently obscure. We derive a simple equation to model the aging process based on scale invariant and increasing change. The solution to this equation indicates that this change itself, irrespective of its quality, is the cause and not simply the effect of aging. This model establishes loss of homeostasis as a fundamental feature of aging. The model is deterministic, but it supports the stochastic nature of age changes. Paradoxically, this model states that a sufficient augmentation of aging processes results in a lack of aging. Experimental evidence in support of this model is presented that spans the levels of population mortality rates, cellular spatial organization, and gene dysregulation.

Differential response to UV stress and DNA damage during the yeast replicative life span

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.

The RAS genes: a homeostatic device in Saccharomyces cerevisiae longevity<

The genetic analysis of the yeast replicative life span has revealed the importance of metabolic control and resistance to stress. It has also illuminated the pivotal role in determining longevity that the RAS genes play by the maintenance of homeostasis. This role appears to be performed by the coordination of a variety of cellular processes. Metabolic control seems to occupy a central position among these cellular processes that include stress resistance. Some of the features of metabolic control in yeast resemble the effects of the daf pathway for adult longevity in Caenorhabditis elegans and the metabolic consequences of selection for extended longevity in Drosophila melanogaster, as well as some of the features of caloric restriction in mammals. The distinction between dividing and nondividing cells is proposed to be less important for the aging process than generally believed because these cell types are part of a metabolic continuum in which the total metabolic capacity determines life span. As a consequence, the study of yeast aging may be helpful in understanding processes occurring in the aging brain.

Evidence for the involvement of a single major species of replicative complex in DNA synthesis from two diverse nuclear replicons in yeast.

The activity that replicates the 2-micron yeast DNA plasmid in vitro can be isolated as a high-molecular weight (approximately 2 X 10(6)) fraction, which possesses many of the features of a multiprotein replicative complex. This fraction also initiates DNA synthesis at the yeast chromosomal replicator ARS1 raising the question whether the preparations discriminate between origins. It was determined that the binding of replicative complex to plasmids containing either 2-microns or ARS1 origins of replication was indistinguishable. The preparations also showed no preference among them for replication. In addition, the DNAs competed with each other to the same extent for binding of replicative complex. These results suggest that these two origins share one major species of replicative complex.