Molecular genetic mechanisms of life span manipulation in Caenorhabditis elegans.

Aging and a limited life span are fundamental biological realities. Recent studies have demonstrated that longevity can be manipulated and have revealed molecular mechanisms underlying longevity control in the soil nematode Caenorhabditis elegans. Signals from both neurons and the gonad appear to negatively regulate longevity. One tissue-specific signal involves an insulin-like phosphatidylinositol 3-OH kinase pathway, dependent upon the DAF-16 forkhead transcription factor. These signals regulate mechanisms determining longevity that include the OLD-1 (formerly referred to as TKR-1) receptor tyrosine kinase. Interestingly, increased resistance to environmental stress shows a strong correlation with life extension.

Increased frequency of deletions in the mitochondrial genome with age of Caenorhabditis elegans.

We have developed a long-extension-PCR strategy which amplifies approximately half of the mitochondrial genome (6.3 kb) of Caenorhabditis elegans using an individual worm as target. We analyzed three strains over their life span to assess the number of detectable deletions in the mitochondrial genome. Two of these strains are wild-type for life span while the third is mutant in the age-1 gene, approximately doubling its maximum life span. At the mean life span in wild-type strains, there was a significant difference between the frequency of deletions detected in the mitochondrial genome compared with the mean number of deletions in young animals. In addition, deletions in the mitochondrial genome occur at a significantly lower rate in age-1 mutants as compared with wild type. We cloned and identified the breakpoints of two deletions and found that one of the deletions had a direct repeat of 8 bp at the breakpoint. This is the largest single study (over 900 individual animals) characterizing the frequency of deletions in the mitochondrial genome as a function of age yet carried out.

Comparing mutants, selective breeding, and transgenics in the dissection of aging processes of Caenorhabditis elegans.

The genetic analysis of aging processes has matured in the last ten years with reports that long-lived strains of both fruit flies and nematodes have been developed. Several attempts to identify mutants in the fruit fly with increased longevity have failed and the reasons for these failures are analyzed. A major problem in obligate sexual species, such as the fruit fly, is the presence of inbreeding depression that makes the analysis of life-history traits in homozygotes very difficult. Nevertheless, several successful genetic analyses of aging in Drosophila suggest that with careful design, fruitful analysis of induced mutants affecting life span is possible. In the nematode Caenorhabditis elegans, mutations in the age-1 gene result in a life extension of some 70%; thus age-1 clearly specifies a process involved in organismic senescence. This gene maps to chromosome II, well separated from a locus (fer-15) which is responsible for a large fertility deficit in the original stocks. There is no trade-off between either rate of development or fertility versus life span associated with the age-1 mutation. Transgenic analyses confirm that the fertility deficit can be corrected by a wild-type fer-15 transformant (transgene); however, the life span of these transformed stocks is affected by the transgenic array in an unpredictable fashion. The molecular nature of the age-1 gene remains unknown and we continue in our efforts to clone the gene.

Transient response to chemotactic stimuli in Escherichia coli.

We have followed by eye and with the tracking microscope the rotational behavior of E. coli tethered to coverslips by their flagella. The cells change their directions of rotation at random, on the average about once a second. When an attractant is added or a repellent is subtracted, they spin clockwise (as viewed through the coverslip, i.e., along the flagellum toward the body) for many seconds, then counter-clockwise for many seconds, and then gradually resume their normal mode of behavior. The time interval between the onset of the stimulus and the clockwise to counter-clockwise transitiion is a linear function of the change in receptor occupancy. The cells adapt slowly at a constant rate to the addition of an attractant or the subtraction of a repellent. They adapt rapidly to the subtraction of an attractant or the addition of a repellent. Responses to mixed stimuli can be analyzed in terms of one equivalent stimulus.