Species comparison of protein synthesis accuracy.

The average accuracy of protein synthesis in reticulocytes from several mammalian species does not correlate with longevity potential from 13 to 90 years. Isoleucine incorporation into highly purified hemoglobin chains which contain no genetically coded isoleucine was used as a direct test of protein synthesis accuracy. Since isoleucine can be incorporated into these molecules by mutations in a few cells as well as errors in most cells, the constant level of isoleucine substitution may also show that the mutation rates are not dramatically different among these species. Isoleucine substitutions in hemoglobin can be used to estimate mutations only above the level of errors, which may be as low as 1/1,000,000, but the probability of seeing a few mutant clones at any time is dependent on the number of stem cells producing reticulocytes. The number of stem cells being expressed is a reflection of the number of cell divisions per clone. If the number of cell divisions per clone is 30 or less, then isoleucine substitutions would increase when the mutation accumulation rose above 30 per million for the mutation to isoleucine at any position in the alpha or beta chain.

Amino acid substitution: its use in detection and analysis of genetic variants.

Techniques of chemical analysis, amino acid sequencing and autoradiography are being used to study the frequency of incorporation of normally noncoded amino acids into hemoglobins and seminal fluid proteins. We are studying, by the sequencing of radiolabeled proteins followed by the recovery of [3H]isoleucine phenylthiohydantoin by high-performance liquid chromatography, the frequency at which normally noncoded isoleucine is incorporated into hemoglobin because of base-substitution mutations versus translational errors. Irradiation increases the isoleucine content of human hemoglobin and the frequency of substitution of isoleucine for specific amino acids in rabbit hemoglobin. Studies to date indicate that these techniques have been developed sufficiently for initial analysis of the potential of drugs and environmental pollutants to induce base-substitution mutations in mammalian somatic cells.

Spontaneous mutations balance reproductive selective advantage and genetically determine longevity.

The theory is presented that the genetic potential for longevity of a given species in a protected environment is determined by the rate of predation in the wild and by the frequency of spontaneous mutations. A common genetically determined lifespan representative of rare, long-lived individuals in the wild results from the slight selective advantage conferred by genotypes which do not cause early death. The selective advantage of reproductive rare survivors is limited by the rate of spontaneous mutations from a common potential longevity to those which limit lifespan to shorter times. The equilibrium frequency of life-limiting genes increases exponentially with increasing age when the rate of mutations is uniform for different degrees of life-shortening.

A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): development and definition of the system.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.