Intragenic complementation, hybrid enzyme formation and dominance in diploid cells of Saccharomyces cerevisiae.

Abstract

1. Intragenic complementation was studied using isoleucine requiring mutants of the is1-structural gene for threonine dehydratase in Saccharomyces cerevisiae. Twenty three mutants were induced with 1-nitrosoimidazolidone-2, four more mutants had been induced with ultraviolet light by other authors. Among the 23 chemical induced mutants there were 5 osmotic remedial mutants which could grow without isoleucine in the presence of 1M KCl, and 3 temperature sensitive mutants which expressed an isoleucine requirement only at elevated temperature. All mutants were shown to be allelic to is1. 2. Intragenic complementation was studied in all possible combinations using isoleucine independent growth as the criterion. All osmotic remedial, temperature sensitive and 6 more nonconditional mutants participated in intragenic complementation. 3. Complementation maps were constructed from data obtained at 25 and 35° C incubation temperature omitting temperature sensitive mutants. Both maps were linear but the 35° C map comprised six complementing groups whereas only four were observed at 25° C. 4. Negative complementation was studied using the three temperature sensitive mutants. It was indicated by a lack of growth at the permissive temperature (25° C) of the diploids formed by crossing temperature sensitive mutants to nonconditional mutants. Three of the five osmotic remedial mutants and seven nonconditional, non-complementing mutants showed negative complementation. 5. Enzyme assays with crude extracts of a representative sample of isoleucine requiring mutants revealed a complete absence of threonine dehydratase activity. 6. Crude extracts of well complementing mutant x mutant heteroallelic diploids were assayed for threonine dehydratase activity. Specific activities were usually very low or there was no activity to be detected at all. In some cases with a sufficiently high activity a further characterization of the enzyme was possible. The most salient feature with some complementing diploids was that feedback inhibition by isoleucine was absent under conditions where wild type enzyme was completely inhibited. 7. Threonine dehydratase activity was also investigated in mutant x wild type heterozygotes. Up to 24% of the total activity in the crude extracts of such heterozygotes was found to be resistant to feedback inhibition. This resistant enzyme fraction differed from wild type by an increased Michaelis constant and altered pH dependence. 8. Intragenic complementation is considered to result from the formation of hybrid enzymes composed of the two different types of subunit polypeptide chains coded for by the two different alleles present in the diploid nucleus. Such hybrid enzymes have properties different from the corresponding two homogeneous aggregates as shown by feedback resistant threonine dehydratase activity in heterozygous cells. 9. True dominance of an active over an inactive allele is considered to be a special case restricted to genes coding for a monomeric enzyme or to mutant alleles which code for no or only a drastically altered polypeptide chain. The normal situation is considered to be an intermediate state with an appreciable amount of hybrid enzyme formation. Hybrid enzymes can cause the appearance of new properties like feedback resistant threonine dehydratase activity. 10. More general problems like heterosis and the role of mutagenesis in diploid organisms are discussed in the light of intragenic complementation and hybrid enzyme formation.