Substrate Stabilization of Enzyme-Forming Capacity During the Segregation of a Heterozygote

Abstract

A character in yeast for slow adaptation in the synthesis of galactozymase is detd. by an allele, gs, which is recessive to normal adaptation (G). When gs cells are grown in the presence of galactose, most of the clones are small and negative, but approx. 1 in 1000 cells yield large positive clones, which remain positive in the presence of galactose but revert to negative between the 6th and 7th generations in the absence of galactose. All G-cells, both adapted and unadapted, yield positive clones on the test plates. In order to determine the mechanism of enzyme synthesis in the two genotypes, adapted and unadapted cytoplasms derived from G-cells were introduced into different gs-cells. Strain 55, which was used, was derived from crosses between S. cerevisiae and S. chevalieri, and was of the genetic constitution Ggs Mm(maltose fermentation) Dd(diploidization). Single spores from ascus dissections were grown in a galactose-glucose broth in a Van Tiegham cell. The resulting micro-clones (500 to 1000 cells) were spread on Eosine Methylene Blue-purified galactose (EMB-PG) agar test plates and the colonies scored as positive or negative. In the control series the cells did not come in contact with galactose until after segregation of the haploid spores, whereas in the experimental series cells were grown in galactose for 24 hrs. before sporulation and subsequently maintained in the presence of galactose. The 8 control asci gave 2 + 2- as expected, whereas in the experimental series 7 asci gave 4 + : 0- and 1 ascus gave 3 + : 1-. For a reversion growth test in the absence of galactose 2 clones were chosen at random from each positive test plate and grown separately in glucose broth for 15 generations, then retested on EMB-PG plates. In the control series none of the positives reverted, but in the experimental series 2 out of 4 spores in the 4 + : 0- asci and 1 out of 3 spores in the 3 + : 1- ascus reverted to negative, restoring the Mendelian ratio and indicating the non-Mendelian nature of the excess positives. For 68 asci all 4 spores were scored and tested for reversion. 34 out of 38 control asci yielded 2 + : 2- spores. 3 exceptional asci were found to have resulted from self-diploidization. In the experimental series, of the 30 asci, 1 gave 2 + : 2- spores, 24 gave 4 + : 0- and 5 gave 3 + : 1- spores. All of the exceptional asci reverted to 2 + : 2- spores except for 2 which were considered self-diploids. In a test run on single spore scoring without regard for tetrad analysis, the control series gave 233 + : 237-, and the experimental series gave 175 + : 13- which reverted to 98 + : 90-. A homozygote gsgs was put through the entire experimental treatment in contact with galactose, but none of the 264 spores recovered were positive, indicating that the dominant allele, G, was necessary for enzyme synthesis. The results of all these experiments are explained by cytoplasmic elements necessary for enzyme formation and produced in the presence of substrate (galactose) and the dominant allele G. Some evidence was obtained that unadapted Ggs cells contained some of the cytoplasmic units, which suggested that they could be formed in the absence of substrate.