Fusion of protoplasts prepared from haploid strains of Saccharomyces yeasts having identical mating type was induced with the aid of polyethylene glycol. Stable fusion products were isolated by complementation of the auxotrophic markers. Of 64 isolates derived by protoplast fusion between two different haploid strains having alpha mating type, 35 fusion products were estimated from their cell volumes to be diploid, 13 to be triploid and 16 to be tetraploid. The isolates showing tetraploid cell size were thought to have resulted from fusion of three protoplasts of one strain and one protoplast of the other (three-to-one fusion) or from two-to-two fusion. In protoplast fusion of three different haploid strains having alpha mating type, all four possible phenotypes of fusion product were recovered. Fusion products of three different protoplasts were obtained in much lower frequency (2.1 x 10(-6)) than those of two different protoplasts (1.2 x 10(-5) to 1.4 x 10(-4)) in the three other combinations. Genetic analyses revealed that triploid fusion products were formed by protoplast fusion of two different strains as well as of three different strains.
Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.
The nonfunctional mutation of the homothallic gene HMLα, designated hmlα, produced two mutant alleles, hmlα-1 and hmlα-2. Both mutant clones were mixed cultures consisting of a mating-type cells and nonmating haploid cells. The frequencies of the two cell types were different, and a few diploid cells able to sporulate were found in the hmlα-2 mutant. Conversions of an a mating-type cell to nonmater, and uice uersa, were observed in both mutants. The conversion of an a mating phenotype to nonmating is postulated to occur by alteration of the a mating type to the sterile mating-type allele in the hmlα-1 mutant. In tetrad dissection of prototrophic diploids that were obtained by rare-mating of hmlα-1 mutants with a heterothallic strain having the MATa ho HMRa HMLa genotype, many mating-deficient haploid segregants were found, while α mating-type segregants were observed in a similar diploid using an hmlα-2 mutant. The mating-type-deficient haploid segregants were supposed to have the sterile a mating-type allele because the nonmating genetic trait always segregated with the mating-type locus. Sporogenous diploid cells obtained in the hmlα-2 mutant clone had the MATa/MAT,α HO/HO HMRa/ HMRa hmlα-2/hmlα-2 genotype. These observations suggested that the hmla-I allele produces a transposable element that gives rise to the sterile su mating type by transposition into the mating-type locus, and that the hmlα-2 allele produces an element that provides,α mating-type information, but is defective in the structure for transposition.
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