Transformation of yeast without the use of foreign DNA

Abstract

Isolated circular molecules of the yeast plasmid 2-micron DNA were converted to linear molecules with restriction endonuclease PstI and ligated with T4 DNA ligase to PstI restriction fragments of total yeast DNA. A haploid strain of Saccharomyces cerevisiae carrying a deletion in thehis4 locus was transformed with the ligated DNA mixture to histidine prototrophy. One unstable histidine prototrophic transformant was obtained. Grown in the absence of histidine, 70–75% of the cells were auxotrophic and this number increased in non-selective medium. The histidine auxotrophic variants carried a deletion in thehis4 locus which had patterns of complementation and UV-induced mitotic recombination identical to the originalhis4 deletion. When the transformant was crossed to ahis4 strain and sporulated, the unstable histidine prototrophy segregated in a non-mendelian way: All five possible segregations from 0∶4 to 4∶0 were observed. When strains carrying the transformed character were crossed to ahis4 karl strain a low frequency of cytoduction of the unstable histidine prototrophy was observed. Nucleic acid from the transformant was able to transform a strain which carried another deletion in thehis4 locus. Treatment of the transformant with ethidium bromide caused an extensive induction of petites without any observable change in the frequency of histidine prototrophic cells.It is concluded that theHIS4 gene function in the transformant is not stably associated with any chromosome. We take the instability as indication that only one or a few copies of the gene conferring the prototrophy are present in the prototrophic cells. The data are consistent with the assumption that the transformant contains a 2-micron DNA in which is inserted a chromosomal DNA region containing theHIS4 gene. A derivative of the transformant with increased stability has been isolated.