STRUCTURAL AND FUNCTIONAL STABILITY OF INCP PLASMIDS DURING STEPWISE TRANSMISSION BY TRANSKINGDOM MATING - PROMISCUOUS CONJUGATION OF ESCHERICHIA-COLI AND SACCHAROMYCES-CEREVISIAE

In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAYl0l, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10-5 to 10-3 per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAYl0l was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells. © 1990, The Genetics Society of Japan. All rights reserved.