High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

Abstract

A set of vector DNA (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, 3 modes of yeast transformation are defined. Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5, type 1) transform yeast cells at low frequency (1-10 colonies/.mu.g) and integrate into the genome by homologous recombination; this recombination is reversible. Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6, type 2) transform yeast cells at much higher frequency (5000-20,000 colonies/.mu.g). Such molecules replicate autonomously with an average copy number of 5-10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7, type 3) transform yeast with an efficiency of 500-5000 colonies/.mu.g; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The use of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events were presented.