Dan T. Stinchcomb

Sterile host yeasts (SHY): A eukaryotic system of biological containment for recombinant DNA experiments

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewers/Bakers yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.

Centromeric DNA from Saccharomyces cerevisiae

We have isolated 29 kilobase-pairs of DNA encompassing the centromere-linked gene, TRP1, of Saccharomyces cerevisiae. Two DNA sequences within the isolated region, ARS1 and ARS4, allow autonomous replication of chimeric DNA molecules upon introduction into yeast. Yeast strains transformed by ARS-containing DNA molecules are unstable; they lose the transformed phenotype and, concomitantly, the hybrid molecules at high frequency. Another function, CEN4, stabilizes the ARS-containing hybrids. CEN4 allows proper segregation of hybrid molecules during mitosis and meiosis. Like its predecessor. CEN3 (Clarke & Carbon, 1980b). CEN4 behaves genetically like a yeast centromere. The centromere-linked sequences we isolated from chromosome IV of yeast are not highly repeated and contain transcriptionally active DNA sequences. We have also isolated other CEN sequences using an enrichment procedure based on the enhanced mitotic stability of molecules carrying both CEN and ARS. Together, the ARS and CEN functions allow DNA molecules to predominantly behave as independent yeast linkage groups. However, improper disjunction of these ARS-CEN hybrids occurs in 3 to 14% of the mitotic cell divisions and ⩽3% of the meiotic divisions while the segregation of other yeast chromosomes, including chromosome IV, is normal. No more than 2% of these monovalent circular chromosomes undergo precocious segregation of sister chromatids during the first meiotic division rather than the second. Thus functional yeast chromosomes have been reconstructed by piecing together fragments of centromeric DNA with fragments that allow autonomous replication in yeast.

A physiological study of functional expression in Escherichia coli of the cloned yeast imidazoleglycerolphosphate dehydratase gene

Abstract Functional expression in Escherichia coli of bacteriophage λ and plasmid hybrids containing the Saccharomyces cerevisiae (yeast) gene for imidazoleglycerolphosphate (IGP) dehydratase ( his3 ) has been characterized in growing E. coli cells lacking the bacterial IGP dehydratase activity and during lytic infection of these cells. his3 expression of an integrated bacteriophage λhis3 hybrid requires transcriptional initiation from a “promoter” in the yeast DNA. A deletion mutant lacking this promoter, but containing the his3 structural gene has been isolated. Fusion of a DNA segment containing this promoter to a segment containing the intact structural gene for tetracycline resistance ( tet r ) but lacking an intact promoter results in expression of the tet r gene. Using four physiological criteria, the level of his3 expression in growing E. coli cells is affected by gene dosage. By these criteria, cells containing multiple copies of the his3 gene produce nearly wild-type E. coli activity levels of yeast IGP dehydratase. There is no evidence for regulation of his3 expression at the gene level as a function of histidine starvation. Expression of the his3 gene during lytic infection by bacteriophage λ hybrids has been assessed by the ability of such hybrids to grow in histidine-starved E. coli cells lacking IGP dehydratase. Phage containing a functional his3 gene grow with a single burst of five under these conditions and also form “plaques without lawns” on the starved cells. Lytic expression depends on transcription from the λ promoter P L ; the level depends on the distance from P L to the his3 gene. The results indicate that expression in E. coli of a eukaryotic gene obeys prokaryotic rules of gene expression and that it can occur at a significantly high level. This suggests that the basic gene recognition signals of eukaryotes and prokaryotes may not be so different.