Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Construction of multicopy yeast plasmids with regulated centromere function.

The promoter region from the cloned glucose-repressible alcohol dehydrogenase II (ADH2) gene has been inserted in front of the centromere III (CEN3) sequence. It has been shown that, in plasmids containing ADH2-CEN3 fusion (YCRp plasmids), the CEN3 function can be regulated by glucose repression. When yeast transformed with YCRp plasmid are grown in the presence of glucose, the ADH2 promoter is repressed and CEN3 functions normally, mitotically stabilizing the YCRp plasmid. When the carbon source in the culture medium is substituted with ethanol, the transcription from the derepressed ADH2 promoter interferes with the CEN3 function, rendering the YCRp plasmids unstable. At that stage YCRp2 plasmids may accumulate to about ten copies per cell whereas YCRp3 copy number increases to about 100. This demonstrates that the CEN3 sequence can be present on high copy number plasmids in yeast with no detectable toxic effects.

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae.

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.

ARS replication during the yeast S phase.

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.