Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae.

We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.

Expression of the Drosophila 70,000 Dalton heat shock protein is translationally controlled in yeast.

Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.

Faithful and efficient translation of homologous and heterologous mRNAs in an mRNA-dependent cell-free system from Saccharomyces cerevisiae.

A cell-free protein synthesizing system from the yeast Saccharomyces cerevisiae has been optimized for the translation of both homologous yeast mRNA and for a number of heterologous eukaryotic mRNAs. A significant increase in protein synthesis was observed when K(OAc) rather than KCl was used as the source of K+ in the in vitro translation system. This was due primarily to an inhibitory effect oif Cl-. The polyamine putrescine hydrochloride stimulated protein synthesis only at low Mg2+ concentrations. Protein synthesis directed by both yeast mRNA and several eukaryotic mRNAs examined in the system was sensitive to the mRNA 5'-cap analogue, 7-methylguanosine 5'-monophosphate. One-dimensional and two-dimensional polyacrylamide gel analysis of polypeptides synthesized in response to yeast polysomal RNA demonstrated faithful translation in vitro. Translational control and post-translational modifications appear to operate normally in vitro. RNA from several eukaryotic viruses (brome mosaic virus, turnip yellow mosaic virus, and tobacco mosaic virus) were found to be faithfully translated in vitro yielding discrete polypeptides. Reticulocyte polysomal RNA directed the synthesis of a single protein that co-migrated with rabbit globin. The prokaryotic RNAs of Q beta and MS2 were translated with a very low efficiency. The yeast cell-free system programmed with yeast polysomal RNA provides an excellent model for the study of translational control in a eukaryote.