David G. Barker

The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription

Using cultures synchronized by elutriator size selection or a feed-starve protocol, we have shown that the CDC8 gene is periodically expressed in the Saccharomyces cerevisiae cell cycle. The transcript level increases some 30-fold in late G1, reaching a peak at approximately the G1/S phase boundary. The timing of this event was compared with those of CDC9 and CDC21, which are already known to be periodically transcribed, and all three genes were found to be expressed at the same time in the cell cycle. In contrast, the histone H2A gene appeared to be expressed distinctly later in the cell cycle than these three genes and this was further investigated by examining expression of all four genes in a cdc4 mutant, held at the restrictive temperature. CDC8, CDC9, and CDC21 were once again expressed together and a complete fluctuation in levels occurred, whereas the histone gene was not expressed, presumably because the cdc4 block point precedes the point of histone expression. The three CDC genes may therefore be coordinately controlled, while the histone gene is regulated separately.

An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae

SummaryA sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast. The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae. Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.

Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents

SummaryUV-irradiation of stationary phase cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a 9-fold and 90-fold increase in transcript levels from the respective DNA ligase genes CDC9 and CDC17, whereas exponential cells show only 3-fold and 2-fold increases. Induction of CDC9 after MMS treatment and γ-irradiation was also observed by using a CDC9-lacZ translational fusion and assaying for β-galactosidase. Surprisingly, irradiation of S. cerevisiae induces only a 50% increase in DNA ligase itself, probably reflecting the extremely high in vivo stability of the enzyme. The UV-induction of ligase may be part of a “fail-safe” mechanism which, together with the enzyme stability, ensures adequate supplies of this essential enzyme.

Characterisation of an autonomously replicating sequence from the fission yeast Schizosaccharomyces pombe.

SummaryA DNA sequence has been isolated from Schizosaccharomyces pombe which promotes high frequency transformation of plasmids in the same organism. It is closely linked to the DNA ligase gene CDC17 and has therefore been named ARS17 although in structure it differs substantially from ARS elements in Saccharomyces cerevisiae. ARS17 spans some 1.8 kb of DNA and deletion of any part of this region affects activity. Moreover, there does not appear to be any short sequence which is, by itself, sufficient for high frequency transformation. ARS17 lies between and partly overlaps two divergently transcribed genes and it is extremely AT rich. It lacks the consensus sequence found in S. cerevisiae ARSs and it has no ARS activity in S. cerevisiae.

DNA ligase-AMP adducts: Identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.

The expression in meiosis of genes which are transcribed periodically in the mitotic cell cycle of budding yeast

The mitotic cell cycle genes CDC 8, 9 and 21 in Saccharomyces cerevisiae, together with the histone H2A gene, are transcribed discontinuously in meiosis. Message from all four genes initially declines in amount, then increases abruptly to reach maximal levels during premeiotic DNA synthesis before again declining. This response occurs only in meiotic cells; in asporogenous diploids the transcript simply declines in amount. In contrast, message from four genes with no known specific meiotic function (including the actin gene) shows the same profile in both sporogenous and asporogenous diploids. In mitotic cells the three CDC genes appear to be transcribed at the same time in the cell cycle, whereas in meiosis their transcripts accumulate with different kinetics, suggesting either that they have different turnover rates in meiotic cells or that the timing of their transcription is different.