We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: ␣ factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at

As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.

In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase. We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity. Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down‐regulated by mating pheromone‐induced G1 arrest. An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle‐specific transcription factor SWI4. In similar experiments, CLN1 is less efficient than CLN3 at activating Start. Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2. To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start. Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control.

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.The sequence of the yeast genome has been determined (9). More recently, the number of mRNA molecules for each expressed gene has been measured (27,30). The next logical level of analysis is that of the expressed set of proteins. We have begun to analyze the yeast proteome by using two-dimensional (2D) gels.2D gel electrophoresis separates proteins according to isoelectric point in one dimension and molecular weight in the other dimension (21), allowing resolution of thousands of proteins on a single gel. Although modern imaging and computing techniques can extract quantitative data for each of the spots in a 2D gel, there are only a few cases in which quantitative data have been gathered from 2D gels. 2D gel electrophoresis is almost unique in its ability to examine biological responses over thousands of proteins simultaneously and should therefore allow us a relatively comprehensive view of cellular metabolism.We and others have worked toward assembling a yeast protein database consisting of a collection of identified spots in 2D gels and of data on each of these spots under various conditions (2,7,8,10,23,25). These data could then be used in analyzing a protein or a metabolic process. Saccharomyces cerevisiae is a good organism for this approach since it has a well-understood physiology as well as a large number of mutants, and its genome has been sequenced. Given the sequence and the relative lack of introns in S. cerevisiae, it is easy to predict the sequence of the primary protein product of most genes. This aids tremendously in identifying these proteins on 2D gels.There are three pillars on which such a database rests: (i) visualization of many protein spots simultaneously, (ii) quantification of the protein in each spot, and (iii) identification of the gene product for each spot. Our first efforts at visualization and identification for S. cerevisiae have been described elsewhere (7,8). Here we describe quantitative data for these proteins under a variety of experimental conditions. ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100) was used (26). ϪMet YNB (yeast nitrogen base) medium was 1.7 g of YNB (Difco) per liter, 5 g of ammonium sulfate per liter, and adenine, uracil, and all amino acids except methionine; ϪMet ϪCys YNB medium was the same but wi...

Cell cycle progression in eukaryotes is controlled by the p34cdc2/CDC28 protein kinase and its short-lived, phase-specific regulatory subunits called cyclins. In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system. Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9. Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA. In ubc9 mutants both CLB5, an S-phase cyclin, and CLB2, an M-phase cyclin, are stabilized. In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage. Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysis.