We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae

Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-containing S. cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2. Overexpression of only TKL1 did not influence growth. The results indicate that the transaldolase level in S. cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites. Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL. The rate of xylose consumption was higher in the presence of glucose. Xylose was used for growth and xylitol formation, but not for ethanol production. Decreased oxygenation resulted in impaired growth and increased xylitol formation. Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation.

Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase

The Drosophila brain tumor (brat) gene encodes a member of the conserved NHL family of proteins, which appear to regulate differentiation and growth in a variety of organisms. One of the founding family members, Caenorhabditis elegans LIN-41, is thought to control posttranscriptional gene expression. However, the mechanism by which LIN-41, or any other NHL protein, acts has not been clear. Using a yeast “four-hybrid” interaction assay, we show that Brain Tumor is recruited to hunchback (hb) mRNA through interactions with Nanos and Pumilio, which bind to the RNA to repress its translation. Interaction with the Nanos/Pumilio/RNA complex is mediated by the Brat NHL domain; single amino acid substitutions in this domain compromise quaternary complex assembly in vitro and hb regulation in vivo. Thus, recruitment of Brat is necessary for translational repression and the normal development of posterior embryonic pattern. In addition to regulating abdominal segmentation, previous genetic analysis has shown that Brat, Nanos, and Pumilio govern a variety of developmental processes. We examined the role of Brat in two of these processes—regulation of maternal Cyclin B mRNA in the embryo and regulation of imaginal disc development. The results of these experiments suggest that NHL domain proteins are recruited to various mRNAs by combinatorial protein–protein interactions.

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Drosophila Brain Tumor is a translational repressor.

There has recently been much controversy surrounding the increased use of antibacterial substances in a wide range of consumer products and the possibility that, as with antibiotics, indiscriminate use of biocides might contribute to the overall pattern of susceptibility in the general environment and in the clinic. Such speculation, based on the isolation of resistant mutants from in vitro monoculture experiments, is not reflected by an emergence of biocide-resistant strains in vivo. This review provides a broad coverage of the biocide and resistance literature and evaluates the potential risks, perceived from such laboratory monoculture experiments, against evidence gathered over 50 years of field studies. An explanation for the continued effectiveness of broad-spectrum biocidal agents against the decline in efficacy of therapeutic agents is provided based on the fitness costs of resistance and the ubiquity of naturally occurring substances that possess antibacterial effect. While we conclude from this review of the literature that the incorporation of antibacterial agents into a widening sphere of personal products has had little or no impact on the patterns of microbial susceptibility observed in the environment, the associated risks remain finite. The use of such products should therefore be associated with a clear demonstration of added value either to consumer health or to the product life. Hygienic products should therefore be targeted to applications for which the risks have been established.

https://cmr.asm.org/content/cmr/16/2/189.full.pdf

Potential Impact of Increased Use of Biocides in Consumer Products on Prevalence of Antibiotic Resistance

We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.

P.A. Lowe

Papers

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.