K.-D. Entian

Complete DNA sequence of yeast chromosome II

In the framework of the EU genome‐sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37‐45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT‐rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT‐rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress

We have isolated several mutants ofSaccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of thepos10 mutation (POS forperoxidesensitivity) to thezwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation,pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in thepos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamedRPE1 (forribulose 5-phosphateepimerase).RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25 966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases ofEscherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus andSolanum tuberosum. We have characterizedRPE1 by testing enzyme activities inrpe1 deletion mutants and in strains that overexpressRPE1, and compared the hydrogen peroxide sensitivity ofrpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

Abstract In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non‐canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.

The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

Abstract In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation group fap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1W191F, which is characterized by a 104-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

The oxidative stress response mediated via Pos9/Skn7 is negatively regulated by the Ras/PKA pathway in Saccharomyces cerevisiae

Abstract Exposure of Saccharomyces cerevisiae to elevated concentrations of hydrogen peroxide induces transcription of several genes involved in the oxidative stress response. Two major transcription factors are involved in this induction, Pos9/Skn7 and Yap1. Fusions of either Yap1 or Pos9/Skn7 with the Gal4 DNA binding domain are active as transcription factors. Gal4-Yap1-dependent reporter gene activity is only weakly regulated by oxidative stress. In contrast, fusion of the Gal4 DNA binding domain to the Pos9/Skn7 protein results in a transcription factor that is independent of the YAP1 gene and is strictly regulated by oxidative stress, indicating that a signaling cascade impinges on the Pos9/Skn7 protein. We have observed that the Ras/PKA (cAMP-dependent protein kinase A) pathway affects this signaling. When PKA activity was low (in the presence of multicopy PDE2 or a cyr1D822→A mutation) maximum reporter gene activity was observed even in the absence of oxidative stress. In contrast, high PKA activity (in strains mutant for either pde2 or bcy1, or expressing the dominant active Ras2Val19) resulted in a complete loss of activation following oxidative stress. The transcription of Pos9/Skn7 target genes was also affected in Ras/PKA pathway mutants. Furthermore, we demonstrated that activated Pos9/Skn7 is necessary for Yap1-dependent reporter gene induction.

Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ

Abstract The closely related lantibiotics epidermin and gallidermin are produced by Staphylococcus epidermidis Tü3298 and S. gallinarum Tü3928, respectively. The epidermin biosynthetic genes involved in maturation, regulation, and immunity have been identified previously. How epidermin or gallidermin is secreted, however, has remained unclear. Here, we characterize two additional genes, epiH and epiT, as well as the homologous gallidermin genes gdmH and gdmT. EpiT and GdmT are similar to one-component ABC transporters that are involved in the secretion of proteins or peptides. EpiH and GdmH are hydrophobic proteins without conspicuous similarities to other proteins. Comparison of the gene sequences revealed that epiT is incomplete, having an internal deletion that causes a frame shift and a second deletion at the 3′-end, while gdmT is intact. Introduction of epiT and epiH into the heterologous host S. carnosus (pTepi14) bearing the maturation and regulation genes had no significant effect on the rather low level of epidermin production. The presence of the homologous gdmT and gdmH, however, resulted in a strong increase (seven- to tenfold) in the production level, which is very likely to be due to increased efficiency of epidermin secretion. Both gdmT and gdmH were necessary for this effect, indicating that the two gene products cooperate in some way. In the epidermin-producing wild-type strain Tü3298, which contains epiH and the disrupted epiT, the addition of gdmT alone led to a two-fold increase in epidermin production. Both gdmT and gdmH and the corresponding epi genes were activated by the transcriptional regulator EpiQ; this is in accordance with the presence of putative EpiQ operator sites in the promoter regions.