An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.     

Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation

Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells.     

Expression,glycosylation and secretion of fungal hydrolases in yeast

Although there are now some systems for transfer and expression of fungal genes, none gives expression at a level useful for production. There are useful expression systems in yeast, however, and these have been used to express genes coding for fungal extracellular hydrolases.This review examines how properties of the genes and gene product affect production and secretion of the enzyme in yeast. Similar considerations apply to expression of other heterologous genes in yeast and in other hosts.     

Evidence that the ribosomal DNA genes of yeast are not on chromosome I

Summary Several workers have reported that most of the ribosomal DNA genes (rDNA) of the yeast Saccharomyces cerevisiae are located on chromosome I. More recently, data indicating that the yeast rDNA genes are located on chromosome XII has been presented. In this report, we present additional evidence indicating that most of the yeast rDNA genes are not on chromosome I. Starting from a diploid yeast strain, we isolated ten strains which were monosomic (2n-1) for chromosome I. We found that each of these ten strains contained two copies of the rDNA-containing chromosome. In addition, we show that the earlier evidence indicating that the yeast rDNA genes were on chromosome I cannot be explained by a difference in the yeast strains which were used in the different experiments.     

Functional genomic studies of aldo-keto reductases

Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.     

Functional genomic studies of aldo–keto reductases

Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo–keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.     

Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs

There exists a similarity among the synonymous codon choice patterns of the yeast nuclear genes that have been sequenced thus far although these genes encode different types of protein molecules, and the patterns are significantly different from those of Escherichia coli genes. Based on constraints caused by the availability of E. coli transfer RNAs and the nature of their codon recognition related to the modified nucleotides at the anticodon wobble position, the characteristic patterns of synonymous codon choice commonly found for E. coli genes have been almost completely explained (Ikemura, 1981a, b). In the present paper, tRNAs of the yeast Saccharomyces cerevisiae were separated by two-dimensional polyacrylamide gel electrophoresis and the relative abundance of purified tRNA molecules was measured on the basis of molecular numbers in cells. A strong correlation between tRNA abundance and codon choice was found for each nuclear gene of yeast, but the correlation was less significant for 2μ plasmid genes. According to the criteria proposed for E. coli genes (Ikemura, 1981b) the order of codon preference in yeast nuclear genes was predicted based on the abundance of yeast isoaccepting tRNAs and on the nature of the modified nucleotides at their anticodons. Clear correlations between predictions and the actual preferences among synonymous codons were revealed, indicating that the codon choices in yeast genes are also constrained by a combination of tRNA availability and nature of its codon recognition. Then the difference in synonymous codon use between the two organisms can be attributed to the difference in these two factors.     

Oncogene homologues in yeast

Two different yeasts have a number of genes bearing striking structural and functional homologies to mammalian oncogenes. In yeast these genes are involved in the control of proliferation and early steps in the cell cycle. Many have putative protein kinase activity and some have been shown to control the activity of the enzyme adenylate cyclase which synthesizes cyclic AMP. Mutant forms of these yeast genes have oncogenic activity in mammalian cells.     

Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

Structure and function of the genes controlling DNA repair in yeast

Recent data on cloning and sequencing of RAD genes controlling DNA repair in yeast are reviewed. The structure of regulatory regions and molecular features of the RAD genes' protein products have been considered. Special attention was paid to the regulation of expression of RAD genes and their functions, differing from those for DNA repair. Examples of homology between yeast RAD genes and their counterparts in bacteria and higher eukaryotes are discussed.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

Zeroing in on zinc uptake in yeast and plants.

Zinc is an essential micronutrient. Genes responsible for zinc uptake have now been identified from yeast and plants. These genes belong to an extended family of cation transporters called the ZIP gene family. Zinc efflux genes that belong to another transporter family, the CDF family, have also been identified in yeast and Arabidopsis. It is clear that studies in yeast can greatly aid our understanding of zinc metabolism in plants.     

Conservative system for dosage-dependent modulation of translational fidelity in eukaryotes.

Variations in dosage of some genes can alter the level of translational fidelity. The Saccharomyces cerevisiae genes that act as dosage-dependent suppressors and/or modulators of suppression, are the following: some tRNA genes (for example, tRNA(Gln)) inducing readthrough by mispairing; genes coding for either translational elongation factor or other proteins taking part in translation; and some genes of unknown function. We suggest that the SUP35 protein is a factor which may play a major role in balance-dependent regulation of translational fidelity. Homologues of this genes have been identified in other yeast genera (Pichia), green algae (Chlamydomonas) and various animals including man. No homologies have been found in the polychaeta (Nereis) or in insects (Drosophila). Rates of evolution differ for two separate parts of the genes; the N-terminal part, which is important for ambiguous translation in Saccharomyces, is markedly variable in the organisms tested. However, the C-terminal part which is required for yeast viability has a common origin but a separate evolution from that of the EF-Tu protein family.     

Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

We report yeast/Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZ gene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication (ori), i.e., of the yeast 2 mu circle and of the ColE1 plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the ApR gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2 mu ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express beta-galactosidase (beta Gal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5'-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by beta Gal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of beta Gal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.