Evolution of the <Emphasis Type="Italic">Groucho</Emphasis>/<Emphasis Type="Italic">Tle</Emphasis> gene family: gene organization and duplication events

The Groucho/Tle family of corepressor proteins has important roles in development and in adult tissue in both Protostomes and Deuterostomes. In Drosophila, a single member of this family has been identified. Unlike in Protostomes, most Deutrostomes contain more than two full-length Tle genes. In this study, I analyse the genomic organization and phylogenetic relationship between the long and short forms of the Groucho/Tle family members in Chordata. The genomic location and sequence similarities suggest that Aes/Grg5 and Tle6/Grg6 arose from duplication of the Tle2 gene; each evolved independently and acquired new functions as negative regulators of the other Tle proteins. Based on these data, a model for Groucho/Tle gene evolution is proposed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amiodarone inhibits multiple drug resistance in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.     

Nonsense mutations in the essential gene<Emphasis Type="Italic"> SUP35</Emphasis> of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.Communicated by C. P. Hollenberg     

Higher Gene Duplicabilities for Metabolic Proteins Than for Nonmetabolic Proteins in Yeast and <Emphasis Type="Italic">E. coli</Emphasis>

Although the evolutionary significance of gene duplication has long been appreciated, it remains unclear what factors determine gene duplicability. In this study we investigated whether metabolism is an important determinant of gene duplicability because cellular metabolism is crucial for the survival and reproduction of an organism. Using genomic data and metabolic pathway data from the yeast (Saccharomyces cerevisiae) and Escherichia coli, we found that metabolic proteins indeed tend to have higher gene duplicability than nonmetabolic proteins. Moreover, a detailed analysis of metabolic pathways in these two organisms revealed that genes in the central metabolic pathways and the catabolic pathways have, on average, higher gene duplicability than do other genes and that most genes in anabolic pathways are single-copy genes.Reviewing Editor: Dr. Rüdiger Cerff     

Ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in the presence of orange-peel oil

Summary Two ethanologenic yeasts, Saccharomyces cerevisiae and Kluyveromyces marxianus, were used to ferment sugar solutions modeling hydrolyzed Valencia orange peel waste at 37°C. Orange stripper oil produced from orange peel was added in various amounts to determine its effect on ethanol production. The minimum peel oil concentration that inhibited ethanol production was determined after 24, 48 and 72 h and the two yeasts were compared to one another in terms of ethanol yield. Minimum inhibitory peel oil concentrations for ethanol production were 0.05% at 24 h, 0.10% at 48 h, and 0.15% at 72 h for both yeasts. S. cerevisiae produced more ethanol than K. marxianus at each time point.     

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Gene <Emphasis Type="Italic">RAD31</Emphasis> is identical to gene <Emphasis Type="Italic">MEC1</Emphasis> of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The earlier identified gene RAD31 was mapped on the right arm of chromosome II in the region of gene MEC1 localization. Epistatic analysis demonstrated that the rad31 mutation is an allele of the MEC1 gene, which allows further designation of the rad31 mutation as mec1-212. Mutation mec1-212, similar to deletion alleles of this gene, causes sensitivity to hydroxyurea, disturbs the check-point function, and suppresses UV-induced mutagenesis. However, this mutation significantly increases the frequency of spontaneous canavanine-resistance mutations induced by disturbances in correcting errors of DNA replication and repair, which distinguishes it from all identified alleles of gene MEC1.     

Reducing haziness in white wine by overexpression of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genes <Emphasis Type="Italic">YOL155c</Emphasis> and <Emphasis Type="Italic">YDR055w</Emphasis>

Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.     

Phylogeny and the Evolution of the <Emphasis Type="Italic">Amylase</Emphasis> Multigenes in the <Emphasis Type="Italic">Drosophila montium</Emphasis> Species Subgroup

Abstract To investigate the phylogenetic relationships and molecular evolution of α-amylase (Amy) genes in the Drosophila montium species subgroup, we constructed the phylogenetic tree of the Amy genes from 40 species from the montium subgroup. On our tree the sequences of the auraria, kikkawai, and jambulina complexes formed distinct tight clusters. However, there were a few inconsistencies between the clustering pattern of the sequences and taxonomic classification in the kikkawai and jambulina complexes. Sequences of species from other complexes (bocqueti, bakoue, nikananu, and serrata) often did not cluster with their respective taxonomic groups. This suggests that relationships among the Amy genes may be different from those among species due to their particular evolution. Alternatively, the current taxonomy of the investigated species is unreliable. Two types of divergent paralogous Amy genes, the so-called Amy1- and Amy3-type genes, previously identified in the D. kikkawai complex, were common in the montium subgroup, suggesting that the duplication event from which these genes originate is as ancient as the subgroup or it could even predate its differentiation. Thc Amy1-type genes were closer to the Amy genes of D. melanogaster and D. pseudoobscura than to the Amy3-type genes. In the Amy1-type genes, the loss of the ancestral intron occurred independently in the auraria complex and in several Afrotropical species. The GC content at synonymous third codon positions (GC3s) of the Amy1-type genes was higher than that of the Amy3-type genes. Furthermore, the Amy1-type genes had more biased codon usage than the Amy3-type genes. The correlations between GC3s and GC content in the introns (GCi) differed between these two Amy-type genes. These findings suggest that the evolutionary forces that have affected silent sites of the two Amy-type genes in the montium species subgroup may differ.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Ultrastructure of yeast cell <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after amiodarone treatment

The ultrastrcutre of Saccharomyces cerevisiae cells (wild-type and ysp2 mutant cells) was studied after amiodarone treatment. Amiodarone is used as a pharmaceutical substance for treating a number of diseases; however, it is known that amiodarone causes structural and functional disturbances in patient tissues. Here, the peculiarities of the amiodarone effect are studied in Saccharomyces cerevisiae yeast, in which amiodarone has been shown to cause apoptosis. Electron-microscopic study of yeast cells after amiodarone treatment reveals a significant increase in the number of lipid particles, which can lead to the formation of a structural complex by interacting with cell membranous organelles. Amiodarone causes the appearance of small and slightly swollen mitochondria. Chromatin displacement to the periphery of the nucleus, nuclear sectioning, and nuclear envelope disturbances are observed in the cells under these conditions. The detected changes int eh ultrastructure of the cell in Saccharomyces cerevisiae are considered to be a specific response to phospholipidosis and apoptosis caused by amiodarone.     

Interaction of gene <Emphasis Type="Italic">HSM3</Emphasis> with genes of the epistatic <Emphasis Type="Italic">RAD6</Emphasis> group in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. This pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G₁ DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.     

Cloning of two cellobiohydrolase genes from <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> and heterogenous expression in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml⁻¹ under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.     

Conditional chromosome splitting in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using the homing endonuclease PI-SceI

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.