The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Deciphering the effects of gene deletion on yeast longevity using network and machine learning approaches

Longevity is one of the most basic and one of the most essential properties of all living organisms. Identification of genes that regulate longevity would increase understanding of the mechanisms of aging, so as to help facilitate anti-aging intervention and extend the life span. In this study, based on the network features and the biochemical/physicochemical features of the deletion network and deletion genes, as well as their functional features, a two-layer model was developed for predicting the deletion effects on yeast longevity. The first stage of our prediction approach was to identify whether the deletion of one gene would change the life span of yeast; if it did, the second stage of our procedure would automatically proceed to predict whether the deletion of one gene would increase or decrease the life span. It was observed by analyzing the predicted results that the functional features (such as mitochondrial function and chromatin silencing), the network features (such as the edge density and edge weight density of the deletion network), and the local centrality of deletion gene, would have important impact for predicting the deletion effects on longevity. It is anticipated that our model may become a useful tool for studying longevity from the angle of genes and networks. Moreover, it has not escaped our notice that, after some modification, the current model can also be used to study many other phenotype prediction problems from the angle of systems biology.     

Applying the genetic theories of ageing to the cytoplasm: cytoplasmic genetic covariation for fitness and lifespan

Two genetic models exist to explain the evolution of ageing – mutation accumulation (MA) and antagonistic pleiotropy (AP). Under MA, a reduced intensity of selection with age results in accumulation of late‐acting deleterious mutations. Under AP, late‐acting deleterious mutations accumulate because they confer beneficial effects early in life. Recent studies suggest that the mitochondrial genome is a major player in ageing. It therefore seems plausible that the MA and AP models will be relevant to genomes within the cytoplasm. This possibility has not been considered previously. We explore whether patterns of covariation between fitness and ageing across 25 cytoplasmic lines, sampled from a population of Drosophila melanogaster, are consistent with the genetic associations predicted under MA or AP. We find negative covariation for fitness and the rate of ageing, and positive covariation for fitness and lifespan. Notably, the direction of these associations is opposite to that typically predicted under AP.     

Selection in yeast II: The fitness distribution of new mutations inSaccharomyces cerevisiae and its use in a computer simulation

Summary The fitness distribution of new mutations inSaccharomyces cerevisiae strain Montrachet was determined for cells on agar irradiated for four periods of time with ultraviolet light. The fitness distributions were obtained by converting a large number of colony diameters into relative fitnesses. The distributions were then used to perform a computer simulation with the purpose of predicting the potential of a stock culture to increase in general fitness through selection, given a frequency and magnitude of mutations.     

核转录因子TR3及其缺失突变体在酵母双杂交系统中转录激活作用的检测

目的:检测TR3及其转录活性域缺失突变体在酵母双杂交系统中的转录自激活作用。方法:采用PCR方法扩增TR3全长序列及其缺失突变体,构建酵母双杂交系统的诱饵载体pGBKT7-TR3和pGBKT7-TR3/Δ1～690,将pGBKT7-TR3转化感受态酵母菌AH109后培养于含缺失培养基的平板上,检测β-半乳糖苷酶活性,判定其是否具有转录自激活作用。结果:构建了包含TR3全长序列和TR3/Δ1～690序列的诱饵载体,转化酵母菌AH109后在双缺和三缺培养基上未能使β-半乳糖苷酶活性滤纸片变蓝,说明β-半乳糖苷酶报告基因未被激活。结论:TR3及其转录活性域缺失突变体没有转录自激活作用,可用于酵母双杂交系统。     

The problem of assessing landmark error in geometric morphometrics: theory, methods, and modifications

Geometric morphometric methods rely on the accurate identification and quantification of landmarks on biological specimens. As in any empirical analysis, the assessment of inter- and intra-observer error is desirable. A review of methods currently being employed to assess measurement error in geometric morphometrics was conducted and three general approaches to the problem were identified. One such approach employs Generalized Procrustes Analysis to superimpose repeatedly digitized landmark configurations, thereby establishing whether repeat measures fall within an acceptable range of variation. The potential problem of this error assessment method (the "Pinocchio effect") is demonstrated and its effect on error studies discussed. An alternative approach involves employing Euclidean distances between the configuration centroid and repeat measures of a landmark to assess the relative repeatability of individual landmarks. This method is also potentially problematic as the inherent geometric properties of the specimen can result in misleading estimates of measurement error. A third approach involved the repeated digitization of landmarks with the specimen held in a constant orientation to assess individual landmark precision. This latter approach is an ideal method for assessing individual landmark precision, but is restrictive in that it does not allow for the incorporation of instrumentally defined or Type III landmarks. Hence, a revised method for assessing landmark error is proposed and described with the aid of worked empirical examples.     

Transmission of the yeast mitochondrial genome to progeny: The impact of intergenic sequences

Summary In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs. In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked, loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin. These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains. Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome. In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker. These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole. More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences. These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome. However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts. Therefore, in a more general sense, variations in the amount and structure of intergenic sequences in various yeasts may reflect processes that have been of selective advantage in the metabolism of individual mitochondrial DNA in a particular environment and that have not drastically interrupted the respiratory phenotype.     

On the relative roles of faster-X evolution and dominance in the establishment of intrinsic postzygotic isolating barriers

The modern theory of speciation assigns a prominent role to the recessivity of genetic incompatibilities in the two rules of speciation, namely Haldane's rule and the large X effect, and considers that the contribution of faster evolution of the X versus the autosomes to those patterns is generally of relatively minor importance. By extending Turelli and Orr's previous analysis of the model of two-locus Dobzhansky–Muller incompatibilities, I first show that when the X and the autosomes evolve at the same rate, the two dominance parameters involved in that model are not equally important for the declaration of a large X effect, but that the degree of recessivity of homozygous–homozygous incompatibilities is the major determinant for such a declaration. When the X evolves faster than the autosomes, the model obviously predicts that the importance of both dominance parameters will progressively vanish. It is then of importance to obtain estimates of the relative evolutionary rate of X-linked incompatibility loci. Several different procedures to obtain such estimates from the perspective of the large X effect are suggested. The application of the appropriate test to the only suitable data from Drosophila hybridizations so far available leads to the conclusion that the X actually evolves at least 2.5 times faster than the autosomes, as far as hybrid male sterility determinants are concerned, thus making dominance considerations absolutely irrelevant. Notwithstanding the necessity of further tests, the relative roles currently assigned to faster-X evolution and dominance in the theory of speciation should be revised, giving due prominence to faster-X evolution, at least for hybrid male sterility in the genus Drosophila.     

On the Analysis and Synthesis of a Four-Field-Table

The real four-field-table measure k is calculable as follows: K₁ for V(a, b, c, d) with k₁ = (arc K₁)/100, K₁₁ for V(a, b, 0, 0) with k₁₁ = (arc K₁₁)/100, K₁₂ for V(c, d, 0, 0) with k₁₂ = (arc K₁₂)/100 and K₁₂₁ for V(0, 0, c, d) with k₁₂₁ = – k₁₂. The equation k₁ = k₁₁ – k₁₂ holds good. It is possible to calculate the probability of error of a four-field-table with small frequencies, indirectly from component values. Two examples are given.     

Regulation of the localization and stability of Cdc6 in living yeast cells

The Cdc6 protein is an essential regulator for initiation of DNA replication. Following the G1/S transition, Cdc6 is degraded through a ubiquitin-mediated proteolysis pathway. In this study, we tagged Cdc6 with green fluorescent protein (GFP) and used site-specific mutations to study the regulation of Cdc6 localization and degradation in living yeast cells. Our major findings are: (1). Cdc6-GFP distributes predominantly in the nucleus in all cell cycle stages, with a small increase in cytoplasmic localization in G2/M cells. (2). This nuclear localization is critical for Cdc6 degradation. When the N-terminal nuclear localization signal (NLS) was mutated, Cdc6-GFP no longer accumulated in the nucleus, and the mutant cdc6 was stabilized compared to wild type. (3). The putative CDK phosphorylation sites are not required for Cdc6 nuclear localization, but are important for protein stability. These observations suggest that the stability of Cdc6 protein is regulated by two factors: nuclear localization and phosphorylation by CDK1.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

Biogeography and population structure of the Neotropical endemic yeast species <Emphasis Type="Italic">Metschnikowia lochheadii</Emphasis>

The genetic structure of populations of the out-crossing haplontic yeast species Metschnikowia lochheadii was investigated. The species is associated with floricolous beetles in Central America and Hawaii. The objective was to determine whether sexual reproduction is prevalent and to what extent the geographic distribution of genotypes can be viewed as historical. The genetic markers examined include the mating type (h (+) or h (-)) and nine polymorphic DNA loci. The data were used to assess population structuring based on F (ST) and linkage disequilibrium and the distribution of alleles using parsimony haplotype networks. In Central America, M. lochheadii is subdivided into sexually active demes between which gene flow is limited. Isolates from five Hawaiian islands had identical haplotypes, confirming that the species has undergone a founder effect concomitant with the recent import of a nitidulid beetle into the archipelago.     

The site of action of 2,4-dinitrophenol and salicylic acid upon the uncoupler-induced K+ efflux from non-metabolizing yeast

Stimulation of K⁺ efflux from non-metabolizing yeast cells by 2,4-dinitrophenol or by salicylic acid occurs only after accumulation of the compounds into the cells, indicating that the site of action of the uncouplers is inside the cells. A correlation is found between the partition ratio of the lipophilic cation dibenzyldimethylammonium between cells and medium and the rate of K⁺ efflux.     

The effect of carbon dioxide,aeration rate and sodium chloride on the secretion of proteins from growing bakers' yeast (Saccharomyces cerevisiae)

Since yeast may be an important microorganism for industrial use when its genes are modified by recombinant DNA techniques to overproduce certain proteins, (particularly those which are glycosylated), it is desirable to study how environmental variables affect its protein secretion ability. It is also of interest to understand how proteins such as proteases, lipases and amylases are excreted in solid matrices to develop a basis for learning more about solid fermentations. With these two applications in mind, the total protein excreted by both aerated and non-aerated Saccharomyces cerevisiae growing in a liquid batch culture (with varying levels of CO₂ and NaCl) was tracked. Using a modified Bradford method (Coomassie Blue dye-binding assay) for the concentration of total proteins in the extracellular fermentation broth, it has been determined that by 24 h of the run, excreted proteins rose to levels of about 10% of the total cell protein (500 μg ml^?1 protein from about 10 g of yeast, containing about 5 g total protein). No cell lysis was observed during the 24 h run. The highest protein levels at the top of the fermentor seemed to be those achieved in response to CO₂ alone. Additions of NaCl did not seem to enhance the secreted protein level. Large inconsistencies in replicating anaerobic runs for protein concentration appeared to be explained by noting that rising CO₂ bubbles may cause ‘foam fractionation’ of the proteins in the broth.     

Liquid holding recovery kinetics in wild-type and radiosensitive mutants of the yeast Saccharomyces exposed to low- and high-LET radiations

Three wild-type diploid yeast strains Saccharomyces ellipsoideus and Saccharomyces cerevisiae and five radiosensitive mutants of S. cerevisiae in the diploid state were irradiated with gamma-rays from 60Co and alpha-particles from 239Pu in the stationary phase of growth. Survival curves and the kinetics of the liquid holding recovery were measured. It was shown that the irreversible component was enhanced for the densely ionizing radiation in comparison to the low-LET radiation while the probability of the recovery was identical for both the low- and high-LET radiations for all the strains investigated. It means that the recovery process itself is not damaged after densely ionizing radiation and the enhanced RBE of the high-LET radiation may be caused by the increased yield of the irreversible damage. A parent diploid strain and all its radiosensitive mutants showed the same probability for recovery from radiation damage. Thus, the mechanism of the enhanced radiosensitivity of the mutant cells might not be related to the damage of the repair systems themselves but with the production of some kind of radiation damage from which cells are incapable to recover.     

Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.