Two types of α-factor receptor determinants for pheromone specificity in the mating-incompatible yeasts S. cerevisiae and S. kluyveri

The specificity of mating-pheromone systems helps prevent mating between different species of yeast. The mechanism of specificity of G protein-coupled receptors in other organisms may be similar. To determine the structural basis of species discrimination between α-factor variants, small extracellular segments of the Saccharomyces cerevisiaeα-factor receptor were substituted with residues from the Saccharomyces kluyveriα-factor receptor. Three substitutions altered specificity towards S. cerevisiae and S. kluyveriα-factor pheromone peptides. A substitution at residues 47–49 affected specificity for pheromone binding but not for pheromone activation of response. Substitution of residues 267–269 affected pheromone specificity for activation of cellular responses, but not for pheromone binding (under the assay conditions used). Substitution of residues 104–123 modestly affected both types of specificity. These results suggest two distinct types of receptor/ligand specificity (perhaps corresponding to ligand binding to G protein-coupled or uncoupled conformational states of the receptor) that evolved independently as S. cerevisiae and S. kluyveri diverged as species. Received: 27 September / 12 November 1996     

Deletion of flanking ARS elements does not affect meiotic recombination at the HIS4 locus in yeast

The HIS4-BIK1 interval on chromosome III of Saccharomyces cerevisiae contains a hotspot for meiotic recombination. Previous reports demonstrated that the initiating lesion is a double-stranded break which is subsequently processed in an asymmetric manner. Data presented here show that the efficiency of initiation of meiotic recombination is unaffected by the deletion of flanking ARS elements, and that the distribution of recombinants is not altered in strains heterozygous for these deletions. These results suggest that the initiation of recombination is not affected by the time of replication of the hotspot at HIS4. The data also indicate that altering the direction of replication-fork movement through the HIS4 region does not affect meiotic recombination. Received: 18 June / 24 September 1996     

Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10⁻² M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected. Received: 21 August / 12 September 1996     

Transient expression of the β-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis

 The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial β-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for β-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the β-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus. Received: 9 July / 18 September 1996     

Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis

 The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other α-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial α-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creA ^d mutants and carbon catabolite repressed by D-glucose. Received: 9 August / 29 August 1996     

Partial sequence of the mitochondrial genome of the crustacean Daphnia pulex

 A 4062-nucleotide (nt) fragment of the mitochondrial genome of the crustacean Daphnia pulex was sequenced and found to contain the complete genes for eight tRNAs and five proteins (ATP6, ATP8, COII, COIII, ND3) and the partial sequence of COI. In combination with data described previously, approximately 50% of the D. pulex mitochondrial genome has been sequenced. The gene order in this half of the genome is identical to that of Drosophila yakuba which differs from that of the other completely sequenced crustacean mitochondrial genome, Artemia franciscana. Comparison of seven mitochondrial proteins among D.␣pulex, A. franciscana, D. yakuba, Anopheles gambiae, Locusta migratoria and Apis mellifera showed that, with one exception, the D. pulex proteins are most similar in length and sequence to the proteins of the dipteran insects. Conversely, patterns of nucleotide bias at third codon positions in fourfold degenerate codon families are very similar in the two crustaceans but differed substantially from the insects. Received: 15 March / 23 September 1996     

Intron sequences provide a tool for high-resolution phylogenetic analysis of volvocine algae

Three nuclear spliceosomal introns in conserved locations were amplified and sequenced from 28 strains representing 14 species and 4 genera of volvocalean green algae. Data derived from the three different introns yielded congruent results in nearly all cases. In pairwise comparisons, a spectrum of taxon-specific sequence differences ranging from complete identity to no significant similarity was observed, with the most distantly related organisms lacking any conserved elements apart from exon-intron boundaries and a pyrimidine-rich stretch near the 3′ splice site. A metric (SI₅₀), providing a measure of the degree of similarity of any pair of intron sequences, was defined and used to calculate phylogenetic distances between organisms whose introns displayed statistically significant similarities. The rate of sequences divergence in the introns was great enough to provide useful information about relationships among different geographical isolates of a single species, but in most cases was too great to provide reliable guides to relationships above the species level. A substitution rate of approximately 3 × 10⁻⁸ per intron position per year was estimated, which is about 150-fold higher than in nuclear genes encoding rRNA and about 10-fold higher than the synonymous substitution rate in protein-coding regions. Thus, these homologous introns not only provide useful information about intraspecific phylogenetic relationships, but also illustrate the concept that different parts of a gene may be subject to extremely different intensities of selection. The intron data generated here (1) reliably resolve for the first time the relationships among the five most extensively studied strains of Volvox, (2) reveal that two other Volvox species may be more closely related than had previously been suspected, (3) confirm prior evidence that particular isolates of Eudorina elegans and Pleodorina illinoisensis appear to be sibling taxa, and (4) contribute to the resolution of several hitherto unsettled issues in Chlamydomonas taxonomy. Received: 7 October / 25 November 1996     

Blood metabolite and catecholamine responses to prolonged exercise following either acute plasma volume expansion or short-term training

 To determine the effect of acute plasma volume (PV) expansion on substrate utilization, blood metabolites and catecholamines to prolonged, moderate intensity cycle exercise, eight untrained men mean maximal oxygen uptake,O_2max 4.10 (SEM 0.32) l · min⁻¹ were infused (10 ml·kg⁻¹) with a 6% dextran (DEX) solution. These responses were also compared to those elicited using a short-term training (TR) protocol involving cycling for 90 to 120 min · day⁻¹ at 60% O_2max for 3 consecutive days. In general DEX, which resulted in a calculated expansion of PV by 23.9%, was without effect in modifying exercise oxygen uptake or the reduction in the respiratory exchange ratio (R) observed during prolonged exercise. In addition, the concentrations of blood glucose, glycerol, alanine and serum free fatty acids, although altered (P < 0.05) by exercise, were not altered by DEX. Blood lactate concentration was only higher (P < 0.05) at 30 min of exercise during DEX compared to the control. With the exception of blood lactate concentration, which was reduced (P < 0.05), TR did not change R or the concentrations of other blood metabolites. The concentrations of nonadrenaline and adrenaline, were depressed (P < 0.05) by DEX and TR at 60 and 90 min of exercise. These results would suggest that mechanisms as yet undefined can compensate for the estimated 10% reduction in arterial oxygen content mediated by acute PV expansion and enable prolonged exercise to be performed without adjustments in substrate selection and substrate mobilization. Accepted: 23 August 1996     

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, δ and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase δ, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase δ and a deletion in the REV3 gene had a complete repair defect, both at permissive (23°C) and restrictive (38°C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase ɛ, nor deletion in the DNA polymerase IV (β₇₀) gene, caused defective repair. The data obtained suggest that DNA polymerases δ and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis. Received: 18 November / 10 December 1996     

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3 end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.     

Two new group-II twintrons in the Euglena gracilis chloroplast are absent in basally branching Euglena species

Studies of the phylogeny and chloroplast intron content of selected Euglena species have led to insights in our understanding of the timing of intron acquisition. In the current study, two new twintrons, found in E. gracilis, have been characterized by the analysis of partially spliced pre-mRNAs. Intron 1 of atpE is a 463-nt group-II intron interrupted by a second group-II intron 320 nt long. Intron 1 of psbD is also a group-II twintron with external and internal introns of 635 nt and 463 nt, respectively. The two introns composing the psbD twintron, as well as six additional group-II introns found in the E. gracilispsbD gene, are not present in several basally branching Euglena species, including E. myxocylindracea, E. stellata and E. viridis. The distribution of psbD introns in Euglena is consistent with a late evolutionary acquisition of group-II introns in this lineage. Received: 4 March / 11 September 1996     

Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae : evidence of a partially overlapping role for thioredoxin

 Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates. However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S. cerevisiae compared to S. pombe. Glutathione metabolism was investigated to understand this delayed growth stasis in S. cerevisiae. The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts. A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied. Yeast thioredoxin was overexpressed in S. cerevisiae and was found to partially relieve the dependence of S. cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell. By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast. Received: 16 November 1999 / 15 February 2000     

Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum : two related ORFs in one loop of a group-I intron, a cox1/2 hybrid gene and an unusually large cox3 gene

 The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined. The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns. The insertion sites of the two group-I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal group-I introns, as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1. Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA. Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively. These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently. Received: 1 May / 16 September 1996     

Reduced performance of male and female athletes at 580 m altitude

This study examined the effect of mild hypobaria (MH) on the peak oxygen consumption (O_2peak) and performance of ten trained male athletes [ (SEM); O_2peak = 72.4 (2.2) ml · kg⁻¹ · min⁻¹] and ten trained female athletes [O_2peak = 60.8 (2.1) ml · kg⁻¹ · min⁻¹]. Subjects performed 5-min maximal work tests on a cycle ergometer within a hypobaric chamber at both normobaria (N, 99.33 kPa) and at MH (92.66 kPa), using a counter-balanced design. MH was equivalent to 580 m altitude. O_2peak at MH decreased significantly compared with N in both men [− 5.9 (0.9)%] and women [− 3.7 (1.0)%]. Performance (total kJ) at MH was also reduced significantly in men [− 3.6 (0.8)%] and women [− 3.8 (1.2)%]. Arterial oxyhaemoglobin saturation (S_aO₂) at O_2peak was significantly lower at MH compared with N in both men [90.1 (0.6)% versus 92.0 (0.6)%] and women [89.7 (3.1)% versus 92.1 (3.0)%]. While S_aO₂ at O_2peak was not different between men and women, it was concluded that relative, rather than absolute, O_2peak may be a more appropriate predictor of exercise-induced hypoxaemia. For men and women, it was calculated that 67–76% of the decrease in O_2peak could be accounted for by a decrease in O₂ delivery, which indicates that reduced O₂ tension at mild altitude (580 m) leads to impairment of exercise performance in a maximal work bout lasting ≈ 5 min. Accepted: 30 July 1996     

Starvation for a specific amino acid induces high frequencies of rho− mutants in Saccharomyces cerevisiae

Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of “adaptive mutation” experiments. Thereby, high proportions of mitochondrially respiratory deficient (rho⁻) mutants accumulated among the cells stressed on selective plates. Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho⁺ and rho⁻ cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes. We show that in spite of the slower growth of rho⁻ cells the late-appearing colonies cannot be interpreted as descending from rho⁻ revertants present before selective plating. Received: 7 November 1996 / 2 February 1997