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Biomedical subjects

P Durrens

Publications and source records attributed to P Durrens.

9 recordsLinked to original sources

Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae.

The aim of this paper is to gather and complete data about four members of a new gene family. Mutation in SUR4 gene was originally selected as a suppressor of defects caused by mutations in RVS161 or RVS167 genes. Cloning and sequencing of the SUR4 gene were performed. The deduced protein contains six putative transmembrane domains. Sequence comparison revealed that two yeast genes, FEN1 and JO343, shared significant similarities with SUR4. Mutants for SUR4 and FEN1 have the same pleiotropic phenotype, including bud localization defects, resistance to an immunosuppressor and resistance to ergosterol biosynthesis inhibitors. The double inactivation of SUR4 and FEN1 genes is lethal. These data and other aspects implicating SUR4 in glucose metabolism, suggest an involvement of these genes in the dynamics of cortical actin cytoskeleton in response to nutrient availability. Moreover, the existence of a fourth homologous gene in C. elegans extends the family to pluricellular organisms.

Amino Acid Sequence

Evidence for a branched pathway in the polarized cell division of Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae can choose a bud site in one of two different spatial patterns (axial or bipolar) determined by their mating type. Genes important for bud-site selection have been identified and a linear model describing the hierarchy of these genes was proposed. We have uncovered a new class of genes which is required only for the bipolar pattern. The phenotype of the corresponding mutants coupled with epistasis experiments with some budding mutants already described suggest the existence of specific genes for the bipolar pathway.

Cell Division

Yeast mutants affected in viability upon starvation have a modified phospholipid composition.

Selection of mutations, based on the suppression of rvs161 delta defects, was performed. Ten mutants were obtained, ranged amongst four complementation groups, named SUR1, SUR2, SUR3 and SUR4. All sur mutations also suppress a mutation in another gene, RVS167, indicating that all six genes are involved in the same biological pathway. The sur mutant cells have abnormal morphologies in stationary phase, i.e. dumbbell-like in sur1, sur2 or sur3 strains and multi-budded in sur4 strains. Several phenotypic characteristics of the physiological suppressors as well as the rvs161 delta strain itself led us to analyse the phospholipid composition of the mutants. The assays show an overall decrease of the phospholipid amounts and modifications in the relative contents of some phospholipid classes in sur mutant cells.

Cell Division

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae.

The gag-myc oncogenic sequence of the avian retrovirus MC29 was first inserted in a multicopy expression vector allowing its expression in Saccharomyces cerevisiae. The oncogene transcripts were detected in yeast by Northern blot hybridization and gag-myc proteins were revealed by immunoprecipitation. On solid medium, the average size of gag-myc transformant colonies was smaller than control. In liquid cultures, the gag-myc strains had a doubling time of 4.7 h compared with 3.1 h in the controls. In one of the recipient strains, and after an initial transient period of 5 days, the gag-myc transformants became physiologically indistinguishable from control. In another recipient strain, the slow-growth phenotype is permanent. Plasmid instability is increased in gag-myc transformants. When a single copy of the gag-myc gene was inserted in a yeast chromosome, no phenotype was observed, showing that slow growth is the consequence of plasmid loss.

Animals

The yeast ATP synthase subunit 4: structure and function.

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.

Amino Acid Sequence

ATP4, the structural gene for yeast F0F1 ATPase subunit 4.

A plasmid containing the gene coding for the Saccharomyces cerevisiae F0F1 ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors. A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da. This subunit presents amphiphilic behaviour with two distinct domains. A high alpha-helix content of 77% was predicted from the sequence. Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.

Amino Acid Sequence

Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans.

The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions.

Aspergillus nidulans

Podospora mutant defective in glucose-dependent growth control.

A mutation (modE), previously described as a membrane mutation, results in several modifications of the female developmental cycle: a high density of protoperithecia, the unscheduled development of protoperithecia into sterile perithecia on the homokaryons of each mating type, and the independence of ascospore outgrowth from the substances normally required for germination. Cultured in liquid medium, the modE strain showed two additional specific features: a higher growth yield than that of wild-type cultures (plus 10% of dry weight) and an extreme reduction of cell life span. Both mutant traits were specific to glucose limitation. Despite the large difference existing in the sensitivity of cells to glucose starvation, the glycogen and trehalose reserves of mutant and wild-type cells were nearly identical. Considered together, these results suggest that the primary effect of the mutation lies in the disruption of a glucose-dependent regulation controlling the transition of the metabolic pattern of cells from growth to quiescence.

Ascomycota

Podospora anserina mutant defective in protoperithecium formation, ascospore germination, and cell regeneration.

A mutant (modx) was selected on the basis of the suppression of self-lysis due to a recessive mutation (modB). modx, a dominant mutation, reduced hyphal branching from nonapical cells, abolished protoperithecium formation, and induced the death of stationary cells only when these were isolated to obtain further development. Mutant ascospores, formed in the fruiting bodies which occasionally occur under specific conditions (32 degrees C on starved medium), showed a delay in the germination process (up to 3 months instead of about 5 h for wild-type ascospores) when submitted to incubation under standard conditions (26 degrees C on germination medium) and failed to germinate at 18 degrees C. Revertants from modx strains, selected on the basis of the suppression of the nonrenewal of growth from stationary cells, were wild type for all the other three defects. Indirect arguments suggested that the modx mutant strain might be defective in the control of a specific class of stable messenger ribonucleic acids which would be essential for the physiology of ascospores and stationary cells.

Alleles