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P Silar

Publications and source records attributed to P Silar.

18 recordsLinked to original sources
PubMed1999

Propagation of a novel cytoplasmic, infectious and deleterious determinant is controlled by translational accuracy in Podospora anserina.

Some mutant strains of the filamentous fungus Podospora anserina spontaneously present a growth impairment, which has been called Crippled Growth (CG). CG is caused by a cytoplasmic and infectious factor, C. C is efficiently transmitted during mitosis but is not transmitted to the progeny after sexual crosses. C is induced by stationary phase and cured by various means, most of which stress the cells. Translational accuracy is shown to tightly regulate the propagation of C during the active growth period, because its propagation in dividing hyphae is restricted to cells that display an increased translational accuracy. However, induction of C in stationary phase proceeds independently from the translational accuracy status of the strain. CG does not seem to be accompanied by mitochondrial DNA modifications, although C activates the action of the Determinant of Senescence, another cytoplasmic and infectious element, which causes a disorganization of the mitochondrial genome. In addition, presence of C drastically modifies the spectrum of the mitochondrial DNA rearrangements in AS6-5 mat- cultures during Senescence. C seems to belong to the growing list of unconventional genetic elements. The biological significance of such elements is discussed.

Ascomycota↗
PubMed1998

Identification of the genes encoding the cytosolic translation release factors from Podospora anserina and analysis of their role during the life cycle.

In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

Alleles↗
PubMed1997

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription.

The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly for acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution of redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Cloning, sequencing, and transgenic expression of Podospora curvicolla and Sordaria macrospora eEF1A genes: relationship between cytosolic translation and longevity in filamentous fungi.

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi, Podospora curvicolla and Sordaria macrospora. These fungi are close relatives of Podospora anserina and also show senescence syndromes. Comparison of the sequences of the deduced proteins with that of P. anserina reveals that the three proteins differ in several positions. Replacement of the P. anserina gene by either of the two exogenous genes does not entail any modification in P. anserina physiology; the longevity of the fungus is not affected. No alteration of in vivo translational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.

Amino Acid Sequence↗
PubMed1997

Contribution of various classes of defective mitochondrial DNA molecules to senescence in Podospora anserina.

The unavoidable arrest of vegetative growth in Podospora anserina (senescence process) is always correlated with rearrangements of the mitochondrial chromosome, mainly consisting in the amplification of particular regions as tandemly repeated circular molecules (senDNAs). One sequence systematically amplified in senescent cultures corresponds precisely to the first intron (intron alpha) of the cox1 gene; nevertheless, other regions (called beta and gamma) are also frequently amplified. The experiments presented in this paper show that cellular death is in some cases associated with the sole presence of large amounts of senDNA beta. In addition, we provide evidence that senDNA beta and senDNA alpha accumulate by different mechanisms, as previously proposed. This suggests that beta senDNAs have a lethal effect on the mycelium on their own and most likely have replicative properties independent of the presence of sequence alpha. These data do not fit well with the current opinion that gives an essential role to intron alpha in the senescence of P. anserina.

Aging↗
PubMed1997

Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing senescence of Podospora anserina.

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNA alpha (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron alpha) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNA alpha seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron alpha plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.

Amino Acid Sequence↗
PubMed1997

[Genetic analysis of two cellular degenerations in filamentous fungus Podospora anserina].

The filamentous fungus Podopsora anserina presents an unavoidable arrest of vegetative growth (Senescence) determined by a cytoplasmic and infectious factor. Senescence is correlated with a disorganization of the mitochondrial DNA. This disorganization is caused by an event which is not the appearance of the first defective DNA molecules. These ones are generated constitutively and their accumulation during Senescence requires the presence of an additional factor. Life span of the strains is under nuclear and cytoplasmic genetic control. At least 600 nuclear genes influence longevity. Our analysis focuses on the role of the genes involved in cytosolic translation, since mutations in these genes seem to display the most drastic effects on longevity but also on the structure of the defective mitochondrial DNA molecules that accumulate during Senescence. We have detected in some Podospora anserina mutant strains (permissive strains) the presence of a novel cytoplasmic and infectious determinant that entails an easily discernible phenotype associated with a severe growth alteration (Crippled Growth). This growth alteration is not associated with mitochondrial DNA modifications. Only the strains that have an increased translational accuracy present Crippled Growth. However, the Crippled Growth Determinant is found in all the strains during the stationary phase; it is eliminated from the non permissive strains during the exit of the stationary phase. The mutants, that have an increased translational accuracy, probably lack a factor which is needed to eliminate the determinant when cells enter the growth phase.

Ascomycota↗
PubMed1996

Genes that control longevity in Podospora anserina.

Two genetic methods were used to estimate the number of genes that potentially modulate longevity in the filamentous fungus Podospora anserina. First, life span of strains carrying mutations selected on criteria unrelated to senescence was measured. Second, strains bearing random mutations were generated by insertional mutagenesis. Life span of these strains was then measured. Surprisingly, both methods lead to the conclusion that a large number of genes (between 600 and 3000) can modulate life span. Among, the mutations that affect longevity, 50% increase life span and 50% diminish it.

Ascomycota↗
PubMed1996

Response of Drosophila metallothionein promoters to metallic, heat shock and oxidative stresses.

The metallothionein system in Drosophila melanogaster is composed of two genes, Mtn and Mto. In order to compare the induction properties of these genes, we transformed D. melanogaster with P-element vectors containing Adh and lacZ reporter genes under the control of Mtn and Mto promoters, respectively. Mtn and Mto transgenes are mainly expressed in digestive tract. However, Mtn expression has been detected also in the fat body. Mtn and Mto transgenes respond differently to metallic, heat-shock and oxidative stresses. These data confirm that both genes are in part functionally different.

Alcohol Dehydrogenase↗
PubMed1994

Increased longevity of EF-1 alpha high-fidelity mutants in Podospora anserina.

Various translation initiation and elongation factors seem to participate in the control of the cellular proliferation and the ageing process in higher eukaryotes. Studies indicate that EF-1 alpha, one of the translation elongation factors, may be one of the major components involved. We here present the cloning of the filamentous fungus P. anserina EF-1 alpha encoding gene and show that strains bearing high fidelity mutations in the EF-1 alpha gene have a drastically increased longevity as well as an impairment in sporulation. This suggests that EF-1 alpha involved in the sexual and senescence processes in lower eukaryotes, through the control of translational errors.

Amino Acid Sequence↗
PubMed1994

Rapid methods for nucleic acids extraction from Petri dish-grown mycelia.

We have developed rapid and economic methods for the isolation of nucleic acids from filamentous fungi. The main advantages of these methods are: (1) the mycelium is directly recovered from a Petri-dish culture, (2) the complete experiment takes place in microfuge tubes, (3) it is very fast and allows for the processing of 24 samples in the same day, and (4) up to 100 micrograms of total DNA or RNA are recovered, both of which are sufficiently pure for most purposes. Of particular interest is the recovery of large amounts of mitochondrial DNA as visualised by electrophoresis in ethidium bromide-stained gels.

Culture Media↗
PubMed1994

Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways.

Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region. CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.5 h. Although a carboxyl-terminal HSF transcriptional activation domain is critical for the activation of CUP1 transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S. cerevisiae hsp70 family. Furthermore, inactivation of the chromosomal SNF1 gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUP1 transcriptional activation in response to glucose starvation without altering heat shock-induced transcription. These studies demonstrate that the S. cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.

AMP-Activated Protein Kinases↗
PubMed1991

ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene.

Copper, zinc superoxide dismutase (SOD1 gene product) (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a copper-containing enzyme that functions to prevent oxygen toxicity. In the yeast Saccharomyces cerevisiae, copper levels exert some control over the level of SOD1 expression. We show that the ACE1 transcriptional activator protein, which is responsible for the induction of yeast metallothionein (CUP1) in response to copper, also controls the SOD1 response to copper. A single binding site for ACE1 is present in the SOD1 promoter region, as demonstrated by DNase I protection and methylation interference experiments, and is highly homologous to a high-affinity ACE1 binding site in the CUP1 promoter. The functional importance of this DNA-protein interaction is demonstrated by the facts that (i) copper induction of SOD1 mRNA does not occur in a strain lacking ACE1 and (ii) it does not occur in a strain containing a genetically engineered SOD1 promoter that lacks a functional ACE1 binding site.

Base Sequence↗
PubMed1991

New shuttle vectors for direct cloning in Saccharomyces cerevisiae.

We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells. The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene. DNA fragments are inserted in a polylinker located after the beginning of the URA3 coding sequence. Transformants are selected either by Trp or Leu prototrophy. Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3+ gene (thus, this method requires the recipient strain to be ura3-); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA. Using these plasmids, we were able to directly reclone the ACE1 gene from genomic DNA by directly transforming a strain deleted for ACE1. These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter.

Base Sequence↗
PubMed1991

Heat shock transcription factor activates transcription of the yeast metallothionein gene.

In the yeast Saccharomyces cerevisiae, transcription of the metallothionein gene CUP1 is induced by copper and silver. Strains with a complete deletion of the ACE1 gene, the copper-dependent activator of CUP1 transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP1 transcription. To identify genes which mediate basal transcription of CUP1 or which activate CUP1 in response to other stimuli, we isolated an extragenic suppressor of an ace1 deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP1 transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp70 gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that HSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.

Amino Acid Sequence↗
PubMed1990

Metallothionein Mto gene of Drosophila melanogaster: structure and regulation.

We report the sequence of the Mto gene, one of the two known metallothionein genes of Drosophila melanogaster, and compare its structure with that of the other metallothionein gene, Mtn. The main structural features are the presence of a small intron (61 base-pairs), the presence of four potential MREs (metal regulatory elements) and the absence of a TATA box in the promoter region. Of all metals tested, Hg2+, Cd2+ and Cu2+ are the most efficient ions for inducing an increase in Mto gene transcription. The Mto and Mtn genes are differentially regulated during normal development. Transcription of Mto is detected early in embryogenesis (0 to 3 h) and persists to the third larval instar, while Mtn expression starts later in embryogenesis (12 to 15 h) and is thereafter maintained throughout larval development and adult stages. Sequencing of the Mto protein is in good agreement with the nucleic acid data. Surprisingly, attempts to isolate and characterize the Mtn protein were unsuccessful. Several lines of evidence suggest that this metallothionein is rapidly incorporated after its synthesis into lysosomes, where it would be processed in a way that would not permit its purification. The function of the Mtn protein thus appears to be mainly related to detoxification processes. The pattern of expression of Mto suggests that this gene may be involved in the control of metal homeostasis during development.

Amino Acid Sequence↗
PubMed1990

Expression of the Drosophila melanogaster metallothionein genes in yeast.

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mtn, that code for distinctly different proteins. In order to compare the properties of Mto and Mtn, we transformed yeast with several fusion plasmids. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper-resistance phenotype to copper-hypersensitive cells. Both Mto and Mtn proteins can be characterized in extracts from transformed yeast cells.

Animals↗