Biomedical subjects

P Silar

Publications and source records attributed to P Silar.

At least 19 recordsLinked to original sources

PubMed2001

Cell degeneration in the model system Podospora anserina.

Podospora anserina is a filamentous fungus used in many studies of fundamental cell biology, including cell ageing. In this organism, ageing is defined as a diminution of cell ability to proliferate and/or differentiate. This may or may not culminate with cell death. Two different ageing processes are intensively studied. The 'Senescence' phenomenon is present in all wild-type strains, results in cell death and is caused by a yet undefined cytoplasmic and infectious element. It is associated with extensive mitochondrial DNA modifications. Longevity of the strains is controlled by a highly complex network of genes. Among these, those involved in cytosolic translation and respiratory metabolism are of special importance. The 'Crippled Growth' phenomenon is present only in strains with elevated translational accuracy. It does not result in cell death but in a severe impairment of cell growth, an acceleration of Senescence and a diminution of differentiation potencies. No mitochondrial DNA modification is associated with Crippled Growth. Another cytoplasmic and infectious element with peculiar properties, C, is causally involved in the set up of this cell degeneration. The study of P. anserina degenerative processes provides a conceptual framework to understand ageing in more complex organisms. Especially, it emphasises the complex control exerted by genes on longevity, the multiplicity of degenerative processes that may occur to cells with identical genotype and the potential role of non-conventional infectious elements in cell ageing.

Animals↗

PubMed2001

eEF1A Controls ascospore differentiation through elevated accuracy, but controls longevity and fruiting body formation through another mechanism in Podospora anserina.

Antisuppressor mutations in the eEF1A gene of Podospora anserina were previously shown to impair ascospore formation, to drastically increase life span, and to permit the development of the Crippled Growth degenerative process. Here, we show that eEF1A controls ascospore formation through accuracy level maintenance. Examination of antisuppressor mutant perithecia reveals two main cytological defects, mislocalization of spindle and nuclei and nuclear death. Antisuppression levels are shown to be highly dependent upon both the mutation site and the suppressor used, precluding any correlation between antisuppression efficiency and severity of the sporulation impairment. Nevertheless, severity of ascospore differentiation defect is correlated with resistance to paromomycin. We also show that eEF1A controls fruiting body formation and longevity through a mechanism(s) different from accuracy control. In vivo, GFP tagging of the protein in a way that partly retains its function confirmed earlier cytological observation; i.e., this factor is mainly diffuse within the cytosol, but may transiently accumulate within nuclei or in defined regions of the cytoplasm. These data emphasize the fact that the translation apparatus exerts a global regulatory control over cell physiology and that eEF1A is one of the key factors involved in this monitoring.

Alleles↗

PubMed2000

In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina.

BACKGROUND: To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. RESULTS: Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. CONCLUSION: Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells. This may contribute to the regulation of cell growth and division.

Cell Cycle↗

PubMed2000

Informational suppressor alleles of the eEF1A gene, fertility and cell degeneration in Podospora anserina.

Mutations that increase readthrough at a UGA stop codon (informational suppressor mutations) were created in the gene (AS4) that encodes translation elongation factor eEF1A in the filamentous fungus Podospora amserina. The results strongly suggest that the net charge of the eEF1A protein controls the accuracy of translation. Physiological analysis of the mutant strains shows that some of the alleles dominantly increase life span, while only one drastically modifies fertility. This exceptional allele (AS4-56) causes a wide array of phenotypes, including a new growth cessation phenomenon that is different from Senescence or Crippled Growth, previously known degenerative syndromes that are both controlled by AS4. The data emphasise the fact that eEF1A exerts a complex control over cellular physiology.

Alleles↗

PubMed2000

Deletion and dosage modulation of the eEF1A gene in Podospora anserina: effect on the life cycle.

eEF1A is encoded by a unique gene in the filamentous fungus Podospora anserina. We show here that (1) this gene is essential for vegetative growth, (2) readthrough at UGA stop codon level is positively correlated with eEF1A level, (3) eEF1A level is regulated in P. anserina. (4) Increasing eEF1A gene dosage does not modify P. anserina life cycle parameters, especially longevity is not changed. These data confirm and extend those previously obtained in yeast and Drosophila.

Gene Deletion↗

PubMed1999

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.

Aging↗

PubMed1999

Non-conventional infectious elements in filamentous fungi.

Old data (most often in French) described phenomena involving non-conventional infectious factors in filamentous fungi. Recently, it was shown that two yeast cytoplasmic determinants are similar to known mammalian prions, in that their different states are attributed to conformational changes of normal cellular proteins. In the light of this discovery, fungal elements are now being reconsidered. This review presents four elements that affect vegetative incompatibility, conidiogenesis, morphology and cell growth. Recently, one element has been shown to be a prion analogue. The status of the others is not clear. We consider the view that non-conventional inheritance might be initiated by the appearance, in the cytoplasm, of a metabolite or a macromolecule whose production involves a positive regulatory loop.

Cytoplasm↗

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↗