Regulation of the fdhF gene encoding the selenopolypeptide for benzyl viologen-linked formate dehydrogenase in Escherichia coli

Summary In Podospora anserina a chromosome walk near the mating type locus was made possible through isolation of genomic sequences linked to a plasmid integrated in this part of the genome. Genetic analysis of 86 transformants obtained from the 5 first cosmids of this walk was performed. These data and those reported elsewhere for cosmids resulting from another chromosome walk allow us to draw two clear-cut rules for transformation with cosmids. First, the large majority of transformants arise from integration at the resident locus, contrasting with the heterologous process which predominates for plasmids. Second, all homologous integrations are highly unstable while all non-homologous integrations are stable. Analysis of the timing of the instability reveals that loss of the selective marker is probably limited to the fruiting body.     

Genetic and biochemical analysis of cycloheximide resistance in the fungus Podospora anserina

Genetic analysis of cycloheximide-resistant mutants has shown that at least three genes control the resistance to cycloheximide in Podospora anserina and that the antibiotic resistance is recessive to sensitivity. In vitro and in vivo studies of protein synthesis indicated that for two mutants cycloheximide resistance is associated with the ribosomes. For one of these mutants, the elongation step in protein biosynthesis is insensitive to cycloheximide over a wide range of concentration. In this mutant the resistance to cycloheximide is a property of the 60S subunit.This work was supported by the Centre National de la Recherche Scientifique ERA No. 485.     

The phenoloxidases of the ascomycete Podospora anserina: The three forms of the major laccase activity

In Podospora anserina three laccase activities (I, II and III) were identified. Present results show the existence of an additional lacaase (an anodic protein; MW 80,000; R_f 0.07). Laccase IV derived from the dissociation at acid pH (4.5) of a protein which showed identical molecular weight (390,000) and R_f (0.1) to the oligomeric laccase I. The recovery of laccase I activity when starting from laccase IV (purified by means of isoelectric focusing) suggests that laccase I itself was the source of laccase IV. In turn, laccase IV gave rise to the laccase III after electrophoresis or dialysis at basic pH (8.5).     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.     

The phenol oxidases of the ascomycete Podospora anserina

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (K _M) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns.Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak.Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o-and p-diphenols, low affinity for m-phenols. The maximal velocity remains largely uninfluenced.This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.     

Search for ribosomal mutants in Podospora anserina: Genetic analysis of mutants resistant to paromomycin

It has recently been shown that paromomycin, an antibiotic of the aminoglycoside family, is also active on eukaryotic cytoplasmic ribosomes. In the fungus Podospora anserina, genetic analysis of ten mutants resistant to high doses of paromomycin shows that this resistance is caused by mutations in two different nuclear genes. These mutants display pleiotropic phenotypes (cold sensitivity, mycelium and spore appearance and coloration, cross-resistance to other antibiotics). Double mutants are either lethal or very altered and unstable. Moreover, the cytochrome spectra of these mutants seem to indicate that cytoplasmic protein synthesis is affected. The mutants also display a slight suppressor effect. We can therefore assume that these mutations affect cytoplasmic ribosomes.This work was supported by a C.N.R.S. Grant (ATP Microbiologie No. 3052) and by a NATO Grant.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

A non-Mendelian MAPK-generated hereditary unit controlled by a second MAPK pathway in Podospora anserina

The Podospora anserina PaMpk1 MAP kinase (MAPK) signaling pathway can generate a cytoplasmic and infectious element resembling prions. When present in the cells, this C element causes the crippled growth (CG) cell degeneration. CG results from the inappropriate autocatalytic activation of the PaMpk1 MAPK pathway during growth, whereas this cascade normally signals stationary phase. Little is known about the control of such prion-like hereditary units involved in regulatory inheritance. Here, we show that another MAPK pathway, PaMpk2, is crucial at every stage of the fungus life cycle, in particular those controlled by PaMpk1 during stationary phase, which includes the generation of C. Inactivation of the third P. anserina MAPK pathway, PaMpk3, has no effect on the development of the fungus. Mutants of MAPK, MAPK kinase, and MAPK kinase kinase of the PaMpk2 pathway are unable to present CG. This inability likely relies upon an incorrect activation of PaMpk1, although this MAPK is normally phosphorylated in the mutants. In PaMpk2 null mutants, hyphae are abnormal and PaMpk1 is mislocalized. Correspondingly, stationary phase differentiations controlled by PaMpk1 are defective in the mutants of the PaMpk2 cascade. Constitutive activation of the PaMpk2 pathway mimics in many ways its inactivation, including an effect on PaMpk1 localization. Analysis of double and triple mutants inactivated for two or all three MAPK genes undercover new growth and differentiation phenotypes, suggesting overlapping roles. Our data underscore the complex regulation of a prion-like element in a model organism.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5 ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors

Summary A high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha.     

<Emphasis Type="Italic">Podospora anserina</Emphasis>: a model organism to study mechanisms of healthy ageing

The filamentous ascomycete Podospora anserina has been extensively studied as an experimental ageing model for more than 50 years. As a result, a huge body of data has been accumulated and various molecular pathways have been identified as part of a molecular network involved in the control of ageing and life span. The aim of this review is to summarize data on P. anserina ageing, including aspects like respiration, cellular copper homeostasis, mitochondrial DNA (mtDNA) stability/instability, mitochondrial dynamics, apoptosis, translation efficiency and pathways directed against oxidative stress. It becomes clear that manipulation of several of these pathways bears the potential to extend the healthy period of time, the health span, within the life time of the fungus. Here we put special attention on recent work aimed to identify and characterize this type of long-lived P. anserina mutants. The study of the molecular pathways which are modified in these mutants can be expected to provide important clues for the elucidation of the mechanistic basis of this type of 'healthy ageing' at the organism level.     

Isolation and characterization of a laccase gene fromPodospora anserina

The genome of the filamentous ascomycetePodospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene ofNeurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with theN. crassa laccase. In shaken cultures,lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance oflac2 mRNA. The promoter region oflac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded bylac2 are discussed.     

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus ‘14’, called segment ‘29’. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.     

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides

Summary Transformation of a Mucor circinelloides Leu^– strain with the plasmid pAD45, harbouring the wild-type allele (leuA⁺) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.