Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina

Summary The existece of plasmid like DNA was demonstrated in senescent mycelia of Podospora anserina (strain s) by biophysical and electronmicroscopic methods. According to their contour length of about 1.4 and 2.7 m respectively the molecular weight for the monomer is in the range of 3·10⁶.This work was supported by the Deutsche Forschungsgemeinschaft. The sojourn of P.A.L. for six months in Bochum was made possible by a Senior U.S. Scientist Award of the Alexander von Humboldt Stiftung (Bonn).     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

A model to explain senescence in the filamentous fungus Podospora anserina by the action of plasmid like DNA

Summary On the basis of genetic, physiological and biochemical data an attempt is made to explain the syndrome of senescence in Podospora anserina. A covalently closed circular DNA (ccc DNA) found only in aging mycelia is considered as the causative agent. It is assumed that during the juvenile phase this species of DNA is an integrated part of the mitochondrial genome. During the presenescent phase it becomes independent from the chondriome and begins to replicate within the mitochondrion. This amplification leads progressively to the senescent phase, where the mitochondria desintegrate and ccc DNA molecules are liberated which are able to propagate autonomously in the cytoplasm and transmit senescence through infection involving cytoplasmic exchange.     

Evidence for a common cytoplasmic determinant of longevity and senescence in the ascomycete Podospora anserina

Summary With the aim of establishing whether a relationship existed between longevity and senescent determinants, three kinds of experiments have been carried out.First, the study of heteroplasmons obtained by mixing together ground mycelia of different longevities, led to observe that the resulting heteroplasmons exhibited the longevity of one parent. Two interpretations were suggested: either the elimination of one sort of determinant, or the dominance, if the two remain together.Second, the use of heteroplasmons obtained by anastomosis allowed to follow the transmission of one type of determinants into a cytoplasm containing another type of determinants. The results are in agreement with the invasion hypothesis.Finally the multiplication of senescent determinants was followed during the incubation period. The number of senescent determinants increased exponentially. The experiments demonstrated that the increase rate is different for strains of different longevities. It can be established a correlation between the increasing rate and the amount of growth necessary before the senescent morphology becomes manifest.A common particle could be responsible of the longevity and senescence characteristics of a given race. The longevity determinant can be transformed into a senescent determinant either by a structural modification of the particle, or through a functional modificationThe correspondence between the longevity determinant and the senescence factor is discussed as the correspondence of the longevity determinant to a known cytoplasmic organelles.     

Linkage group-chromosome correlation in Podospora anserina

Summary Two reciprocal translocations have been studied in an attempt to establish a one-to-one assignment of the seven linkage groups of P. anserina to specific chromosomes. Genetical data demonstrated clearly the relationships of the two translocations with three linkage groups. Cytological observations at meiosis in crosses heterozygous for the chromosomal interchanges showed the characteristic cross-like figures and confirmed the fact that the two translocations had one chromosome in common. The correspondence of the three largest chromosomes, including the one bearing the nucleolar organizer, with three well known linkage groups has thus been established. Evidence is also given that loci exhibiting a percentage of second division segregation as high as 98% are not at the end of the chromosomes.     

Cold-sensitivity of a double mutant strain combining two ribosomal mutations in the ascomycete Podospora anserina

Summary A double mutant strain combining two ribosomal mutations conferring resistance to cycloheximide exhibits a cold-sensitive phenotype. At low temperature the biosynthesis of the 60S subunit is impaired. Genetic analysis of cold-resistant revertants have shown that this double mutant strain can be used efficiently to isolate new ribosomal mutations.     

Transformation by integration in Podospora anserina

Summary A new transformation system for spheroplasts of Podospora anserina has been developed. The recipient leu1-1 strain is auxotrophic for leucine. The plasmid DNA does not carry the wild-type allele leu ⁺.but a tRNA suppressor: su4-1 or su8-1. The following protocol for genetic analysis has been developed: the [leu ⁺transformants are crossed with another mutant strain, carrying the 193 mutation. This mutation prevents the pigmentation of the spores and is also suppressed by the cloned suppressor. Thus, the genetic analysis of the transformants can be performed directly on ordered tetrads by the observation of pigmentation restoration. The first application of the method is described comparing the integration points when different suppressors are used. Integration of the plasmid DNA in the homologous site was not the rule; in most cases the integration point was located elsewhere.     

Genes involved in caryogamy and meiosis in Podospora anserina

Summary Ten new mutants affected during caryogamy and first meiotic prophase have been isolated in Podospora anserina. They belong to nine loci, and only one mutant is allelic with a gene previously known. The loci are distributed on six of the seven linkage groups. The precise moment where meiosis is blocked or altered has been studied by light microscopy for each mutant. Several of them have a pleiotropic phenotype which suggests that the altered functions involved in meiotic process in these mutants are also involved in vegetative growth.The systematic search of meiotic mutants in P. anserina permitted the identification of twelve genes involved during first meiotic prophase. The time of gene action and the nature of the controled steps are discussed.     

The incompatibility relationships between geographical races of Podospora anserina

Summary The physiological action of heterogenic incompatibility between the geographical races s and S, which differ by a single gene and a corresponding cytoplasmic factor, has been studied. Microscopical, cytochemical, and enzymatic investigations have revealed that the cell disintegration occurring after anastomosis of incompatible hyphae under the immediate influence of the cytoplasmic factors leads to a destruction of cellular compartments which is followed by a liberation of catabolic enzymes. These incidents are counteracted by regenerative processes.Supported with grants from the Deutsche Forschungsgemeinschaft, Bad Godesberg, and from the Landesamt für Forschung, Düsseldorf.     

Increase of translational fidelity blocks sporulation in the fungus Podospora anserina

Summary AS7-1 and AS7-2 are antisuppressor mutations reducing the miscoding capacity of ribosomes. Strains carrying and AS7 mutation do not sporulate. We have investigated whether the sporulation deficiency is due to the decrease of translational ambiguity. Two major findings argue in favour of this assumption. First, a significant sporulation level is restored in the presence of paromomycin. Second, three mutations which restore the sporulation of AS7-2 increase the ribosomal misreading in vitro. They define two new loci for ribosomal suppressors, su11 and su12. The two ribosomal proteins altered by su11-1 and su12-1 have been identified by electrophoresis. The results are discussed in the context of a more general hypothesis proposed by Picard-Bennoun (1982).     

Lethal mitochondrial genotypes in Podospora anserina: A model for senescence

Summary Crosses between spg1 and spg2, two mitochondrial mutants of Podospora anserina, yield a new type of strain, called pseudo wild-type (PSW), in addition to wild-type recombinants. PSW strains are characterized by a variable phenotype for germination of ascospores and a variable longevity. By autofecondation, PSW strains yield early lethal strains (which die soon after the germination of the spores and so cannot be used for further studies), short-lived strains (which stop their vegetative growth after several centimeters) and long-lived strains (which grow longer than 16 cm). Genetic analysis of the last two categories shows that the PSW phenotype corresponds to a new mitochondrial genotype resulting from the interaction of the two parental mitochondrial genomes.Variability in the longevity of PSW strains is interpretated as the result of a high rate of mutation of their mitochondrial genome into a lethal and suppressive genome, similar to that of the mitochondrial rho ^- suppressive mutant of yeast. Furthermore, on the basis of the striking similarities observed between short-lived PSW strains and senescent cultures of Podospora anserina, we propose that commitment and development of senescence in wild-type strains of Podospora anserina would result, in a similar way, of spontaneous suppressive rho ^--like mitochondrial mutations.     

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.     

丝状真菌Podospora anserina中光敏色素基因的鉴定及功能分析

光敏色素在细菌和植物发育中起着关键作用,但它们在真菌中的生物学功能尚不完全清楚。【目的】探究光敏色素基因PaPhy1和PaPhy2在Podospora anserina有性生殖和无性发育中的作用及其调控机制。【方法】利用同源重组方法对P.anserina中2个光敏色素基因PaPhy1和PaPhy2进行定点敲除,获得光敏色素基因缺失菌株ΔPaPhy1和ΔPaPhy2,并通过遗传杂交构建双重突变体ΔPaPhy1ΔPaPhy2;分析突变型菌株和野生型菌株在不同光照下有性生殖、无性发育、生长速率和活性氧代谢等方面的差异,明确光敏色素基因在P.anserina中的主要功能。【结果】白光和蓝光诱导P.anserina子实体的形成,ΔPaPhy在光照下产生子实体的数量减少,ΔPaPhy的生命周期延长。【结论】光敏色素基因与P.anserina有性生殖密切相关;ΔPaPhy的衰老延迟和活性氧代谢有关。本研究的结果为进一步探索光照对丝状真菌繁殖调控机制以及抗衰老研究提供了新的思路。     

转录因子PaPho与丝状真菌Podospora anserina中磷元素的吸收和利用研究

作为生物体必需的营养元素之一,磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用,可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P.anserina中2个磷代谢相关转录因子PaPho1和PaPho2,遗传杂交构建双重突变体ΔPaPho1ΔPaPho2;通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化;利用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上,ΔPaPho1ΔPaPho2无法生长;在添加有机磷的培养基中,ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中,ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%,ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P...     

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1 ⁺ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron. Received: 26 April 1996 / Accepted: 22 August 1996     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids. Received: 13 November 1997 / Accepted: 17 February 1998     

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).