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A genetic block, effective only with the addition of mutations in two genes, suppresses both the incompatibility process in strain confrontation (“barrage”) and the formation of female organs. Investigations on this block lead us to propose that it operates at the ribosomal level and prevents the translation of the messenger of a proteolytic enzyme, a postulated early protein in protoperithecia differenciation.

Summary: The growth of a mycelium of Podospora anserina ceases after a period of time characteristic for a given race. As the mycelium grows out from the germinated spore it passes through two physiological states; a ‘non-senescent’ state where the growth potential of the various regions of the growing mycelium remains constant and a ‘senescent’ state where the growth rate remains constant but the growth potential of the mycelium progressively decreases. The ‘senescent’ state is apparently due to the presence of a variant cytoplasmic determinant which is discontinuously distributed among the cells of the mycelium and whose concentration increases exponentially during growth. The rate of transformation from the ‘non-senescent’ to ‘senescent’ state is directly proportional to the number of hyphae per mycelium. The median length of growth can be greatly extended by short periods of growth in the presence of cycloheximide or cyanide, but not by a variety of other drugs which inhibit mitochondrial functions.

SUMMARYForty-one suppressors obtained after NG and EMS mutagenesis of two ‘polar’ mutants of segment ‘29’ inPodospora anserinawere genetically analysed. Three classes of suppressor could be distinguished on spectrum pattern criteria. One representative suppressor of each class was demonstrated to be non-gene specific. The class I suppressor was dominant and only suppressed polar mutants in segment ‘29’ and non-ICR-induced ones in genes where polarity cannot be determined. Class II and III suppressors were partially dominant and they suppressed polar, non-polar and even ICR-induced mutants. The difference between classes II and III seems to be only quantitative. According to whether class II and III suppressors are considered strongly or weakly allele-specific, two hypotheses are considered. First,tRNAs could be involved in all three classes of suppression: class I would be nonsense-specific and classes II and III would be nonsense-missense suppressors. Secondly,tRNA could be involved only in class I suppression, while ribosomal ambiguity could be responsible for class II and III suppression.

The present study deals with conversion analysis within the polycistronic unit of transcription of locus “14” in Podospora anserina. Data from 74 two-point crosses involving 33 different mutants are in favour of a conversion polarity exhibiting two reversals (Fig. 1a, b), one being entirely located within the distal cistron. This pattern of polarized recombination suggests the existence of an intracistronic recombinational discontinuity. Two, three, four and five-point (within the polycistronic unit) crosses allow to draw several conclusions concerning the conversion event: it involves a continuous chromosomal segment; it can take place in two or several sites belonging to the same cistron or to different contiguous cistrons; it can overlap the region corresponding to the postulated discontinuity; its maximum dimension is smaller than that of the polycistronic unit. Many two-point crosses give double-site conversions occurring as two separate but correlated events, and involving two homologous chromatids (non associated conversions). The frequency of such recombination events is lower than that of a simultaneous conversion. All these results are briefly discussed in terms of different recombination models.

In order to learn the internal conditions for the production of the various phenoloxidases produced by the Ascomycete Podospora anserina the wild strain has been grown under controlled conditions in a fermenter for a period of 34 days. Samples were withdrawn at regular intervals and assayed for mycelial yield and intra- and extracellular phenoloxidase production. Maximal yield was obtained at the following age of the culture: Mycelial production 9 d, tyrosinase 4 d, the high molecular weight laccase I between 9 and 19 d. The low molecular weight laccases II and III, initially present in medium concentrations, dropped to an early minimum after 4 days, followed by an increase with a maximum in the late autolytic phase. The changes in the phenoloxidase spectrum and the antiparallel production curve for the high molecular weight against the low molecular weight laccases are discussed in relation to the earlier observed genetical and physiological control of phenoloxidase synthesis and in relation to the possibility of laccase I being composed of active subunits of low molecular weight laccases.

Summary: Reciprocal crosses and repeated backcrosses have shown that the distance of growth before the first mycelium of a population exhibits ‘senescence’ and the rate at which the remaining mycelia exhibit ‘senescence’ are cytoplasmically inherited in several races of Podospora anserina. A nuclear gene (the plus mating type allele of race S) was found to influence the timing of ‘senescence’ in interracial hybrids carrying the cytoplasmic determinants of race A. Known nuclear genes segregated normally in the interracial crosses employed in the work.

The behaviour of the division spindle and centrosomal plaques is described in four species of Ascomycetes (Ascobolus immersus, Ascobolus stercorarius, Podospora anserina and Podospora setosa) studied by light and electron microscopy. Two unique features of the kinetical apparatus were observed: presence of centrosomal plaques and intranuclear location of the spindle. In all types of mitoses (mycelium, crosier and postmeiotical mitosis) the apparatus is structurally identical to that found in meiosis. The centrosomal plaques, present in all divisions, are always contiguous with the nuclear envelope and never show centrioles similar to those commonly found in Metazoa and Protozoa. During metaphase and anaphase the plaque is constituted of two zones situated on each side of the nuclear envelope: an electron opaque outer zone and inner one less opaque in which most of the microtubules end. In Podospora the outer zone appears in sections as consisting of two dark layers separated by a clear one. Two dispositions of plaques are possible: either they are entirely contiguous with the nuclear envelope (Ascobolus) or only partially so, the remainder being perpendicular to the nuclear envelope (Podospora). — The localisation of the plaques in the ascus was determined by light and electron microscopy. The nuclear envelope was shown to remain intact during division. It was possible to observe that the sporal wall of each spore originated from the same unique double membrane formed in the ascus during the meiotic second division and postmeiotical mitosis. This fact is of genetical interest for the study of morphological and physiological characters of the spores.

SUMMARY: The ascus cytology of Podospora anserina (Ces.) Rehm, a secondarily homothallic Pyrenomycete fungus, has been followed under the light microscope from the crozier, through karyogamy and the three nuclear divisions in the ascus (the first two constituting a meiosis and the third a mitosis) to the first spore mitosis and spore maturation. The spindles remain widely separated at division II, during which segregation for mating type is said to occur. After division III, in which the spindles are transverse, the ascus contains four pairs of sister nuclei. The realignment of the nuclei so that two non-sister nuclei are included in each of the four spores is brought about by the movements of the centrosomes or, more likely, of their outer portions. Spore delimitation, which starts from these organelles, appears as a thin cleavage line separating the spore plasm from the epiplasm of the ascus. A mitosis in the spore initial followed by degeneration of one of the four daughter-nuclei in the primary appendage leads to an imbalance in the mating-type factors in the spore. The wall of the mature spore is separable into three layers, the middle one of which is pigmented. The mucilaginous secondary appendage formed at each end of the spore is already present before the primary appendage is cut off.

Der cis-trans-Positionseffekt in Heterokaryen der nicht gekoppelten morphologischen Mutanten z und oct-1 von Podospora anserina wird durch quantitative Kernunterschiede vorgetauscht.

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.

SUMMARYDouble cross-overs were detected in a few crosses involving segment ‘29’ mutants of locus ‘14’ in Podospora anserina. The occurrence of such recombination events made it necessary to reconsider the localization of three mutants. Use of an outside marker and the analysis of many other crosses allowed these mutants to be mapped unambiguously. The change in the localization of one of these mutants together with complementation tests on a new mutant leads to the conclusion that the polycistronic unit of transcription contains only four cistrons instead of the five previously thought to exist.

Laccase of the wild strain of Podospora anserina was purified by subsequent treatment with protamine sulfate, precipitation with ammonium sulfate and column chromatography on DEAE-Sephadex and hydroxylapatite. The purity was confirmed by sedimentation and electrophoresis (molecular weight about 361 000, isoeletric point at pH 5.1).

SUMMARY: The ascospores of Podospora anserina (Ces.) Rehm are delimited by a double membrane system. The primary spore wall develops within this, the outer part of the double membrane being pushed out to form the spore membrane and the inner part forming the plasmalemma of the spore. Starting in the middle of the matrix of the expanding primary wall, a secondary wall is laid down and gradually extends to the outer periphery of the spore wall. Later, a thick tertiary wall is formed at the inner side of the secondary wall by blocks of electron-dense material between which channels of the primary wall matrix remain. This is the pigmented layer of the spore wall. On the innermost side of the spore wall, a part of the original primary wall remains. The primary appendage at the base of the spore arises as part of the spore initial, but, after it has been cut off by a septum, its contents degenerate and it is bounded only by the primary and secondary wall layers. The secondary appendages, formed at the apex of the spore and at the bottom of the primary appendage, are considered to be actively growing processes bounded by the spore membrane.

Incompatibility in the ascomycete Podospora anserina results in death of cells where the products of two antagonistic genes are meeting. These genes can be alleles or non alleles. In all cases incompatibility, like in graft incompatibility in mammals, results from an heterogenic mechanism. From the study of numerous wild type strains, 9 incompatibility loci were identified. For two of them—c and e loci—allelic series are known (Table 1).

Aus dem Wildstamm des Ascomyceten Podospora anserina wurden zwei Aminopeptidasen isoliert, die immunologisch nicht verwandt sind: P1 hat ein MG von ung. 140000, sie kann durch Hitzebehandlung aktiviert werden (z. B. 8 min bei 80° C um 100%) und wird von DFP (5 mM) vollstandig gehemmt. Wie Hydrolyseversuche mit uber 30 Oligopeptiden zeigten, hat sie eine ziemlich enge Substratspezifitat (z. B. fur Ala-Pro, Gly-Pro-Ala, Glu-Gly-Phe und Valinpeptide). P1 wird durch Behandlung mit 0,1% SDS nicht in Untereinheiten gespalten.

In the fungus Podospora anserina , the interaction between the nonallelic incompatible R and V genes has two consequences: a lytic reaction due to the synthesis of specific proteolytic enzymes, and a quenching in protein and ribonucleic acid synthesis. The incompatibility reaction when vegetative or sexual R and V cells fuse is asymmetric: it is induced only in the R protoplasm. The cessation in ribonucleic acid and protein synthesis was investigated in heterokaryotic strains carrying the antagonistic R and V genes and their “neutral” r and v alleles. Asymmetry between R and V genes lies in the fact that the strains homozygous for the R genes are the only strains that cannot grow. From these results it is postulated that the V -gene product is a diffusible cytoplasmic factor and that the R -gene product, which is nonautonomous, is a ribosomal component.

1. A model is presented which may help to elucidate the growth pattern of the clock-mutant zonata of Podospora anserina. The model consists of two main parts. The superficial hyphae grow with an increasing change of the branching pattern and a decreasing rate of elongation. The submerged hyphae do not exhibit this development for they are growing under oxygen deficiency. They emerge in front of the retarding superficial hyphae and thus initiate the next growth band. 2. The model was supported by the following results: Absence of oxygen from the surface of the cultures prevented the rhythmic growth; mycelia separated from the medium by a cellophane cover produced no zonations or rings and grew at a decreasing rate; the presence of a sufficient supply of oxygen in the total medium, as achieved in vigorously shaken cultures, prevented the formation of the successive growth phases. 3. Further evidence for this model was found in the literature. Various aspects of this rhythmic type of growth in other fungi are explained in terms of this model.

Genetic analysis of 61 mutations of the median segment of the complex locus “14” in Podospora anserina is presented. These mutations affecting spore pigmentation have been screened following various mutagenesis (UV, Nitrosoguanidine, ICR-170) and they display several phenotypes. Comparison of complementation and genetic maps shows that this genetic region exhibits the most conspicuous genetic properties of classical operons: polarized complementation, asymmetric localization of non complementing mutants and suppressibility of some such ones. This polycistronic unit of transcription would contain five cistrons. The products coded by at least three of these genes would be involved in a multiprotein complex.

Three meiosis-deficient mutants of gene mei2 (mei2-1, mei-2-2 and mei2-3) are blocked during the prophase I of meiosis, before normal pachytene. The mutant mei-2-2 is leaky and there is a partial complementation in crosses mei2-2xmei-2-1 and mei2-2xmei2-3. It has thus been possible to analyse descendants of these crosses. This analysis shows an important alteration in recombination frequencies on at least three different linkage groups. Recombination frequencies appear to be increased near the centromere and decreased in other regions of the chromosomes. This coincides with a decrease in chiasma interference. Intergenic recombination is increased in a locus located very near to the chromosome II centromere. Moreover, the relative proportion of crossovers among the recombination events is stronger than in the control. Though it is impossible at present to formulate a precise hypothesis for the action of the mei2 gene at the molecular level, it is proposed that it might well control a stage of the DNA repair or synthesis.

Summary. 1 . By using an improved method for purification of the laccase I of the wild type of Podospora anserina, yield and specific activity of this enzyme have been increased, whereas its stability decreased. 2. Freshly prepared laccase I seems to be homogeneous, according to its behavior in hydroxylapatite-chromatography, in sedimentation-analysis and in disc-electro-phoresis. 3. The catalytic, electrophoretic, spectral and molecular properties and the carbohydrate-composition of laccase I have been investigated. Laccase I exhibits a double-pH-optimum for its reaction with dopa (at pH 5.5 and 7.5), the isoelectric point being at pH 4.9. The enzyme is reversibly inhibited by sodium azide (Kt = 1.9xlO -6 M) and has absorption-maxima at 280 and 610 nm. The content of nitrogen, copper and carbohydrate is 13.6%> 0.3 °/o and 23% of the dry weight, respectively. Hexosamine, mannose, glucose and galactose have been found in the carbohydrate moiety of the enzyme. The following molecular data w

Electron microscopy of three intracellular laccases from the Ascomycete Podospora anserina showed that the high molecular weight laccase I (Mr = 390 000) appears mainly as tetramers with the four subunits arranged at the corners of rectangles (13.3 nm × 8.5 nm). In size and shape the subunits of laccase I equal the monomeric molecules of laccase II (Mr = 70 000, 6.8 nm × 5.5 nm) and laccase III (Mr = 80 000, 7.0 nm × 5.8 nm). The dimensions of the monomeric laccases estimated from the electron micrographs are in good agreement with the hydrodynamic measurements of their molecular weight.

THE failure to form heterocaryons between non-isogenic strains in fungi, which has been reported for many species, results from protoplasmic disintegration that quickly follows hyphal fusion. Genetic control of this heterogenic incompatibility has been investigated especially in the Ascomycetes Neurospora crassa1 and Podospora anserina2. Cellular (or protoplasmic) incompatibility always arises from a very specific interaction between two genes. Allelic mechanisms, when antagonistic genes are allelic, are found in the two species. In Podospora anserina, however, three incompatibility mechanisms, c/d, c/e and r/v, involve genes of unlike loci3,4.

The experimental study of wild and mutant strains of the two ascomycetes Ascobolus immersus Pers. ex Fr. and Podospora anserina (Cesati) Niessl shows that their pattern of growth, smooth or wavy, depends on the working of definite nuclear genes. It is the same with the monopodial or sympodial arrangement of hyphae. But the co-ordination of the developmental phenomena within a mycelium is achieved by the circulation of internal cytoplasmic agents of hormonal nature.