Jacques Labarère

Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants.

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per μg of DNA. The phenotype of the stable Ura+transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.

Isolation of transcripts preferentially expressed during fruit body primordia differentiation in the basidiomycete Agrocybe aegerita.

SummaryAn Agrocybe aegerita cDNA library, constructed from fruit body primordia poly(A)+ RNAs, was screened by differential colony hybridization. Clones which preferentially hybridized to poly(A)+ RNA sequences from fruit body primordia, versus poly(A)+ RNAs from mycelium, were isolated. Eight of these clones (EMAa-1 to EMAa-8) encoded eight different poly(A)+ RNAs which were demonstrated to be undetectable in the four stages preceding primordia formation and to be concomitantly accumulated when primordia differentiate, suggesting that EMAa gene products are closely involved in the morphogenesis of primordia. The eight EMAa cDNAs hybridize to at least seven unique regions distributed randomly in the A. aegerita genome. The expression of two EMAa cDNA sequences in E. coli led to the isolation of their gene products as fusion proteins.

Isozyme characterization of dikaryotic strains of the edible basidiomyceteAgaricus bitorquis (Quel.) Sacc. (syn.Agaricus edulis)

Abstract Seven isozyme activities were studied by isoelectric focusing and blotting of total protein extracts of dikaryotic strains of Agaricus bitorquis (Quel.) Sacc. (syn. Agaricus edulis ) to characterize strains and varieties and to provide information for subsequent protection of new putative commercial varieties. Isoelectric focusing gave reproducible patterns and blotting onto nitrocellulose membrane increased the sensitivity of enzyme detection. Five activities showed high variability: alcohol dehydrogenases, dopa-reacting enzyme phenoloxidases, tolidine-reacting enzyme phenoloxidases, esterases, and peroxidases. Two activities, diaphorases and acid phosphatases, presented low variability. Compilation of patterns obtained for the seven isozyme activities allowed the distribution of the closely genetically related varieties into separate groups. The five enzyme activities with high variability were sufficient to discriminate all the A. bitorquis varieties tested and to define a method for characterization and protection of new strains. Analysis of these patterns and comparison with other higher fungi showed that the variability in A. bitorquis is comparable with those described for Pleurotus eryngii, Coprinus congregatus , and ·Lentinus edodes , and higher than the variability found for Agaricus bisporus .

Incompatibility in the fungus Podospora anserina

SummaryIn the Ascomycete Podospora anserina the incompatibility reaction due to the interaction of non allelic genes exhibits some sensitivity to the antibiotic dihydrostreptomycin as well as to high levels of Magnesium. This incompatibility reaction can be suppressed or made more sensitive to the Magnesium or dihydrostreptomycin effect by mutations in the same genes mod1 and mod2. Properties of mutant strains suggest that mod1 and mod2 are ribosomal genes whose products seem to regulate, in a positive way for the first gene and in a negative way for the second gene, the translation of specific messengers, especially that of some proteolytic enzymes.

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning, physical mapping and gene location

SummaryThe mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.

Evidence for viral and naked double-stranded RNAs in the basidiomycete Agrocybe aegerita

SummaryDouble-stranded (ds) RNAs were purified from the vegetative mycelium of the commercial basidiomycete Agrocybe aegerita, by gel-filtration chromatography, and their dsRNA nature was demonstrated. They were shown to be distributed in two different RNase-resistant complexes which were separated by sucrose density gradient ultracentrifugation: the first complex is the encapsidated genome (6200 bp) of an isometric mycovirus; the second consists of three naked dsRNA molecules (1900-1800-1700 bp respectively) associated with large vesicles or mitochondria. This is the first report of the presence of naked dsRNA, unencapsidated, viral genome fragments which have co-evolved with their host, or else represent new replicating genetic elements (dsRNA “plasmids”) of unknown function, in a non pathogenic higher fungus.

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.

Isolation ofSpiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction, which consisted essentially of extended sheets of membranes, exhibited membrane-boundpara-nitrophenylphosphatase specific activity 1.5-fold over that of the whole-cell lysate. Only traces of soluble NADH oxidase were present in the membrane preparation. The latter fraction appeared homogeneous upon sorbitol density gradient centrifugation and banded at an equilibrium density of 1.107 g/ml. The plasma membrane proteins were then analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 40 different proteins were detected in the membrane preparations. By comparison with the patterns obtained for whole-cell extracts and cytoplasmic fractions, a protein map ofS. citri could be established in which membrane and cytoplasmic proteins were identified.

Involvement of a large inverted repeated sequence in a recombinational rearrangement of the mitochondrial genome of the higher fungus Agrocybe aegerita

SummarySouthern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two singlecopy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.