Gérard Barroso

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.

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.

Influence of genetic markers and of the fusing agent polyethylene glycol on chromosomal gene transfer in Spiroplasma citri.

Gene transfer-recombination involving three different chromosomal markers (ArsR, VanR, and XylR) and influence of the fusing agent PEG on transfer efficiency were investigated in matings between single-resistant or single and double-resistant strains ofSpiroplasma citri. Efficiency and kinetics vary, inS. citri, with the markers involved in the crosses and are not enhanced by the membrane fusion effect of PEG. These features allow one to distinguish the chromosomal gene transfer inS. citri from the closely related transfer by protoplast fusion in Gram-positive bacteria. However, the results may be explained by a spontaneous high frequency of the membrane fusion events ofS. citri cells and the existence of variations in the recombination frequency of the markers. Indeed, inS. citri, the formation of the heterozygotic polyploid structure formed after membrane fusion may be considered as a nonlimiting step of the mechanism and allows one, accordingly, to point out differences in recombination frequencies of the markers.

Molecular organization and heredity of the mitochondrial genome in Basidiomycetes

ConclusionThe studies on basidiomycete mitochondrial genomes provide numerous amounts of information with important repercussions on fundamental as well as aplied fields of mycology. This information is particularly interesting to furnish performant molecular markers (RFLPs) which allow a precise characterization of species and, within a species, constitute strain-specific markers.Moreover, these markers will be useful to correlate sequences of the mitochondrial genome with physiologically important and, at this time, seemingly unrelated functions.The analysis of the complex mitochondrial heredity of basidiomycetes will allow a better understanding of the transmission of mitochondria in eukaryotic life cycles and especially a characterization of the molecular mechanisms involved in mitochondria selection after plasmogamy in these somatogamic species and, certainly, in other eukaryotic cells.

The intraspecific variability of mitochondrial genes of Agaricus bisporus reveals an extensive group I intron mobility combined with low nucleotide substitution rates.

Intraspecific mitochondrial variability was studied in ten strains of A. bisporus var. bisporus, in a strain representative of A. bisporus var. eurotetrasporus and in a strain of the closely related species Agaricus devoniensis. In A. bisporus, the cox1 gene is the richest in group I introns harboring homing endonuclease genes (heg). This study led to identify group I introns as the main source of cox1 gene polymorphism. Among the studied introns, two groups were distinguished according to the heg they contained. One group harbored heg maintained putatively functional. The other group was composed of eroded heg sequences that appeared to evolve toward their elimination. Low nucleotide substitution rates were found in both types of intronic sequences. This feature was also shared by all types of studied mitochondrial sequences, not only intronic but also genic and intergenic ones, when compared with nuclear sequences. Hence, the intraspecific evolution of A. bisporus mitochondrial genome appears characterized by both an important mobility (presence/absence) of large group I introns and by low nt substitution rates. This stringent conservation of mitochondrial sequences, when compared with their nuclear counterparts, appears irrespective of their apparent functionality and contrasts to what is widely accepted in fungal sequence evolution. This strengthens the usefulness of mtDNA sequences to get clues on intraspecific evolution.