Intron mobility results in rearrangement in mitochondrial DNAs of heterokaryon incompatible Aspergillus japonicus strains after protoplast fusion.

Mitochondrial transmission was carried out under selective conditions between incompatible Aspergillus japonicus strains always using an oligomycin-resistant mitochondrial donor and selecting for recipient nuclei and oligomycin-resistant mitochondria. All attempted intraspecific mitochondrial transmissions were successful, but the transmission between closely related A. japonicus and A. aculeatus failed. Under selection pressure, resistant progeny harbor the mitochondrial DNA (mtDNA) of the donor strain, which may remain unchanged or may be modified by the introns of the recipient mitochondrial genome. Detailed analysis of a certain strain harboring rearranged mtDNA suggests that the mtDNA profiles of recombinant-like progeny are strongly influenced by the characteristics and mobility of introns of both parental mtDNAs. Both intron loss and intron acquisition play a role in the rearrangement of mtDNA. In certain parental combinations, a particular intron was lost very frequently.

Interaction between mitochondria derived from incompatible black Aspergillus isolates.

As black Aspergillus isolates are highly heterokaryon-incompatible mitochondrial transmissions were performed by protoplast fusion. Donor strains with oligomycin-resistant mitochondria and sensitive recipient partners of various A. japonicus isolates were applied and the progeny were selected for oligomycin resistance and for recipient nuclear phenotype. These strains basically inherited the mitochondrial DNA of the donor strain, which might remain unchanged (substituted progeny) or might be modified by specific sequences of the recipient mtDNAs (recombinant progeny). Different mobile elements characteristic of the recipient parents were exclusively responsible for the development of the feature of recombinant mtDNAs. Substituted progeny were either stable wild-type-like strains as a result of compatible co-operation between donor mitochondria and recipient nuclei, or aconidial strains with a reduced fitness, exhibiting a certain instability. The latter type was probably due to the less compatible communication between nuclear and extrachromosomal genetic systems originating from different parents. These progeny were able to undergo some developmental (segregation) processes during subsequent cultivation, resulting in a stable, wild-type phenotype which possessed a new type of mtDNA resembling that of the acceptor parents.

Genetic diversity in the red yeast Cryptococcus hungaricus and its phylogenetic relationship to some related basidiomycetous yeasts.

Cryptococcus hungaricus is a basidiomycetous yeast with the abilities to synthesize carotenoid pigments and to grow under psychrophile conditions. Six C. hungaricus strains have been isolated so far from different habitats. In this study we wished to clarify the relationships amongst them. Morphological and physiological characters, mitochondrial DNA restriction profiles, and the presence of mycoviruses were examined. Internal transcribed spacers together with the 5.8S rDNA, the D1/D2 region of 26S rDNA, and partial sequences of the 18S rRNA gene were also analysed. On the basis of the phylogenetic analyses the type strain CBS 4214(T) together with four other C. hungaricus isolates were closely related to Bullera armeniaca and Bullera crocea, while strain CBS 6569 was much more similar to Cystofilobasidium than to the other C. hungaricus isolates.

Interpretation of variability of mitochondrial genomes in the species Aspergillus carbonarius.

For interpretation of intraspecific polymorphism and the considerable differences in the size of mtDNAs among three groups of A. carbonarius, restriction maps were constructed from several enzymes. Functional maps were also developed to compare genome organisations and gene content. The appearance of various mtDNAs of A. carbonarius strains are different in size, but their gene content is almost identical. The 1.1 kb size difference between two closely related subgroups (1a, 1b) can be attributed to the presence or absence of an intron in cox2 gene. This phenomenon demonstrates that the migration of introns is possibly responsible for the development of variable mitochondrial genomes in nature. The striking differences in size and restriction patterns between two main mtDNA groups might derive from both the intronal variations and the altered intergenic organisation.

Recombination of mitochondrial DNA without selection pressure among compatible strains of the Aspergillus niger species aggregate.

Previous mitochondrial transmission experiments between oligomycin-resistant and oligomycin-sensitive incompatible strains of the A. niger aggregate bearing various mtDNA RFLP profiles resulted in a great variety of mitochondrial recombinants under selection pressure. Apart from the recombinant mtDNAs, resistant clones harbouring unchanged RFLP profiles of resistant donor mtDNAs with the recipient nuclear backgrounds were rarely isolated. These strains were anastomosed with nuclearly isogenic oligomycin-sensitive recipient partners and the mitochondria of the resulting progeny were examined under non-selective conditions. These experiments provide insights into events which are possibly similar to those occurring in nature. The heterokaryons obtained formed both oligomycin-resistant and -sensitive sectors, most of which were found to be homoplasmons. Progenies harbouring oligomycin-resistant and -sensitive mtDNAs may originate either from individual recombination events or be due to parental segregation. MtDNA recombination might take place in the heterokaryons without selection by oligomycin. The most frequent recombinant types of mtDNA RFLP profiles were indistinguishable from those recombinant mtDNAs which were frequently obtained under selection pressure from directed transfer experiments between incompatible strains. We present evidence that mixed mitochondrial populations may influence the compatibility reactions in the presence of an isogenic nuclear background, that recombination may take place without selection pressure, and that the process does not require specific nuclear sequences of both parental strains.

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli.

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a-1e and 2a-2f). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a-->2 and 1a-->3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data.

Molecular and phenotypic characterization of Aspergillus japonicus and Aspergillus aculeatus strains with special regard to their mitochondrial DNA polymorphisms.

Forty Aspergillus japonicus and A. aculeatus strains, most of them wild-type isolates, were examined using various molecular and phenotypic techniques. The rDNAs proved to be invariable (even strains of the species A. aculeatus exhibited the same restriction profile), while the strains could be classified into seven different mtDNA RFLP groups. Hybridisation data suggest that six of these mtDNA types have certain common restriction sites, while mtDNA type 7, which was exhibited by some A. aculeatus strains, probably has quite different mtDNA organisation and their size was smallest among the strains studied. The RAPD technique and isoenzyme analysis revealed some variabilities within these RFLP groups and strain specific features could also be recognised. Carbon source assimilation spectra were found to be very distinctive for strains of A. japonicus, A. aculeatus and A. niger, providing a useful tool for pre-characterising new wild-type isolates of black Aspergilli. Only a limited correlation was observed between the dendrograms based on genotypic and phenotypic characters.

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius.

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.

Immunochemical detection of ochratoxin A in black Aspergillus strains.

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A. japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger.