Ilona Pfeiffer

Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Sexual activity was induced in the basidiomyceteous Phaffia rhodozyma (Xanthophyllomyces dendrorhous) by depletion of nitrogen from the culture medium. This activity involved both mating between two yeast cells and the formation of basidiospores. Mating is possibly started by a G1 phase arrest of the cell cycle, as in other yeasts. The life cycle exhibited homothallic features. Crosses between genetically marked strains, and pulse-field gel electrophoresis of the chromosomal DNA of cells derived from individual spores revealed evidence of karyogamy, meiosis and even recombination. The segregation ratio in tetrads pointed to diploid vegetative cells, which formed tetraploid zygotes and the immediate meiosis then gave rise to diploid progenies again. Apart from the type strain Phaffia rhodozyma CBS 5905, all the examined strains were able to sporulate.

Variability and inheritance of double stranded RNA viruses in Phaffia rhodozyma

Abstract The present survey demonstrates polymorphism in both the length and the number of double-stranded RNAs (dsRNAs) among six Phaffia rhodozyma strains. Strains with one-, three- and four-types of dsRNA molecules were found, while two strains proved to be dsRNA-free. Elongated icosahedral virus-like particles (VLPs) 34×26 nm in size were detected in strains carrying four- or three-types of dsRNAs. One 3.7-kb dsRNA molecule was found not to form part of the VLP genome. Transmission of the VLPs of strain ATCC 24203 was followed through the basidiospores during the sexual cycle. Cytoplasmic inheritance was observed.

The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5' termini.

As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5′ ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element.

Differences in mitochondrial genome organization of Cryptococcus neoformans strains.

The organization of the mitochondrial genomes in two strains belonging in different varieties of Cryptococcus neoformans was analysed. Physical maps of the mtDNA of the IFM5844 (var. neoformans) and IFO410 (var. grubii) strains were constructed by using EcoRI and EcoRV restriction enzymes; functional maps were constructed by hybridization, cloning and sequencing. Most of the genes important in the mitochondrial function (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, ATP6, ATP9, COX1, COX2 and COB) and protein synthesis (SsrRNA and LsrRNA) were localized. We did not find any differences between the strains in the order of these genes. However, they differed significantly in the sizes of the mtDNAs: 32.6 kb for IFM5844, and 24.1 kb for IFO410. This can be attributed to two large regions of the mtDNA. In these regions, differences were found in the numbers of introns in COX1 (no intron in var. grubii, 5 introns in var. neoformans), COB (1 intron in var. grubii, 2 introns in var. neoformans), LsrRNA (no intron in var. grubii, 2 introns in var. neoformans), and ND5 (no intron in var. grubii, 1 intron in var. neoformans) genes. In several introns of the COB and COX1 genes LAGLIDADG motifs were found. Differences were also observed in the nucleotide sequences of some genes and in the sizes and sequences of intergenic regions. The nucleotide sequences of the genes of the IFM and IFO strains were compared with those of the H-99 and JEC 21 strains from the database. Surprisingly high similarities were found between the strains belonging in var. grubii (IFO 410 and H-99) and var. neoformans (IFM 5844 and JEC 21).

Mycocin production in Trichosporon pullulans populations colonizing tree exudates in the spring.

Mycocin production was demonstrated in Trichosporon pullulans, which is a dominant member of the yeast community in tree exudates released in the early spring (spring sap). Mycocin synthesis was associated with dsRNA-containing virus-like particles. Natural strains of Tr. pullulans free of dsRNA have a sensitive phenotype, and a mycocinogenic strain cured of small dsRNA becomes sensitive to its own mycocin. The mycocin studied was active against isolates from tree exudations only but not against Tr. pullulans strains isolated from other habitats. No activity was found against any other yeast species. The competitive advantage of mycocin production at the population level was exemplified by the predominance of mycocinogenic strains both in laboratory model communities and in natural populations.

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.

Isoenzyme, restriction fragment length polymorphism, and random amplified polymorphic DNA characterization of Phaffia rhodozyma Miller et al.

The validity of the species concept was examined with strains of Phaffia rhodozyma by comparing the isoenzyme profiles, ribosomal DNAs, and random amplified polymorphic DNA (RAPD) patterns of the strains. The isoenzyme profiles appeared to be more stable than the RAPD patterns or the electrophoretic karyotypes determined previously (A. Nagy, N. Garamszegi, C. Vágvölgyi, and L. Ferenczy, FEMS Microbiol. Lett., in press). The ribosomal DNA patterns revealed only a limited degree of variability, while RAPD analysis proved to be the most useful method for differentiating the strains studied. Strain CBS 5905T (T = type strain) produced characteristic RAPD patterns, which were different from those produced by the other strains. However, despite the high degree of variability observed, the isoenzyme data and the slightly variable ribosomal DNA hybridization profiles confirmed that the strains which we examined belong to one species.

Comparative analysis of the complete mitochondrial genomes of Aspergillus niger mtDNA type 1a and Aspergillus tubingensis mtDNA type 2b.

To understand the differences in the organization of mitochondrial genomes of the very closely related Aspergillus niger and Aspergillus tubingensis species, we determined the complete genome sequence of the 1a mtDNA type of A. niger and 2b mtDNA type of A. tubingensis and now we provide a comparative analysis of the two mtDNAs. We found that (1) the organization (gene content and order) of the two genomes is almost identical and (2) the size difference between them is principally attributed to the different intron content of their cox1, atp9 and ndh4L genes.

Mycocin production in Cryptococcus aquaticus

Double-stranded RNA viruses of about 35 nm in diameter were isolated from a mycocin-secreting strain of Cryptococcus aquaticus. A derivative of this strain, lacking small dsRNA, was non-mycocinogenic and sensitive to its own toxin. The killing pattern of this mycocin was restricted to some species of the Cystofilobasidiales clade. Despite the differences in genome size of dsRNA viruses in mycocinogenic strains of Cryptococcus aquaticus, Cystofilobasidium sp. CBS 6569, Cystofilobasidium bisporidii, Cystofilobasidium infirmominiatum, Trichosporon pullulans and Xanthophyllomyces dendrorhous and killing patterns of their mycocins, the viral genomes showed homology in hybridisation experiments.