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Letter↗
Biomedical subjects
Jozef Nosek
Publications and source records attributed to Jozef Nosek.
Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants.
We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular-mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome.
Base Sequence↗
On the origin of telomeres: a glimpse at the pre-telomerase world.
Chromosomes may be either circular or linear, the latter being prone to erosion caused by incomplete replication, degradation and inappropriate repair. Despite these problems, the linear form of DNA is frequently found in viruses, bacteria, eukaryotic nuclei and organelles. The high incidence of linear chromosomes and/or genomes evokes why and how they emerged in evolution. Here we suggest that the primordial terminal structures (telomeres) of linear chromosomes in eukaryotic nuclei were derived from selfish element(s), which caused the linearization of ancestral circular genome. The telomeres were then essential in solving the emerged problems. Molecular fossils of such elements were recently identified in phylogenetically distant genomes and were shown to generate terminal arrays of tandem repeats. These arrays might mediate the formation of higher order structures at chromosomal termini that stabilize the linear chromosomal form by fulfilling essential telomeric functions.
Animals↗
Mitochondrial chromosome structure: an insight from analysis of complete yeast genomes.
Recent progress in the analysis of protein components of the mitochondrial nucleoid and replisome of baker's yeast, Saccharomyces cerevisiae, opens a unique opportunity for understanding the molecular principles of mitochondrial inheritance. In this work we identified homologs of proteins involved in the mitochondrial DNA packaging and replication in the complete genome sequence of the petite-negative yeast Kluyveromyces lactis. Comparative analysis of their counterparts from phylogenetically diverse yeast species revealed conserved as well as diverged features of the organellar chromosome structure and its replication strategy. Moreover, it provides a basis for subsequent functional studies of the structure and dynamics of the mitochondrial nucleoids.
Amino Acid Sequence↗
Amplification of telomeric arrays via rolling-circle mechanism.
Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.
Animals↗
Taz1 binding to a fission yeast model telomere: formation of telomeric loops and higher order structures.
Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.
DNA↗
Alternatives to telomerase: keeping linear chromosomes via telomeric circles.
Recombination is often capable of lengthening telomeres in situations where telomerase is absent. This recombinational telomere maintenance is often accompanied by telomeric instability including the accumulation of extrachromosomal telomeric circles (t-circles). Recent results of in vivo and in vitro experiments have suggested that t-circles can lead to the production of extended stretches of telomeric DNA by serving as templates for rolling-circle synthesis. This implies that t-circles can provide an efficient means of telomere elongation. The existence of t-circles in both nuclear and mitochondrial compartments of distantly related species suggests that they may be important contributors to an evolutionary conserved telomerase-independent mechanism of maintenance of telomeric tandem arrays.
Chromosomes↗
Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis.
The yeast species Candida parapsilosis, an opportunistic pathogen, exhibits genetic and genomic heterogeneity. To assess the polymorphism at the level of mitochondrial DNA (mtDNA), the organization of the mitochondrial genome in strains belonging to the three variant groups of this species was investigated. Although these analyses revealed a group-specific restriction fragment pattern of mtDNA, strains belonging to different groups appear to have similar genes in the same gene order. An extensive survey of C. parapsilosis isolates uncovered surprising alterations in the molecular architecture of their mitochondrial genome. A screening strategy for strains harbouring mtDNA with rearranged architecture showed that nearly all strains from groups I and III possess linear mtDNA molecules terminating with arrays of tandem repeat units, while most of the group II strains have a circular mitochondrial genome. In addition, it was found that linear genophores in mitochondria of strains from different groups differ in the sequence of the mitochondrial telomeric repeat unit. The occurrence of altered forms of mtDNA among C. parapsilosis strains opens up the unique possibility to address questions concerning the evolutionary origin and replication strategy of linear and circular genomes in mitochondria.
Candida↗
High-efficiency transformation of the pathogenic yeast Candida parapsilosis.
A recently developed transformation system for the pathogenic yeast Candida parapsilosis opened a venue for studying the biological phenomena of this species at the molecular level. However, the standard chemical method yielded only about 1x10(3) transformants/microg of DNA, which is insufficient for certain types of experiment. With the aim of increasing the transformation efficiency, we employed two alternative methods for the introduction of plasmids into the recipient cells. Whereas biolistics resulted in about 5x10(2) transformants/microg of plasmid DNA, electroporation was an order of magnitude more efficient than the chemical method. Pretreatment of cells with 100 mM lithium acetate or 10 mM dithiothreitol resulted in a 5-fold (5x10(4)) or a 10-fold (1x10(5)) increase in transformation efficiency, respectively. This high-efficiency transformation method should be suitable for experiments such as the screening of DNA libraries.
Acetates↗
Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy.
Mitochondrial genomes in organisms from diverse phylogenetic groups vary in both size and molecular form. Although the types of mitochondrial genome appear very dissimilar, several lines of evidence argue that they do not differ radically. This would imply that interconversion between different types of mitochondrial genome might have occurred via relatively simple mechanisms. We exemplify this scenario on patterns accompanying evolution of mitochondrial telomeres. We propose that mitochondrial telomeres are derived from mobile elements (transposons or plasmids) that invaded mitochondria, integrated into circular or polydisperse linear mitochondrial DNAs (mtDNAs) and subsequently enabled precise resolution of the linear genophore. Simply, the selfish elements generated a problem - how to maintain the ends of a linear DNA - and, at the same time, made themselves essential by providing its solution. This scenario implies that insertion or deletion of such resolution elements may represent relatively simple routes for interconversion between different forms of the mitochondrial genome.
DNA Replication↗
An overlooked riddle of life's origins: energy-dependent nucleic acid unzipping.
The imposing progress in understanding contemporary life forms on Earth and in manipulating them has not been matched by a comparable progress in understanding the origins of life. This paper argues that a crucial problem of unzipping of the double helix molecule of nucleic acid during its replication has been underrated, if not plainly overlooked, in the theories of life's origin and evolution. A model is presented of how evolution may have solved the problem in its early phase. Similar to several previous models, the model envisages the existence of a protocell, in which osmotic disbalance is being created by accumulation of synthetic products resulting in expansion and division of the protocell. Novel in the model is the presence in the protocell of a double-stranded nucleic acid, with each of its two strands being affixed by its 3'-terminus to the opposite sides of the membrane of a protocell. In the course of the protocell expansion, osmotic force is utilized to pull the two strands longitudinally in opposite directions, unzipping the helix and partitioning the strands between the two daughter protocells. The model is also being used as a background for arguments of why life need operate in cycles. Many formal models of life's origin and evolution have not taken into account the fact that logical possibility does not equal thermodynamic feasibility. A system of self-replication has to consist of both replicators and replicants.
Cell Membrane↗
Genetic manipulation of the pathogenic yeast Candida parapsilosis.
Candida parapsilosis is an important human pathogen, responsible for severe cases of systemic candidiasis and one of the leading causes of mortality in neonates. In this report, we describe the first system for genetic manipulation of C. parapsilosis. We isolated and subsequently determined DNA sequences of genes encoding galactokinase ( CpGAL1) and orotidine-5'-phosphate decarboxylase ( CpURA3) from a genomic DNA library of C. parapsilosis by functional complementation of corresponding mutations in Saccharomyces cerevisiae. The predicted protein products, Gal1p and Ura3p, displayed a high degree of homology with corresponding sequences of C. albicans and S. cerevisiae, respectively. A collection of galactokinase-deficient ( gal1) strains of C. parapsilosis was prepared using direct selection of mutagenized cells on media containing 2-deoxy-galactose. Additionally, we constructed a plasmid vector carrying CpGAL1 as a selection marker and a genomic DNA fragment with an autonomously replicating sequence activity that transforms the C. parapsilosis gal1 mutant strain with high efficiency. This system for genetic transformation of C. parapsilosis may significantly advance the study of this human pathogen, greatly improving our understanding of its biology and virulence, with implications for drug development.
Candida↗
Isolation of genes coding for Ade2 and Ura3 homologues from the multinuclear yeast Dipodascus magnusii.
Dipodascus magnusii is a nonconventional yeast species with giant multinuclear cells. We constructed two genomic DNA libraries in plasmid vectors and isolated the first two D. magnusii protein-encoding genes, DmADE2 and DmURA3, coding for phosphoribosylaminoimidazole carboxylase and orotidine-5'-phosphate decarboxylase, respectively. Both genes represent functional orthologues, since they complement ade2 and ura3 mutations in Saccharomyces cerevisiae and their putative products possess conserved sequences important for enzymatic activities. Moreover, the results of Southern blot analysis indicate that the genome of D. magnusii contains additional, paralogous sequences of the DmADE2 and DmURA3 genes.
Blotting, Southern↗
t-Loops in yeast mitochondria.
Mitochondria of several yeast species contain a linear DNA genome possessing specific terminal DNA structures dubbed mitochondrial telomeres. Several tandemly repeated units and a 5' single-stranded extension characterize mitochondrial telomeres in Candida parapsilosis, Pichia philodendra and Candida salmanticensis. Resemblance of this type of mitochondrial telomeres to typical nuclear telomeres suggests that they might form t-loop structures. Therefore we adopted a protocol for stabilization of potential t-loops in the mtDNA of C. parapsilosis and observed several loops at the ends of the mtDNA. A potential role of t-loops in protection of the ends of mtDNA and/or in mitochondrial telomere dynamics is discussed.
Journal Article↗
Mitochondrial telomeres as molecular markers for identification of the opportunistic yeast pathogen Candida parapsilosis.
Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen Candida parapsilosis. The strategy for identification of C. parapsilosis strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy.
Candida↗