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J Piskur

Publications and source records attributed to J Piskur.

At least 37 records · Page 2Linked to original sources
PubMed2000

Bacterial toxin-antitoxin gene system as containment control in yeast cells.

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae. Expression of the relE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.

Antitoxins↗
PubMed1999

A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila.

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.

Adenosine Triphosphate↗
PubMed1999

A synthetic combination of mutations, including fs(1)pyrSu(b), rSu(b) and b, causes female sterility and reduces embryonic viability in Drosophila melanogaster.

A Drosophila melanogaster mutant, fs(1)pyrSu(b), carrying a mutation that maps to the tip of the X chromosome, has been isolated. The mutation, when present alone, does not confer a detectable phenotype. However, this mutation causes female sterility and reduces embryonic viability when combined with mutations which deregulate the pyrimidine and beta-alanine pools. Embryos that are homozygous for the mutations fs(1)pyrSu(b), rSu(b) [previously designated as Su(b)] and b, and originate from a female parent homozygous for the three mutations show severely reduced viability. Newly laid eggs begin development normally, but the majority of the embryos die just before the eggs are due to hatch.

Alleles↗
PubMed1999

Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases.

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.

Aspartate Carbamoyltransferase↗
PubMed1999

Karyotypes of Saccharomyces sensu lato species.

An improved pulsed-field electrophoresis program was developed to study differently sized chromosomes within the genus Saccharomyces. The number of chromosomes in the type strains was shown to be nine in Saccharomyces castellii and Saccharomyces dairenensis, 12 in Saccharomyces servazzii and Saccharomyces unisporus, 16 in Saccharomyces exiguus and seven in Saccharomyces kluyveri. The sizes of individual chromosomes were resolved and the approximate genome sizes were determined by the addition of individual chromosomes of the karyotypes. Apparently, the genome of S. exiguus, which is the only Saccharomyces sensu lato yeast to contain small chromosomes, is larger than that of Saccharomyces cerevisiae. On the other hand, other species exhibited genome sizes that were 10-25% smaller than that of S. cerevisiae. Well-defined karyotypes represent the basis for future genome mapping and sequencing projects, as well as studies of the origin of the modern genomes.

Chromosomes, Fungal↗
PubMed1999

A natural chimeric yeast containing genetic material from three species.

The Saccharomyces sp. CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes. The data show that Saccharomyces sp. CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species. Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp. IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule. These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.

Base Sequence↗
PubMed1999

Horizontal transfer of genetic material among Saccharomyces yeasts.

The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or alpha-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.

Cell Nucleus↗
PubMed1998

Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase.

In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5'-fluorodeoxyuridine, were phosphorylated. There was negligible 3'-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides.

Animals↗
PubMed1998

Structure and genetic stability of mitochondrial genomes vary among yeasts of the genus Saccharomyces.

Several yeast species/isolates belonging to the genus Saccharomyces were examined for the organization of their mtDNAs and ability to generate petite mutants. A general characteristic for all of the mtDNAs tested was that they were very A+T-rich. However, restriction patterns and inducibility of petite mutations revealed a great diversity in the organization and genetic behaviour of mtDNAs. One group of yeasts, Saccharomyces sensu stricto, contains mtDNA ranging in size from 64 to 85 kb. mtDNAs form these yeasts contain a high number of restriction sites that are recognized by the enzymes Haelll and Mspl, which cut specifically in G+C clusters. There are three to nine ori/rep sequences per genome. These yeasts spontaneously generate respiration deficient mutants. Ethidium bromide (Et-Br), at low concentrations, induces a majority of cells to give rise to petites. A second group of yeasts, Saccharomyces sensu lato, contains smaller mtDNAs, ranging in size from 23 to 48 kb, and probably only a few intergenic G+C clusters and no ori/rep sequences. These yeasts also generate petite clones spontaneously. but Et-Br, even when present at high concentrations, does not substantially increase the frequency of petites. In most petite clones from these yeasts only a small fragment of the wild-type molecule is retained and apparently multiplied. A third group, represented by Saccharomyces kluyveri, does not give rise to petite mutants either spontaneously or after induction.

Base Composition↗
PubMed1998

New hybrids between Saccharomyces sensu stricto yeast species found among wine and cider production strains.

Two yeast isolates, a wine-making yeast first identified as a Mel+ strain (ex. S. uvarum) and a cider-making yeast, were characterized for their nuclear and mitochondrial genomes. Electrophoretic karyotyping analyses, restriction fragment length polymorphism maps of PCR-amplified MET2 gene fragments, and the sequence analysis of a part of the two MET2 gene alleles found support the notion that these two strains constitute hybrids between Saccharomyces cerevisiae and Saccharomyces bayanus. The two hybrid strains had completely different restriction patterns of mitochondrial DNA as well as different sequences of the OLI1 gene. The sequence of the OLI1 gene from the wine hybrid strain appeared to be the same as that of the S. cerevisiae gene, whereas the OLI1 gene of the cider hybrid strain is equally divergent from both putative parents, S. bayanus and S. cerevisiae. Some fermentative properties were also examined, and one phenotype was found to reflect the hybrid nature of these two strains. The origin and nature of such hybridization events are discussed.

Alleles↗
PubMed1998

[Genetic analysis of Saccharomyces castellii, S. exiguus and S. martiniae yeasts].

Genetic lines of three biological yeast species of the Saccharomyces sensu lato group--S. castellii, S. exiguus, and S. martiniae--were created. Homo- and heterothalmic monosporic fertile strains were marked by different auxotrophic mutations. Conditions for the hybridization of the Saccharomyces sensu lato yeast were established. The mating of spores or haploid cells of auxotrophic complementary mutants was performed on the complete YPD medium. Prototrophic diploid hybrids were selected on a minimal medium. In S. castellii, the hybridization of diploid cells was demonstrated. All three species studied were suitable for tetrad analysis.

DNA, Fungal↗
PubMed1997

The transmission disadvantage of yeast mitochondrial intergenic mutants is eliminated in the mgt1 (cce1) background.

A trans-acting element, MGT1 (also called CCE1), has previously been shown to be required in Saccharomyces cerevisiae for the preferential transmission of petite mitochondrial DNA (mtDNA) molecules over wild-type mtDNA molecules. In the present study a possible role of this nuclear gene in the transmission of mtDNA from various respiration-competent mutants was studied. Several of these mutants, lacking one or the other of two biologically active mitochondrial intergenic sequences, were employed in genetic crosses. When these deletion mutants were crossed to the parental wild-type strain in the MGT1/CCE1 background, the progeny contained predominantly wild-type mtDNA molecules. When crosses were performed in the mgt1/cce1 background, the parental molecules interacted in zygotes and underwent homologous recombination but wild-type and intergenic-deletion alleles were transmitted with equal frequencies.

Crosses, Genetic↗
PubMed1997

Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2.

Sex determination in Drosophila melanogaster is regulated by a cascade of splicing factors which direct the sex-specific expression of gene products needed for male and female differentiation. The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. Here we have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in D. virilis testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in D. melanogaster and D. virilis is accomplished under the control of similar regulatory pathways.

Alternative Splicing↗
PubMed1995

Aggregated DNA in ethanol solution.

A recently developed mechanochemical method has provided a new, efficient tool for studies on the thermal stability and structure of aggregated DNA in ethanol-water solutions. At low ethanol concentrations DNA is fully soluble and is in the B form. However, with increasing ethanol concentration the melting temperature of DNA, Tm, decreases. At a critical ethanol concentration, dependent on the nature and concentration of the counterion, aggregation of the DNA molecules sets in. This is reflected in a marked increase in Tm indicating that the aggregated DNA molecules are thermally more stable than the dissolved ones. However, they are still in the B form. In general, Tm of aggregated DNA also decreases with further increasing ethanol concentration and is dependent on the nature of the counterion, but Tm is not affected by the concentration of the counterion (excess salt) in the ethanol-water solution. When the ethanol concentration reaches the range of 70-80% (v/v), the B-to-A conformational transition occurs in the case of Na-, K- and CsDNA. Above this transition point the A form is more stable than the B form due to the reduced water activity and to increased interhelical interactions. At very high ethanol concentrations, above 85% and dependent on the nature of the counterion, a drastic change in the thermal behaviour is observed. Apparently such a strong interhelical interaction is induced in the aggregated DNA that the DNA is stabilized and cannot adopt a random coil state even at very high temperatures. This stability of DNA in the P form is fully reversed if the ethanol concentration is lowered and the activity of water, thereby, is restored.

Cations, Monovalent↗
PubMed1995

A mitochondrial molecular marker, ori-rep-tra, for differentiation of yeast species.

Yeasts exhibit various mechanisms for the inheritance of their mitochondrial genomes. Differences among these mechanisms are based on variations within nuclear as well as mitochondrial genetic elements. Here we report diagnostic differences in the presence of biologically active mitochondrial intergenic sequences, ori-reptra, among related yeasts in the genera Saccharomyces, Arxiozyma, Debaryomyces, Kluyveromyces, Pachytichospora, Torulaspora, and Zygosaccharomyces. A molecular probe containing ori-rep-tra can be employed specifically for the differentiation and identification of isolates belonging to the species complex Saccharomyces sensu stricto.

DNA, Mitochondrial↗