Molecular cloning of the ARG7 gene of Schizosaccharomyces pombe encoding argininosuccinate lyase

Summary A gene bank of Sau3A partially restricted Schizosaccharomyces pombe DNA in YEp13 was used to transform an arg4 mutant of Saccharomyces cerevisiae. One colony was recovered which contained the YEp13 plasmid bearing a large insert complementing the argininosuccinate lyase (ASL) mutation. As shown by restriction mapping and subcloning experiments, the DNA sequence required for complementation is localized on a 2 kb BamHI-BamHI fragment. The plasmid complemented several S. cerevisiae arg4 mutants of independent origin and a S. pombe arg7 mutant lacking ASL. Low but significant ASL activities were detected in crude extracts of these transformants. No complementation of the E. coli argH mutant was observed. Southern blot hybridizations showed that the insert originates from the S. pombe genome. No cross-hybridization was found between this sequence and S. cerevisiae DNA. It can be concluded that the cloned DNA fragment bears the S. pombe ARG7 gene coding for ASL.Abbreviations ASL argininosuccinate lyase - bp base pair - kb kilobase pair     

The primary structure of the leul + gene of Schizosaccharomyces pombe

Summary A DNA fragment which carries the leul gene encoding beta-isopropylmalate dehydrogenase in Schizosaccharomyces pombe has been isolated by complementation of an E. coli leuB mutation. This 1.5 kb DNA fragment complements not only the S. pombe leul mutation, but also the S. cerevisiae leu2 mutation. The nucleotide sequence of the essential part of the leul gene and its flanking regions was determined. This sequence contains an open reading frame of 371 codons, from which a protein having a Mr = 39,732 can be predicted. The deduced amino acid sequence and its codon usage were compared with those of the S. cerevisiae LEU2 protein. The cloned DNA will be a useful marker when transforming S pombe.     

Transformation of Candida maltosa and Pichia guilliermondii by a plasmid containing Saccharomyces cerevisiae ARG4 DNA

Summary Saccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii have been transformed by the plasmid pYe(ARG4)411, which contains the S. cerevisiae ARG4 gene inserted into pBR322. In all transformants argininosuccinate lyase as well as -lactamase were detected. The ARG⁺ phenotype of transformants is mitotically unstable. Closed circular pYe(ARG4)411, DNA was detected in transformant DNA preparations by hybridization to pBR322 DNA and by transformation of E. coli to ampicillin resistance.     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

The ade4 gene of Schizosaccharomyces pombe: cloning,sequence and regulation

We report the isolation and sequence of the Schizosaccharomyces pombe ade4 gene which encodes the glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of the purine nucleotide de-novo biosynthetic pathway. The enzyme contains 533 amino acids and its sequence exhibits homologies to the corresponding enzymes of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, chicken, rat, and human. In contrast to the situation in S. cerevisiae, adenine does not repress ade4 expression at the mRNA level and also other nutritional signals seem not to affect its expression.     

The Saccharomyces cerevisiae HIS3 and LYS2 genes complement the Schizosaccharomyces pombe his5-303 and lys1-131 mutations,respectively: new selectable markers and new multi-purpose multicopy shuttle vectors,pSP3 and pSP4

Three new S. pombe plasmids are described. Plasmids pSP3 and pSP4 are two Schizosaccharomyces pombe ars1 multicopy vectors with the Saccharomyces cerevisiae HIS3 or LYS2 genes as selectable markers. They complement the S. pombe his5-303 or lys1-131 mutations, respectively. Plasmid pSPars1 is a vector carrying the S. pombe ars1 and a unique NdeI site which allows the introduction of any selectable marker therefore bringing a unified vector backbone for the construction of new S. pombe/S. cerevisiae/E. coli shuttle vectors. These plasmids permit classical molecular genetic techniques to be performed directly.     

As in Saccharomyces cerevisiae,aspartate transcarbamoylase is assembled on a multifunctional protein including a dihydroorotase-like cryptic domain in Schizosaccharomyces pombe

The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.     

Genetic relationships among <Emphasis Type="Italic">Trichinella pseudospiralis</Emphasis> isolates from Australian,Nearctic, and Palearctic regions

The study of the genetic polymorphism of pathogens is important for phylogenetic and biogeographic studies and, in the case of foodborne pathogens, to trace the origin of food infection. Since its discovery in 1972, the nonencapsulated species Trichinella pseudospiralis has been detected in mammals and birds, and human infection has occurred, in some cases resulting in death. We studied DNA polymorphism among ten T. pseudospiralis isolates from the Palearctic, Nearctic, and Australian regions, screening the sequences of nine genes [18sRNA, a random amplified polymorphism DNA derived sequence, mitochondrial cytochrome oxidase subunit I (COI), cytochrome P450, cynate lyase, epithelial fusion failure-1, and three unknown genes of Tp3, Tp8, and Tp26]. A high identity of sequence for the nine gene loci was obtained among the seven isolates from the Palearctic region and between the two isolates from the Nearctic region. Genetic identity analysis indicated the distinct polymorphism among the three geographical origins. To easily identify T. pseudospiralis genotypes, a polymerase chain reaction-restriction fragment length polymorphism analysis of COI gene was performed, and the results confirmed the DNA polymorphism within T. pseudospiralis, corresponding to the three regions of origin. We have named the three genotypes as "T. pseudospiralis Palearctic genotype" (code T4P), "Nearctic genotype" (code T4N), and "Australian genotype" (code T4A). To further investigate polymorphism among the nonencapsulated Trichinella species, the sequences of four gene loci (COI, P450, cynate lyase, and SB147D) of T. pseudospiralis, T. papuae, and T. zimbabwensis were analyzed, and the results showed high polymorphism among the three species, strongly supporting their classification as separate species.