Summary

URA4, the gene coding for orotidine monophosphate decarboxylase (OMPdecase), has been cloned from the fission yeast by homologous complementation and restricted in an Escherichia coli-Schizosaccharomyces pombe (E. coli-S. pombe) replicative plasmid to a 1.76 kb HindIII fragment. This plasmid is maintained at a high copy number in S. pombe and allows OMPdecase expression in Saccharomyces cerevisiae (S. cerevisiae) as well as in E. coli. After characterisation by restriction mapping and Southern hybridisation, the cloned gene was used as a probe to measure URA4 transcription and to examine its regulation. Messenger RNA levels were measured by DNA/RNA filter-hybridisation with pulse labelled RNAs during 6-azauridine (6-AUR) inhibited growth in wild type and 6-AUR sensitive strains. We found that in S. pombe the OMP analogue 6-AUR does not regulate the level of OMPdecase formation as it does in S. cerevisiae but rather modifies the ratio of total polyA⁺ to polyA⁻ RNAs in the cell. Based on these results and on corresponding enzyme activities this study demonstrates divergent pyrimidine pathway regulation in the two yeasts S. cerevisiae and S. pombe. Finally, we propose the use of the URA4 gene as a convenient selective marker for genetic engineering in S. pombe.

We have cloned and sequenced human and bovine cDNAs for the β subunit of the ATP synthase (ATP-synß), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsynß was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsynß proved to be 1.9 × 10⁻⁹ substitutions per site per year (substitutions × site⁻¹ × year⁻¹) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the “selective constraint ratio”. This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.

Summary

Barley β-glucans present in wort reduce beer filtrability and cause hazes and precipitates in the finished beer. The endo-β-1,4-glucanase enzyme, EGI, found in the filamentous fungus Trichoderma reesei, is capable of efficiently hydrolyzing these β-glucans. The cDNA copy of the eg11 gene, which codes for the EGI enzyme, was coupled to yeast regulatory sequences and transferred to a brewer's yeast using the yeast copper chelatin gene CUP1 as a selection marker in the transformation. The eg11 gene was transferred to the yeast both on a multicopy plasmid and on an integrating plasmid. In both cases, highly glycosylated, active EGI enzyme was secreted into the medium. Barley β-glucans present in wort were efficiently hydrolyzed by the recombinant brewer's yeast.

Summary

The ade1 gene of the fission yeast Schizosaccharomyces pombe encodes a bifunctional polypeptide with glycinamide ribotide synthetase (GARSase) and aminoimidazole ribotide synthetase (AIRSase) enzeyme activities. These enzyme activities carry out the 2nd and 5th steps, respectively, of the purine synthetic pathway. We report the cloning of the ade1 gene on a 4.4 kb Sau3A insert in the yeast shuttle vector pWH5. Integration of this genomic insert at or near the ade1 locus and its ability to complement, by transformation, three different types of ade1 mutants proved that it contains the ade1 chromosomal gene. Analysis of the nucleotide sequence of this insert revealed the presence of an uninterrupted open reading frame of 2,367 pb. This sequence, and the predicted 789 amino acid sequence encoded, both show a high degree of homology with the functionally equivalent ade5,7 gene sequence of Saccharomyces cerevisiae (approx. 60% overall in both cases) and Gart gene sequences of Drosophila melanogaster. The size of the ade1 RNA transcript is about 2.7 kb.

Summary

The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.

Summary

Passaging on selective media of yeast strains transformed with complete 2 micron vectors carrying TRP1, LEU2 or URA3 selective markers leads to curing of the resident endogenous 2 micron DNA in a majority of the population. Vector plasmids defective in FLP function are fixed as populations of A, A + B or B forms after 2 micron loss. Transformation with these plasmids offers a general method of obtaining cir^o derivatives of any yeast strain.

Summary

Mutations in the qutD gene of Aspergillus nidulans cause the loss of ability to grow upon quinic acid as sole carbon source in media at normal pH 6.5 and failure to induce three enzyme activities specifically required for metabolism to protochatechuic acid. All 9 qutD mutants recovered are recessive and have been found to be pH sensitive, growing strongly on quinic acid media at pH 3.5 and producing significant induced enzyme acitivities. These properties are consistent with the hypothesis that the QUTD gene encodes an essential component of a permease required for transport of quinate ion into mycelium at pH 6.5. The QUTD gene has been located within the cloned QUT gene cluster of A. nidulans by transformation of qutD mutants with fragments of cloned sequences from phage λ-Q1. The QUTD locus is in a region distinct from other QUT genes and which contains sequences homologous to the QA-Y gene in the corresponding QA gene cluster of Neurospora crassa.

Summary

Neurospora crassa wild type genome shows DNA sequences which are homologous to the sequences present in the rRNA processing genes of the yeast Saccharomyces cerevisiae. Five such processing genes from yeast, viz., RNA1 through RNA5, cloned in plasmid pBR322 were transformed in Escherichia coli strain LE392. Southern blots containing DNAs from these clones were restricted with several restriction endonucleases along with DNAs from lambda phage, rice (plant) and neuroblastoma (animal), were hybridized with ³²P-labelled nick-translated N. crassa nuclear DNA under very stringent conditions. Autoradiograms of these blots revealed that four yeast rRNA processing genes (RNA1, RNA2, RNA3, and RNA4) showed homology with N. crassa nuclear DNA but such analogs were not found in DNAs representing prokaryotes, phages, higher plants and animals.

Summary

The MRS3 gene cloned in the multicopy plasmid YEp13 suppresses the mitochondrial splice defect exerted by mutation M1301 in the group II intron bIl. In this article we report on the behavior of the MRS3 gene cloned in the integration vector pEMBLYi27 and in the CEN4-ARS vector YCp50. Transformation of mutant M1301 cells with these recombinant vectors produced transformants, the majority of which showed the original splice defect and contained the recombinant vectors in single or low copy; a minority, however, was splicing competent and showed exceptionally high copy numbers of the MRS3 gene. These latter transformants had either the pEMBLYi27/MRS3 sequence repeated at least 20 times in tandem at the chromosomal site of the MRS3 gene or they had the YCp50/MRS3 sequence established as a multicopy plasmid lacking the copy number control usually exerted by the CEN4 sequence in this plasmid.

Summary

HindIII digested DNA from various mutant strains of Saccharomyces cerevisiae probed with a 340 by nucleotide sequence in M13mp8 derived from a mouse liver cDNA clone p1581 showed strong hybridization to a 4.1 kb DNA fragment class. This was limited to the DNA of cells of alpha mating type but the fragments concerned apparently do not originate from chromosome III. The pattern of hybridization was modified in strains carrying the HO gene consistent with there being extra copies of the Bkm-homologous sequence in these cells. Northern analysis of RNA from cells synchronised in various stages of the mitotic and meiotic cell cycle probed with MI 3mp8/p1581 indicated related transcripts in meiotic cells.