Conversion of homothallic yeast to heterothallism through HO gene disruption

A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho::neo disrupted allele was constructed by cloning a dominant selectable marker, the bacterial neo gene, within the HO gene. Transformation of a homothallic diploid S. cerevisiae strain with plasmid DNA containing the ho::neo allele yielded G418-resistant yeast transformants in which one of the HO alleles was replaced by the disrupted ho::neo allele. Meiotic tetrad analysis of four-spored asci from these G418-resistant transformants gave rise to haploid heterothallic and diploid homothallic tetrad progeny. The presence of the ho::neo and HO alleles in the heterothallic and homothallic progeny was confirmed by Souther-blot analysis.     

Failure to detect an antimutator phenotype following disruption of the Saccharomyces cerevisiae DDR48 gene

The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway. We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator. However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2–1 allele, or forward mutation at the CAN1 locus. Interestingly, the latter gene had been reported previously to be subject to the antimutator effect. DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes. Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype.     

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase

Summary The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.     

Characterization of the <Emphasis Type="Italic">Arxula adeninivorans AHOG1</Emphasis> gene and the encoded mitogen-activated protein kinase

Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol. As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway. In the present study, we isolated and characterized as a key component of this pathway the A. adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa. The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron. The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi. Under stress-free conditions, the inactive Ahog1p is present in low levels. When exposed to osmotic stress, Ahog1p is rendered active by phosphorylation. In addition, AHOG1 expression is increased. Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species. This specific property could account for the fast adaptation and high osmo-resistance encountered in this species.     

Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase

Summary The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48 500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.     

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

Transient inhibition of histone deacetylase activity overcomes silencing in the mating-type region in fission yeast

We have investigated the effects of inhibition of histone de-acetylase activity on silencing at the silent mating-type loci in fission yeast. Treatment of exponentially growing cells with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in derepression of a marker gene inserted 150 bp distal from the silent mat3-M locus. The natural targets for the silencing mechanism in this region were only partially derepressed and the activation appeared to be asymmetric, i.e. the mat2-P cassette remained silent at concentrations that clearly partially derepressed the mat3-M cassette. We further noted that treatment of wild-type h ⁹⁰ cells resulted in the generation of altered sporulation phenotypes, indicating that the treatment affected the expression of mating-type genes and/or mating-type switching. The results are discussed in the light of recent accumulated data regarding the role of deacetylation for silencing in other species. Received: 8 September 1998 / 5 January 1999     

Characterization of bacteriophages fromtox-containing, non-toxigenic isolates ofCorynebacterium diphtheriae

Non-toxigenic strains ofCorynebacterium diphtheriaecontinue to cause disease within immunized populations. A subset of these corynebacteria carry the diphtheria toxin gene but in a cryptic form. To determine whether such strains might contribute to the re-emergence of functional toxin genes, the phages andtoxmutations within three clone types were examined.tox-containing, β-related phages were isolated from two of the strain types. The third isolate appeared to harbour a defective prophage. One of thetox⁻phages encoded truncated, yet enzymatically-active, forms of diphtheria toxin, suggesting that it had sustained a point mutation within the latter half of its toxin gene. In contrast, the other mutant phage did not elicit the production of either a cross-reacting material or an ADP-ribosylating activity. Complementation tests employing a series of double lysogens confirmed that the mutations responsible for the non-toxigenic phenotype of all of the phages werecisdominant. Given these findings, it is reasonable to hypothesize thattox⁺genes can arise within human populations by either homologous recombination between two distincttox⁻phages or spontaneous reversion within a single mutant allele.     

Genetic regulation of isocitrate lyase in the yeastYarrowia lipolytica

Summary Isocitrate lyase-less (Icl^–) mutants were selected from genetically defined strains ofYarrowia lipolytica to investigate the regulation of synthesis of isocitrate lyase (ICL). Eighteen mutations were localized in the geneICLI, which is most probably the structural gene of ICL in this yeast. Atrans-acting positive element (ICL2) could be identified. This gene is not linked toICL1. One mutation was detected in anotherICL gene (ICL3) which is linked to geneICL1. This mutation lowered the ICL activity incis but not intrans position to geneICLI.     

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations

Summary We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely neccessary for glucose fermentation in yeast.     

Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae

In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae. In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12. Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere. Subcloning experiments revealed that a centromerelike activity was included in a small segment of DNA immediately downstream from the DNM1 gene. Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid. Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region. The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres.     

Effective long range mapping in Schizosaccharomyces pombe with the help of swi5

The switching gene swi5 has a function in mating-type switching. In addition, the swi5 mutation causes an increased radiation sensitivity and reduces meiotic recombination about ten-fold. Based on the latter property, an experimental protocol was developed for using swi5 in long-range mapping in S. pombe. It is suitable for a speedy mapping of any new gene which has not yet been cloned. The procedure was used to clarify the map positions of some genes.     

A mutated swi4 gene causes duplications in the mating-type region of Schizosaccharomyces pombe

Summary Efficient mating-type (MT) switching in homothallic strains of Schizosaccharomyces pombe is significantly reduced if they have a mutation in any of the eleven known swi genes. The swi4 mutation causes heterothallic as well as homothallic segregants, both of which have duplications in the MT region. In contrast to homothallic strains, h ⁺ swi4 strains yield only a few duplications. The duplications originate in the process of MT switching, presumably by mistakes in the resolution of DNA intermediates. They always consist of one cassette and one of the intervening sequences, L and K respectively. Strains with up to seven cassettes in the MT region were found. The possible modes of their origins are discussed.     

Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene

Summary In the further analysis of a cross in which the mis-sense allele, am ³, of the Neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the RIP process whereas the am ³ allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. The am ³ allele had also acquired a new HindIII restriction site. It had no detectable methylation. The mutations responsible respectively for the new restriction site and the modified enzyme properties were separated from each other, and from the original am ³ mutation, by selecting for intragenic recombination on either side of the am ³ site. In this way two new effectively wild-type alleles were generated, one characterised by its heat-sensitive and kinetically modified enzyme product and the other by a new HindIII site. These results demonstrate that the RIP phenomenon can be a source of new functional alleles.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Two tightly linked silent cassettes in the mating-type region of Schizosaccharomyces pombe

Summary Genetic evidence is presented for the presence of two silent cassettes mat2-P and mat3- M, which both map to the right of the expressible site mat1 of the mating-type region in Schizosaccharomyces pombe. During a switch of mating type, the resident cassette at mat1 is replaced by a copy of opposite mating-type information from one of the silent loci. Usually the switch becomes effective in one of two daughter cells, thus allowing for efficient sister-cell conjugation. In swi mutants, mating-type switching can be observed as early as for the first division after spore germination, albeit at a lower frequency. Genetically the two silent cassettes are linked so tightly that no crossovers were observed between mat2 and mat3 at a resolution of 10^–3 cM.     

Novel mutations of <Emphasis Type="Italic">EVER1/TMC6</Emphasis> gene in a Japanese patient with epidermodysplasia verruciformis

Germline mutations of the EVER1/TMC6 gene are associated with epidermodysplasia verruciformis (EV), which is characterized by an abnormal susceptibility to human papillomaviruses that were considered to be innocuous for the general population. In this study, we have employed polymerase chain reaction and DNA sequencing analysis to characterize the EVER1 gene in a 65-year-old Japanese EV patient. Direct sequence analyses resulted in the identification of two novel mutations. One nonsense mutation consisting of a (C>A) transversion at nucleotide 744 in exon 8 in one EVER1 allele resulted in the introduction of a premature termination codon (Y248X). Another mutation was identified in the splice acceptor site of intron 8 (892-2, IVS8-2, A>T) in another allele. This is the second report of EVER1/TMC6 mutations in EV.     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).