Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae MNN9 gene

A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.     

Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3

The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.     

Cloning and sequencing of the ornithine carbamoyltransferase gene from Pachysolen tannophilus

A fragment of DNA from a yeast Pachysolen tannophilus, bearing the ornithine carbamoyltransferase gene (OCTase, EC 2.1.3.3) has been cloned from a genomic library by functional complementation of the Escherichia coli OCT-negative mutant. The gene was located within the cloned segment of DNA and its coding sequence identified by DNA sequencing. This has indicated that P. tannophilus OCT gene encodes a 347 amino acid polypeptide, which shows 60% identity to the homologous Saccharomyces cerevisiae protein. The amino acid composition of its N-terminus indicates that this protein is translocated across the mitochondrial membrane. The gene can be expressed in E. coli as well as in S. cerevisiae. Comparison with other OCTases confirms a high degree of conservation among these proteins.     

Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1Delta strain of Saccharomyces cerevisiae

The HOG1 gene encodes a MAP kinase that plays an essential role in maintaining water homeostasis in the yeast Saccharomyces cerevisiae. A gene homologous to S. cerevisiae HOG1 has been isolated from a highly salt-tolerant yeast, Debaryomyces hansenii, by phenotypic complementation. DNA sequencing of the clone revealed the presence of an open reading frame encoding a protein 387 amino acids long. The deduced amino acid sequence showed very high similarity with homologous genes identified from S. cerevisiae, Candida albicans and Zygosaccharomyces rouxii. In addition, it has also TGY motif characteristics of hyperosmolarity-activated MAP kinases. The Genbank Accession No. of this sequence is AF185278.     

Cloning and sequence of the LYS2 homologue gene from the osmotolerant yeast Pichia sorbitophila

We have isolated the Pichia sorbitophila LYS2 (PsLYS2) gene by complementation of a lys2 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 4197 bp and the deduced translation product shares a global identity of 66% and 58% to the Lys2 protein homologues of Candida albicans and S. cerevisiae, respectively. Analysis of PsLYS2 sequence suggests that, similarly to S. cerevisiae LYS2, it codes for a polypeptide having two separate enzymatic activities which reside in different domains of the protein, including an adenylate domain, an acyl-carrier site and a short-chain reductase domain. Several GCN4- and NIT2-binding motifs have been matched in the promotor sequence of PsLYS2. In addition, upstream of the sequenced PsLYS2 sequence, we have found the 3'-terminal half of a gene of same orientation encoding a RAD16-like protein, a genomic organization similar to that of C. albicans.     

A single nucleotide change in the MNR1 (VCX1/HUM1) gene determines resistance to manganese in Saccharomyces cerevisiae

Several spontaneous Mn2+-resistant mutants were isolated from Saccharomyces cerevisiae strain W303-1b. All displayed an identical semidominant resistance phenotype. The gene responsible for this phenotype from one of these mutants, as well as that from the wild-type, was cloned and sequenced, which allowed the identification of a single nucleotide change in the former. Further sequencing work with the remaining mutants, as well as with others on the MNAR1 gene (Bianchi et al., 1981), indicated that all displayed the same mutation (mnr1 allele). In addition, MNR1 was shown to correspond to VCX1 and HUM1, which determine a vacuolar membrane protein. The nucleotide sequence has been deposited at EMBL, with Accession No. AJ001272.     

Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative beta-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.     

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.     

MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses.

We have isolated a new Saccharomyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the MOL1 gene (for MOLasses-inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M(r) 35 kDa that is closely related to stress-inducible proteins of similar size from two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 disruption strain showed no apparent phenotype under a variety of growth conditions. The 5' region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA-gly (UCC) gene, which has been mapped to chromosome VII.     

Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.     

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.     

Sequencing and functional analysis of the Hansenula polymorpha genomic fragment containing the YPT1 and PMI40 genes

A 6.0 kb genomic DNA segment was isolated by its ability to rescue the temperature-sensitive growth defect and the hypersensitivity to sodium deoxycholate of a spontaneous vanadate-resistant mutant derived from Hansenula polymorpha DL-1. The genomic fragment contains four open reading frames homologous to the Saccharomyces cerevisiae genes YPT1 (which codes for a GTP-binding protein; 75% amino acid identity), PMI40 (encoding phosphomannose isomerase; 61% identity), YLR065c (30% identity) and CST13 (28% identity). The H. polymorpha YPT1 homologue (HpYPT1) was found to be responsible for the complementation of the temperature-sensitive phenotype and the sodium deoxycholate sensitivity of the mutant strain. Disruption of the H. polymorpha PMI40 homologue (HpPMI40) resulted in the auxotrophic requirement for D-mannose. The heterologous expressions of HpYPT1 and HpPMI40 were able to complement the temperature-sensitive phenotype of S. cerevisiae ypt1-1 mutant and the mannose auxotrophy of S. cerevisiae pmi40 null mutant, respectively, indicating that the H. polymorpha genes encode the functional homologues of S. cerevisiae YPT1 and PMI40 proteins. The nucleotide sequence has been submitted to GenBank under Accession No. AF454544.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca²⁺-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.     

LEU2 gene homolog in Kluyveromyces lactis.

A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis. The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.