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.     

DNA sequence of the beta-isopropylmalate dehydrogenase gene and phylogenetic analysis of the yeast Saccharomyces exiguus Yp74L-3

The beta-isopropylmalate dehydrogenase (LEU2) gene from a homothallic wild-type yeast, Saccharomyces exiguus Yp74L-3, was analyzed to estimate the phylogenetic position of this strain in yeasts. The beta-isopropylmalate dehydrogenase gene of Yp74L-3 was first isolated as a clone complementing the leu2 mutation of Saccharomyces cerevisiae, and then confirmed to complement the haploid leu2 mutant derived from strain Yp74L-3 through genetic transformation. The nucleotide sequence of the cloned DNA revealed an open reading frame (ORF) encoding the beta-isopropylmalate dehydrogenase composed of 365 amino acids. The beta-isopropylmalate dehydrogenase coding sequence from the Yp74L-3 strain displayed 76.7% similarity to that of S. cerevisiae. Candidates for a UAS and a TATA-box in the 5'-upstream region and for a poly-A attachment site in the 3'-downstream region were found. A phylogenetic tree constructed from the nucleotide sequences of the beta-isopropylmalate dehydrogenase coding regions revealed that Yp74L-3 is located between S. cerevisiae and the Kluyveromyces yeasts. The LEU2 gene cloned from Yp74L-3 will serve as an effective genetic marker for constructing the transformation system in S. exiguus Yp74L-3.     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

Identification of a Candida albicans homologue of the PHO85 gene, a negative regulator of the PHO system in Saccharomyces cerevisiae

In a screen for the protein kinase genes of the human pathogenic yeast Candida albicans, a putative homologue (CaPHO85) of PHO85, a negative regulator of the PHO system of Saccharomyces cerevisiae, which is one of the cyclin-dependent protein kinases (CDKs), was isolated. An open reading frame (ORF) of this gene was identified encoding a predicted protein of 326 amino acids with a calculated molecular weight of 37.6 kDa. The amino acid sequence is highly homologous to S. cerevisiae Pho85 (62% identity) and its Aspergillus nidulans homologue (70% identity), but less homologous to Cdc28 (50% identity) of S. cerevisiae and to its C. albicans homologue CaCdc28 (49% identity), both of which are also CDK. The coding region for the C. albicans gene was interrupted by an intron of 81 nucleotides near the sequence encoding the N-terminal region, similarly to the case of the S. cerevisiae PHO85 gene. Alignment of CaPho85 with various yeast CDKs revealed that most of the domains for ATP-binding and protein kinase activity are conserved among fungal species. Southern blot analysis indicated that CaPHO85 is most likely present as a single copy gene. This gene complemented the pho85 mutation of S. cerevisiae by transformation.     

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).     

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 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.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.     

I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.     

Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system

The Saccharomyces cerevisiae TIM10 gene encodes one of the few essential mitochondrial proteins that are required for the import of nuclear-encoded precursor proteins from the cytosol and their subsequent sorting into the different mitochondrial compartments. We have isolated and characterized a putative homologue of TIM10 from the halotolerant yeast Pichia sorbitophila. The Pichia TIM10 gene encodes a protein of 90 amino acids with 66% identity to S. cerevisiae Tim10p. It was capable of suppressing the temperature sensitivity of tim10-1 mutant in S. cerevisiae, suggesting that Pichia TIM10 is both a functional and structural homologue of S. cerevisiae TIM10. The putative Pichia TIM10 gene product contains all the four conserved cysteine residues and the two CX(3)C motifs typical of the Tim family proteins in the mitochondrial intermembrane space. Using anti-Tim10p serum, Western blots detected a protein of about 10 kDa, suggesting that the Pichia Tim10p is a mitochondrial protein. The results suggest that mitochondrial import and sorting systems might be also strongly conserved in other fungi. The coding sequence of the P. sorbitophila TIM10 has been deposited in the EMBL Nucleotide Sequence Database under Accession No. AJ243940.     

A two-step cloning-free PCR-based method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae

We describe a new cloning-free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3 Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used to determine the minimum amount of homology required for efficient homologous recombination by double crossing-over of a linear DNA fragment restoring URA3 expression. This amount was estimated to 200 bp on each side of the DNA fragment. These data constituted the basis of the development of a strategy to construct DNA cassettes for gene deletion by a cloning-free overlapping PCR method. Two cassettes were necessary in two successive transformation steps for the complete removal of a gene of interest. As an example, we report here the deletion of the LEU2 gene. The first cassette was constituted by the URA3 gene flanked by two large fragments (500 bp) homologous to the 5' and 3' non-coding regions of LEU2. After transformation of an ura3 Δ recipient strain and integration of the cassette at the LEU2 locus, the URA3 gene was removed by a second transformation round with a DNA cassette made by the fusion between the 5' and 3' non-coding regions of the LEU2 gene. The overall procedure takes less than 2 weeks and allows the creation of a clean null mutant that retains no foreign DNA sequence integrated in its genome.     

Molecular cloning and sequence analysis of the Zygosaccharomyces rouxiiLEU2 gene encoding a beta-isopropylmalate dehydrogenase.

A DNA fragment carrying the LEU2 gene of osmotolerant yeast Zygosaccharomyces rouxii was isolated. The sequenced DNA fragment (2630 bp) contained two ORFs; one of them (1086 bp long, predicting a protein of 362 amino acids) shared a high degree of similarity with LEU2 genes of other yeast species. The cloned DNA fragment fully complemented the leu2 mutations of Saccharomyces cerevisiae and Z. rouxii.     

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.     

Molecular cloning and DNA analysis of a gene encoding alpha mating pheromone from the yeast Saccharomyces naganishii

A DNA fragment encoding the precursor peptide for alpha mating pheromone was isolated from the S. naganishii genome based on the amino acid sequence of the mature pheromone. The precursor peptide contains three copies of the pheromone. Hydrophobicity analysis of the precursor peptide revealed an N-terminal signal sequence for translocation into the lumen of the endoplasmic reticulum and several signals for a series of secretion-related processes. However, upstream regulatory sequences necessary for expression of the S. cerevisiae alpha mating pheromone gene were not found, suggesting the divergence of systems that regulate alpha mating pheromone gene expression in S. naganishii and S. cerevisiae. Hybridization of a probe corresponding to the S. naganishii alpha mating pheromone nucleotide sequence to S. naganishii chromosomal DNA revealed a single gene located on either chromosome VI or VII. The S. naganishii alpha mating pheromone sequence has been deposited in the DDBJ/EMBL/GenBank data library under Accession No. AB086431.     

Disruption and mapping of IDI1, the gene for isopentenyl diphosphate isomerase in Saccharomyces cerevisiae.

Isopentenyl diphosphate isomerase catalyses an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for isomerase, IDI1, was recently isolated and characterized (Anderson et al. J. Biol. Chem. 1989a, 264, 19169-19175). Wild-type IDI1 was disrupted with a LEU2 marker, and the resulting DNA was used to transform a yeast leucine auxotroph. Southern blots of EcoRI fragments of chromosomal DNA from the diploid strain showed the expected fragments for intact and disrupted IDI1. Dissection and analysis of tetrads demonstrated that IDI1 is an essential single-copy gene. A CHEF gel and clone grid filter analysis, followed by chromosomal mapping indicated that the gene is located on chromosome XVI approximately 55 kb centromere proximal to PEP4.     

TDH2 is linked to MET3 on chromosome X of Saccharomyces cerevisiae.

The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al. Gene 34, 269-281, 1985) and nucleotide sequence (Cherest et al. Mol. Gen. Genet. 210, 307-313, 1987). Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C-based library had the same restriction map as the chromosomal DNA from both of the strains. Comparison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact. In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J. Biol. Chem. 255, 2596-2605, 1980). The chromosomal orientation of the two genes was determined to be MET3-TDH2-CEN10.