Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker

The nucleotide sequence of the URA3 gene encoding orotidine-5'-phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking. Deduced amino acid sequence showed strong homologies (59-85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains. Very close upstream from the URA3 gene, the 3'-end of a gene encoding a Gea2-like protein was identified. A non-revertible C. lusitaniae ura3 mutant was selected on the basis of 5-fluoroorotic acid resistance. The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism. The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100-200 transformants per micro g of DNA. Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus. The GeneBank Accession No. for C. lusitaniae URA3 gene is AF450297.     

Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19

A gene designated RPL19A has been identified in the region downstream from the 3′-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C₁-tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57·5% identity with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5′-end. The 5′-flanking region contains one similar and one complete UAS_rpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^tm/EMBL data bank with the accession number Z36751.     

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.     

Cloning of orotidine-5′-phosphate decarboxylase (URA3) gene from sourdough yeast Candida milleri CBS 8195

The nucleotide sequence of the URA3 gene encoding orotidine-5′-phosphate decarboxylase (OMPDCase) in sourdough yeast Candida milleri CBS 8195 was determined by degenerate PCR and genome walking. Sequence analysis revealed the presence of an openreading frame of 810 bp, encoding 269 amino acid residue protein with the highest identity to the OMPDCase of the yeast Saccharomyces cerevisiae. Phylogenetic analysis of deduced amino acid sequence revealed that it shares a high degree of identity with other yeast OMPDCases. The cloned URA3 gene successfully complemented the ura3 mutation in S. cerevisiae, indicating that it encodes a functional OMPDCase in C. millieri CBS 8195.     

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.     

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.     

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.     

Functional analysis of the Zygosaccharomyces rouxii Fps1p homologue

The osmotolerant yeast Zygosaccharomyces rouxii accumulates the polyols glycerol and D-arabitol intracellularly in response to hyperosmotic stress, but the membrane transport proteins regulating polyol accumulation have not been studied. We have cloned and characterized a FPS1 homologue in Z. rouxii NRRL Y2547, and its sequence revealed a 2709 bp open reading frame encoding a peptide of 692 deduced amino acids with 56.9% identity to the Saccharomyces cerevisiae Fps1p. The role of this putative membrane channel protein in polyol accumulation and release during osmoregulation was investigated. The Z. rouxii FPS1 (ZrFPS1) complemented the S. cerevisiae fps1Delta growth defect and glycerol release upon hypo-osmotic shock. Deletion of ZrFPS1 did not affect growth on glycerol as sole carbon source, suggesting that other transport proteins are involved in the uptake of glycerol. However, mutants lacking ZrFPS1 exhibited a significant decrease in glycerol and D-arabitol efflux and poor growth during hypo-osmotic conditions, suggesting that ZrFPS1 might be involved in D-arabitol transport in addition to glycerol. This is the first demonstration of a yeast gene that affects D-arabitol transport. The full-length ZrFPS1 gene sequence including upstream promoter has been deposited in the public database under Accession No. AY488133.     

Gene regulation in response to protein disulphide isomerase deficiency

We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue, revealed a cis-acting element responding to deficiency of protein disulphide isomerase activity (designated CERP). The presence of the sequence element is necessary and sufficient for the upregulation in response to disulphide isomerase deficiency, as measured by a minimal promoter containing the CERP element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes.     

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,sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.     

Identification and characterization of a novel yeast omega3-fatty acid desaturase acting on long-chain n-6 fatty acid substrates from Pichia pastoris

A cDNA sequence putatively encoding a omega(3)-fatty acid desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-fatty acid desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino acid sequence exhibited omega(3)-fatty acid desaturase activity. The newly identified omega(3)-fatty acid desaturase, named Pp-FAD3, is novel because it showed broad n-6 fatty acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 fatty acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-fatty acid desaturase to act on long-chain n-6 fatty acid substrates. Heterologous expression of the newly identified omega(3) desaturase in different hosts will be an alternative method to increase the flow of n-6 fatty acid intermediates into their n-3 derivatives.     

Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.     

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.