Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene

The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h. Nucleotide sequence data reported are available in the DDBJ/EMBL/Genbank databases under the accession number DQ173437.     

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.     

Characterization of the Saccharomyces cerevisiaeFMS1 gene related to Candida albicans Corticosteroid-binding protein 1

 In order to investigate ergosterol metabolism in S. cerevisiae we studied the CM8 mutant strain defective in the regulation of this pathway. A genomic multicopy library was screened to reverse the CM8 phenotype. This allowed us to characterize a new gene, FMS1, which relieves mutant phenotype by extragenic functional complementation. FMS1 may encode a 508 amino-acid protein. The predicted protein shares 35% identity with Cbp1p, a Candida albicans corticosteroid binding-protein. Fms1p also shows a weaker homology with monoamine oxidases. The construction of a FMS1 null-allele yeast strain demonstrated that this gene is not essential for yeast in normal usual laboratory culture conditions. The existence of a gene related to CBP1 of C. albicans in S. cerevisiae strongly suggests a possible function of steroid-binding proteins in yeast general physiology rather than in a process related to pathogenicity. Received: 10 December 1995/22 March 1996     

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.     

Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34–37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino-and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.     

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.     

Heterologous gene expression of the glyphosate resistance marker and its application in yeast transformation

Summary The E. coli aroA gene was inserted between yeast promoter and terminator sequences in different shuttle expression plasmids and found to confer enhanced EPSP synthase activity as well as resistance to glyphosate toxicity. Subsequently, a transformation system using these newly constructed vectors in yeast was characterized. The efficiency of the glyphosate resistance marker for transformation and selection with plasmid pHR6/20-1 in S. cerevisiae laboratory strain SHY2 was found to be relatively high when compared with selection for LEU2 prototrophy. The fate of the recombinant plasmid pHR6/20-1 in the transformants, the preservation of the aroA E. coli DNA fragment in yeast, mitotic stability, EPSP synthase activity, and growth on glyphosate-containing medium have been investigated. As this plasmid also allows direct selection for glyphosate resistant transformants on rich media, the glyphosate resistance marker was used for transforming both S. cerevisiae laboratory strain SHY2 and brewer's yeast strains S. cerevisiae var. uvarum BHS5 and BHS2. In all cases, the vector pHR6/20-1 was maintained as an autonomously replicating plasmid. The resistance marker is, therefore, suitable for transforming genetically unlabeled S. cerevisiae laboratory, wild, and industrial yeast strains.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate     

Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpn1 (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpn1 are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpn1 in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Evolutionary conservation of genomic sequences related to the GGP1 gene encoding a yeast GPI-anchored glycoprotein

Summary The GGP1 gene encodes the only GPI-anchored glycoprotein (gp115) that has been purified todate in the budding yeast Saccharomyces cerevisiae. It is a single-copy gene whose deduced amino-acid sequence shares no significant homology to any other known protein. In this paper we report a Southern hybridization analysis of genomic DNA from different eukaryotic organisms to identify homologues of the GGP1 gene. We have analyzed DNA prepared from a unicellular green alga (Chlamydomonas eugametos), from two distantly related yeast species (Candida cylindracea and Schizosaccharomyces pombe), and from the common bean Phasoleus vulgaris. The moderate stringency of the experimental conditions and the high specificity of the probes used indicate that a single-copy of GGP1-related sequences exists in all these eukaryotic organisms. The chromosomal localization of the GGP1 gene in S. cerevisiae has also been determined.     

Genetic control of Yarrowia lipolytica fatty acid synthetase biosynthesis and function

Yarrowia lipolytica, like other lower fungi, has a fatty acid synthetase complex (FAS) with an alpha 6 beta 6 molecular structure. Both subunits are multifunctional proteins each with a molecular weight of more then 200,000 daltons. A collection of FAS-deficient) Y. lipolytica mutants was isolated and characterized by both genetic complementation and enzyme activity measurements. It was found that the three acyl transferases (acetyl-, malonyl- and palmityl-transacylation) together with the enoyl reductase domain are located on subunit beta and, therefore, are encoded by the gene locus FAS1. beta-Ketoacyl reductase, beta-ketoacyl synthase and acyl carrier protein functions are part of the FAS2-encoded subunit alpha. Thus, the functional organization of FAS1 and FAS2 is identical in both yeasts, Saccharomyces cerevisiae and Yarrowia lipolytica. Nevertheless, the two yeasts differ significantly with respect to the intragenic complementation characteristics of fas1 and fas2 mutants. This finding is discussed in terms of a specific inter- or intramolecular reaction mechanism within the oligomeric FAS complex. The pentafunctional Y. lipolytica FAS1 gene was isolated from a lambda gt11 expression library using polyclonal antisera against the purified FAS complex. At present, sequencing of FAS1, which is more than 5 kilobases long, is almost completed. Available data indicate approx. 60 percent sequence homology together with an identical order of catalytic domains within subunit beta of the two yeasts, Y. lipolytica and S. cerevisiae.     

Isolation of a Trichoderma reesei cDNA encoding GTP: a-d-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation

A cDNA coding for GTP: α-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant. Received: 21 July 1997 / 24 April 1998     

Basidiomycetousras cDNA functionally replaces its homolog genes in yeast

It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomyceteLentinus edodes (namedLeras cDNA) can functionally replace its homolog genes (ScRAS1 andScRAS2) in the yeastSaccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of bothRAS genes. The strain replaced by aLeras–cDNA-carrying plasmid, however, grew slower than the strains replaced by aScRAS1– or aScRAS2–carrying plasmid. The intracellular level of cAMP in the strain harboring theLeras–cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying theS. cerevisiae cAMP-dependent protein kinase catalytic subunit C₁ gene,TPK1, but was lower than that in a strain harboring anScRAS2–carrying plasmid. These results suggest that theLeras cDNA can complement theras1 ^– ras2^– mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing theScRAS2 gene.     

Disruption of the MRP-L23 gene encoding the mitochondrial ribosomal protein L23 is lethal for Kluyveromyces lactis but not for Saccharomyces cerevisiae

The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated KlL23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a “petite-positive” strain carrying a ρ^o-lethality suppressor atp mutation rendered cells unable to grow on a non-fermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis. Received: 2 September / 20 October 1999     

Expression and analysis of the green fluorescent protein gene in the fission yeast Schizosaccharomyces pombe

This report demonstrates that the Aequorea victoria green fluorescence protein (gfp) gene product will fluoresce in the fission yeast Schizosaccharomyces pombe when expressed from an episomal expression vector. Fluorescence was readily detectable at both the colony and single cell level. Application of fluorescence-activated cell sorting (FACS) techniques showed that gfp-expressing cells could be detected when they were as rare as 1% of a total yeast population. Quantitative analysis of gfp-expressing cells constituting as little as 5% of a total population was possible. These observations establish the suitability of the gfp gene for use in S. pombe and, in combination with FACS, offers an experimental strategy for quantitative analysis of gene expression in yeast populations.     

The Escherichia coli recA gene increases UV-induced mitotic gene conversion in Saccharomyces cerevisiae

The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceeding mitotic conversion events in yeast.     

Structural heterozygosis at genes IL V2 and IL V5 in Saccharomyces carlsbergensiss

Summary Chromosomes XII and XIII of a Saccharomyces carlsbergensis brewing strain were analysed after their transfer into Saccharomyces cerevisiae by kar1-mediated single chromosome transfer. The lager yeast was found to be heterozygous for the isoleucine-valine biosynthesis genes IL V2 (encoding acetohydroxy acid synthase) and IL V5 (encoding acetohydroxy acid reductoisomerase). In both cases, Southern analysis showed restriction site polymorphisms, and that one allele hybridizes more strongly to that of S. cerevisiae than the other. The alleles with limited nucleotide sequence homology are located on chromosomes which recombine poorly with the corresponding S. cerevisiae chromosomes (XIII and XII) during meiosis. A cluster of ribosomal RNA genes is located on the chromosome XII with the S. cerevisiae-like IL V5, but not on the homoeologous chromosome. The present analysis supports the view that S. carlsbergensis is an amphiploid hybrid.     

Allelism of SNQ1 and ATR1, genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide an aminotriazole

Summary SNQ1 gene function is required for the expression of resistance to 4NQO in wild-type yeast. The sequence of a 3.7 kb yeast DNA containing the gene SNQ1 was determined. The SNQ1 gene consists of an open reading frame of 1641 bp and encodes, according to the hydrophobicity analysis of the putative protein, a transmembrane protein of 547 amino acids. Homology searches in yeast genome databanks revealed a 100% sequence homology with gene ATR1 which controls resistance to aminotriazole in S. cerevisiae. Pre-treatment of wild-type yeast, but not of snq1-0::LEU2 disruption mutants, with sublethal doses of aminotriazole induced hyper-resistance to 4-nitroquinoline-N-oxide. Partial deletion of the nucleotide sequence coding for a putative ATP-binding site has no, or little, influence on resistance to 4NQO whereas total deletion of the region coding for this ATP-binding domain leads to 4NQO-sensitive nullmutants.Abbreviation 4NQO 4-nitroquinoline-N-oxide - aminotriazole 3-amino-1,2,4-triazole - HYR hyper-resistance