Sequence comparison of the ARG4 chromosomal regions from the two related yeasts,Saccharomyces cerevisiae and Saccharomyces douglasii

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.     

Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.     

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.     

Heterologous HIS3 Marker and GFP Reporter Modules for PCR-Targeting in Saccharomyces cerevisiae

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1·4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His⁺ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0·9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2·4 kb-long double modules flanked by 40–45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter. © 1997 by John Wiley & Sons, Ltd.     

Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).     

The sequence of a 23·4kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals CLB6, SPT6, RP28A and NUP57 genes,a ty3 element and 11 new open reading frames

The DNA sequence of 23427bp from the right arm of chromosome VII of Saccharomyces cerevisiae is reported. The sequence contains 18 open reading frames (ORFs). Four of these are identical to genes already known. G5970 corresponds to the CLB6 gene. G6169 is identical to the SPT6 gene. G6178 represents the RPS28A gene and G6320 corresponds to the 3′-region from the NUP57 gene. Four ORFs (G5978, G5982, G5984, G5995) belong to a Ty3-1 element. A further ORF (G5975) encodes a tRNA^cys. The other ORFs revealed no significant similarity to any known gene. The DNA and protein sequences have been deposited in the EMBL Data bank. They are available under the following accession numbers: ORF G 5970, G 5975, G 5978, G 5982, G 5984, G 5995, G 5999, G 6140, G 6145, G 6150, G 6153, G 6163, G 6166, G 6169, G 6172, G 6178, G 6320; DNA sequence accession Z72894/ X70436/ X72890, M34549, —, Z72895, Z72896, Z72897, Z72898, Z72899, Z72899, Z72899/ M34391, Z72902, Z72903/ M96570, Z72904/ X83099/ X81155; Protein sequence accession S64417/ S43736, S41736, —, S64417, S64419, S64420, S64421, S64422, S64423, S64423/ A36468, S64425, S64426/ A46703, S64428 /S51799/ S55976.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe. Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca²⁺-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca²⁺-binding motifs. The KlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. © 1998 John Wiley & Sons, Ltd.     

Identification and Expression of the Saccharomyces cerevisiae Cytoplasmic Tryptophanyl-tRNA Synthetase Gene

The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site. The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiae enzyme. The HRE342 ORF plus about 250 bp of 5′ and 3′ flanking sequence was amplified by polymerase chain reaction, cloned into a 2 μ based vector, and transformed into a host strain, S. cerevisiae JG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert. Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1. A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetases suggests there is sufficient similarity to identify both class I and class II genes. © 1997 by John Wiley & Sons, Ltd.     

The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae

The polymorphic extracellular glucoamylase-encoding genes STA1 (chr. IV), STA2 (chr. II) and STA3 (chr. XIV), from Saccharomyces cerevisiae var. diastaticus probably evolved by genomic rearrangement of DNA regions (S1, S2 and SGA1) present in S. cerevisiae, and subsequent translocation to unlinked regions of chromosomal regions. S1, encoding a homologue to the threonine/serine-rich domain of STA glucoamylases (GAI-III), mapped to the right arm of chromosome IX. S2, encoding the hydrophobic leader peptide of GAI-III, was also mapped on the right arm of chromosome IX, next to S1, close to DAL81. The SGA1 sporulation-specific, intracellular glucoamylase-encoding gene is located on the left arm of chromosome IX, 32 kb proximal of HIS5.     

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.     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1α-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82·4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part of a region homologous to EF-1α. The comparison of amino acid sequences of the Sup2 proteins reveals high conservations (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae where found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.     

PetCR46, a gene which is essential for respiration and integrity of the mitochondrial genome

In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one-step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303. The deletion has been checked by meiotic segregation and Southern blot analyses. Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites. Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho⁻ clones) while others did not (possibly rho° clones). The wild-type gene has been cloned and shown to complement the deletion. The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.     

Yeast sequencing reports. CAN1, a gene encoding a permease for basic amino acids in Candida albicans

The first gene coding for an amino-acid permease of Candida albicans was sequenced. The DNA fragment complementing the lysine-permease deficiency was 3385 bp long. An open reading frame of 1713 nucleotides was found encoding a protein of 571 amino acids, with a calculated molecular weight of 63 343. Analysis of the deduced primary structure revealed ten membrane spanning regions and three potential N-glycosylation sites. The protein sequence is strongly homologous to both permeases for basic amino acids (Can1 and Lyp1) of Saccharomyces cerevisiae. C-terminal part of another ORF (105 aa), highly homologous to the gene HAL2 of S. cerevisiae, was found 133 bp downstream, and in tail-to-tail orientation to the permease gene. The sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number X76689.     

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology. The complete nucleotide sequence was submitted to the EMBL Data Library under Accession Number X83690.