bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames, including a homologue of the KIN1/KIN2 and SNF1 protein kinases
Authors: Pallier Chantal , Valens Michèle , Puzos Valérie , Fukuhara Hiroshi , Chéret Geneviève , Sor Frédéric , Bolotin‐Fukuhara Monique
Published: May 2005

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5′ part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine‐rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.
Ribosome synthesis during the growth cycle of Saccharomyces cerevisiae
Authors: Ju Qida , Warner Jonathan R.
Published: May 2005

We have measured the content of ribosomes, the rate of synthesis of ribosomal RNA, and the level of the mRNA for ribosomal proteins as a culture of Saccharomyces cerevisiae passes through the growth cycle. The transcription of both ribosomal RNA and ribosomal protein genes disappears at an unexpectedly early stage in the growth cycle, accompanied by a decline in the total RNA content of the culture by nearly 50% and a decline in the number of ribosomes per cell to less than 25% of the maximum value. During this time the cells continue to grow through more than two doublings, initially at the normal log growth rate, which then decline gradually for several hours. The data suggest that the cell can sense an unfavorable change within the medium and responds by employing regulation of both synthesis and degradation of its ribosomes. We conclude that the cell regulates ribosome synthesis and content according to its estimate of the potential for growth.
XI. Yeast sequencing reports. The complete sequence of an 18,002 bp segment of Saccharomyces cerevisiae chromosome XI contains the HBS1, MRP‐L20 and PRP16 genes, and six new open reading frames
Authors: García‐Cantalejo Jesús , Baladrón Victoriano , Esteban Pedro F. , Santos M. Angeles , Bou Germán , Remacha Miguel A. , Revuelta José L. , Ballesta Juan P. G. , Jiménez Antonio , Del Rey Francisco
Published: May 2005

We report the sequence of an 18,002 bp DNA fragment from the right arm of Saccharomyces cerevisiae chromosome XI. This segment contains nine complete open reading frames (ORFs), YKR401 to YKR409, and part of another ORF, YKR400, covering altogether 87·2% of the entire sequence. One of them, YKR400, encodes an NAD‐dependent 5,10‐methylene‐tetrahydrofolate dehydrogenase. YKR404, YKR405 and YKR406 correspond to the previously characterized HBS1, MRP‐L20 and PRP16 genes, coding for a translation elongation factor, a mitochondrial ribosomal protein and an ATP‐binding protein, respectively. The putative product of YKR407 contains the zinc‐binding region signature of neutral zinc metallopeptidases. The five other ORFs do not show significant homology to any known protein. The sequence data reported here have been assigned EMBL accession number Z27116.
III. Yeast sequencing reports. Corrected sequence for the right telomere of Saccharomyces cerevisiae chromosome III
Authors: Louis Edward J.
Published: May 2005

A comparison of the sequences of telomere regions from several yeast chromosomes revealed an apparent cloning artifact for the right end of chromosome III. An integrating vector containing G1–3T telomere sequences was used to clone the right end of chromosome III from a strain related to S288C. The sequence of this clone confirmed that the published sequence was incorrect and demonstrated that the right telomere region of chromosome III is similar to other telomeres.
XIII. Yeast sequencing reports. Cloning and sequencing of the NES24 gene of Saccharomyces cerevisiae
Authors: Yoshida Miho , Shimma Yoh‐Ichi , Uno Isao , Toh‐E Akio
Published: May 2005

We have cloned NES24 using a temperature‐sensitive nes24‐1 mutant as a host and sequenced a 3162 bp XhoI‐EcoRI DNA fragment containing the NES24 gene. Computer analysis revealed that this segment contains a 1806 bp open reading frame which is needed for complementation of the nes24‐1 mutation. We found SUP8 in the region upstream of the NES24 gene, placing the NES24 gene on chromosome XIII. A protein homology search indicated that NES24 encodes a new protein. The disruption of the NES24 gene resulted in temperature‐sensitive growth. The sequence has been deposited in DDBJ/EmBL/GenBank data bases under Accession Number D15052.
Vectors for Cu2+‐inducible production of glutathione S‐transferase‐fusion proteins for single‐step purification from yeast
Authors: Ward Alister C. , Castelli Laura A. , Macreadie Ian G. , Azad Ahmed A.
Published: May 2005

We describe six new yeast episomal vectors which encode glutathione S‐transferase (GST) affinity tags. These allow for the production of GST‐fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione‐Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single‐step purification of both GST and a functional GST‐metallothionein fusion from yeast are shown as an example of the application of these vectors.
Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe
Authors: Viljoen Marinda , Subden Ron E. , Krizus Aldis , van Vuuren Hendrik J. J.
Published: May 2005

Sequence analysis of a 4·6‐kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2·0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD‐ and NADP‐binding sites.
XI. Yeast sequencing reports. The complete sequencing of a 24·6 kb segment of yeast chromosome XI identified the known loci URA1, SAC1 and TRP3, and revealed 6 new open reading frames including homologues to the threonine dehydratases, membrane transporters, hydantoinases and the phospholipase A2‐activating protein
Authors: Tzermia Maria , Horaitis Ourania , Alexandraki Despina
Published: May 2005

We report the entire sequence of a 26·4 kb segment of chromosome XI of Saccharomyces cerevisiae. Identification of the known loci URA1, TRP3 and SAC1 revealed a translocation compared to the genetic map. Additionally, six unknown open reading frames have been identified. One of them is similar to catabolic threonine dehydratases. Another one contains characteristic features of membrane transporters. A third one is homologous in half of its length to the prokaryotic hydantoinase HyuA and in the other half to hydatoinase HyuB. A fourth one is homologous to the mammalian phospholipase A2‐activating protein. A fifth one, finally, is homologous to the hypothetical open reading frame YCR007C of chromosome III. The sequence has been deposited in the EMBL data library under Accession Number X75951.
VIII. Yeast mapping reports. Genetic mapping of STE20 to the left arm of chromosome VIII
Authors: Horecka Joe , Sprague George F.
Published: May 2005

STE20 is a newly‐discovered element of the Saccharomyces cerevisiae pheromone response pathway. We have isolated a recessive ste20 mutation and have used it to map the gene to the left arm of chromosome VIII, establishing the gene order STE20‐CEN8‐GPA1‐ARG4.
A new nuclear suppressor system for a mitochondrial RNA polymerase mutant identifies an unusual zinc‐finger protein and a polyglutamine domain protein in Saccharomyces cerevisiae
Authors: Bröhl Stefanie , Lisowsky Thomas , Riemen Gudula , Michaelis Georg
Published: May 2005

A yeast strain with a point mutation in the nuclear gene for the core subunit of mitochondrial RNA polymerase was used to isolate new extragenic suppressors. Spontaneously occurring phenotypical revertants were analysed by crosses with the wild‐type and tetrad dissection. One of the new nuclear suppressor mutants was characterized by temperature‐sensitive growth on non‐fermentable carbon sources. This mutant was transformed with a genomic yeast library. Two independent types of DNA clones were isolated which both complemented the temperature‐sensitive defect. Subcloning and DNA sequencing identified two novel yeast genes which code for proteins with the characteristic features of transcription factors. Both factors exhibit highly structured protein domains consisting of runs and clusters of asparagine and glutamine residues. One of the proteins contains in addition zinc‐finger domains of the C2H2‐type. Therefore the genes are proposed to be named AZF1 (asparagine‐rich zinc‐ffinger protein) and PGD1 (polyglutamine domain protein). Gene disruption of both reading frames has no detectable influence on the vegetative growth on complete glucose or glycerol media, indicating that the genes may act as high copy number suppressors of the mutant defect. Additional transformation experiments showed that AZF1 is also an efficient suppressor for the original defect in the core subunit of mitochondrial RNA polymerase. The DNA sequences for the AZF1 and PGD1 genes were submitted to the EMBL data base (Accession Numbers: Z26253 and Z26254).
II. Yeast sequencing reports. Nucleotide sequence analysis of an 11·7 kb fragment of yeast chromosome II including BEM1, a new gene of the WD‐40 repeat family and a new member of the KRE2/MNT1 family
Authors: Mallet Laurent , Bussereau Françloise , Jacquet Michel
Published: May 2005

This paper reports the DNA sequence and analysis of an 11·7 kb segment localized on the right arm of Saccharomyces cerevisiae chromosome II. This fragment contains one incomplete and five long and non‐overlapping open reading frames (ORFs) designated from centromere to telomere‐proximal side as: YBR1406, 1409, 1410, 1411, 1412 and 1413. YBR1406 corresponds to the 5′ end to PGI1 encoding phosphoglucoisomerase. YBR1410 encodes a polypeptide of 798 amino acids whose C terminus contains five repeats (WD‐40 repeat) similar to those found in the β‐subunits of G proteins and different yeast proteins such as Tup1, Prp4 and Cdc4. The higher similarity score is obtained with dTAFII80, a component of the RNA polymerase II transcriptional complex TFIID. YBR1411 encodes a polypeptide of 464 amino acids which belongs to the family of α‐mannosyltransferases: KRE2/MNT1, KTR1, KTR2, YUR1 and the product of previously sequenced ORF YBR1445. YBR1412 corresponds to BEM1. The two ORFs, YBR1409 and YBR1413, which do not exhibit significant similarity with any known coding sequences, define new genes. The sequence has been deposited in the EMBL Data Library under Accession Number Z21487.
Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small‐subunit rRNA gene sequences
Authors: James S. A. , Collins M. D. , Roberts I. N.
Published: May 2005

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99·8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora.

The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.
X. Yeast sequencing reports. Sequence and function analysis of a 9·46 kb fragment of Saccharomyces cerevisiae chromosome X
Authors: Miosga Thomas , Witzel Andrea , Zimmermann Friedrich K.
Published: May 2005

In the framework of the European yeast genome sequencing project, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9464 base pairs of this cosmid located on the left arm of Saccharomyces cerevisiae chromosome X. This sequence contains two new open reading frames and includes the published sequences of the RADH gene (also identified as SRS2/HPR5) and the 3′‐end of the gene BCK1/SLK1/SSP31. Deletion mutants of the two unknown genes J0909 and J0911 are viable. The sequence has been deposited in the EMBL data library under accession number X77087.
A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white
Authors: Ram Arthur F. J. , Wolters Aly , Hoopen Rogier Ten , Klis Frans M.
Published: May 2005

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White‐hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups.

The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild‐type cell walls contained about equal amounts of mannose and glucose. This indicates that both low‐mannose and low‐glucose cell wall mutants had been obtained. Further characterization showed the presence of three low‐mannose cell wall mutants with a mnn9‐like phenotype, affected, however, in different genes. In addition, four new killer‐resistant (kre) mutants were found, which are presumably affected in the synthesis of β1,6‐glucan. Most low‐glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of β1,3‐glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with β1,3‐glucan synthesis, and four cwh mutants were temperature‐sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.
II. Yeast sequencing reports. The two genes encoding yeast ribosomal protein S8 reside on different chromosomes, and are closely linked to the hsp70 stress protein genes SSA3 and SSA4
Authors: Logghe Marc , Molemans Francis , Fiers Walter , Contreras Roland
Published: May 2005

A 7·4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone α1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI=11·1; molecular weight=22·5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG) S8. Consensus splice signals were found in the 5′ leader sequence and also potential RPG‐specific sequences. Chromoblot analysis revealed a second copy of the S8 RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene, SSA4. The sequence has been deposited in the EMBL data library under accession number Z26879.
Physical constitution of ribosomal genes in common strains of Saccharomyces cerevisiae
Authors: Rustchenko Elena P. , Sherman Fred
Published: May 2005

Several recent investigations, employing restriction endonucleases that do not cleave within rDNA units, revealed that a number of laboratory strains of Saccharomyces cerevisiae apparently contain a single tandem array of approximately 50 to 200 rDNA units on each chromosome XII homolog. The number of these rDNA units varies from strain to strain, among subclones of the same strain, and after different conditions of growth. In contrast, the commonly‐used strain S288C and its derivatives contain two clusters on each chromosome XII homolog. Although the two clusters are stably maintained, the number of rDNA units within each cluster can vary as in strains with single clusters.
Isolation and partial purification of mannose‐specific agglutinin from brewer's yeast involved in flocculation
Authors: Straver Marika H. , Traas Vincent M. , Smit Gerrit , Kijne Jan W.
Published: May 2005

Yeast cell‐agglutinating activity, designated agglutinin (possible lectin), was isolated from cell walls of both non‐flocculent and flocculent brewer's yeast cells. Agglutinin‐mediated aggregation of yeast cells in a manner similar to flocculation with respect to specific mannose‐sensitivity, pH‐dependence and calcium‐dependence. Agglutinating activity was found to be heat‐stable and protease‐insensitive. Furthermore, addition of agglutinin to flocculent cells strongly stimulated the flocculation ability of the cells, whereas addition to non‐flocculent cells rendered these cells weakly flocculent.

Agglutinin was found to be released from flocculent cells during the course of a flocculation assay, but not from non‐flocculent cells. Presence of mannose during the assay inhibited release of agglutinin. Our results suggest that (i) mannose‐specific agglutinin is continuously synthesized during growth of brewer's yeast cells, (ii) agglutinin is present in cell walls of non‐flocculent cells but is unable to bind its ligand on other cells, and (iii) the ability of yeast cells to flocculate in a flocculation assay depends, among other factors, on release of agglutinin from the cells. A 10‐kDa polypeptide might represent one form of agglutinin.
II. Yeast sequencing reports. A putative P‐type Cu2+‐transporting ATPase gene on chromosome II of Saccharomyces cerevisiae
Authors: Rad Massoud Ramezani , Kirchrath Lutz , Hollenberg Cornelis P.
Published: May 2005

We have sequenced a gene on the right arm near the telomere of chromosome II of Saccharomyces cerevisiae which codes for a putative P‐type cation‐transporting ATPase (PCA1). The gene codes for a 1216 amino acids protein. The PCA1 gene expresses a 3·5 kb message in both haploid and diploid cells when grown in glucose‐based rich medium YPD. The gene product is most similar at the C‐terminal region to a human copper‐transporting ATPase and Enterococcus hirae copper‐transporting ATPases and also an N‐terminal dithiol region that was proposed to be a ‘metal‐binding motif’. Cells lacking PCA1 display no obvious phenotype when tested under standard conditions; whereas they cease growth much earlier than the isogenic wild‐type cells in a minimal medium with high copper concentration. Overexpression of PCA1 under GAL1/10 promoter in yeast cells causes poor growth. We also show that yeast strains carrying PCA1 in multiple copies grow slower than isogenic wild‐type strains in a minimal synthetic medium containing 0·3 mM‐CuSO4. The sequence has been deposited in the EMBL data library under Accession Number Z29332.
Rapid protein extraction from Saccharomyces cerevisiae
Authors: Horvath Anton , Riezman Howard
Published: May 2005

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)–gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2‐mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.
Yeast sequencing reports. Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA‐protein family
Authors: Guelin Emmanuel , Rep Martijn , Grivell Leslie A.
Published: May 2005

A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature‐sensitive respiratory‐deficient mutant. DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA‐protein family (ÃPases associated with diverse cellular activities). The members of this family are involved in very different biological processes. Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post‐translational processing of PBP3, a protein necessary for septation during cell division. This newly sequenced gene, which we have designated AFG3 for ÃPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively. The sequence has been deposited in the EMBL data library under Accession Number X76643.
The in vivo activation of Saccharomyces cerevisiae plasma membrane H+‐ATPase by ethanol depends on the expression of the PMA1 gene, but not of the PMA2 gene
Authors: Monteiro G. A. , Sá‐Correia I. , Supply P. , Goffeau A.
Published: May 2005

The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1‐lacZ and PMA2‐lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene. The presence of sub‐lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1. The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays. Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1. On the other hand, ethanol activated the plasma membrane H+‐ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase. These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post‐translational mechanism and that the PMA2 ATPase is not involved.
The peroxisomal membrane proteins of Candida boidinii: Gene isolation and expression
Authors: Moreno Myrthala , Lark Richard , Campbell Kimberly L. , Goodman Joel M.
Published: May 2005

Candida boidinii is a methylotrophic yeast in which several growth substrates can cause vigorous peroxisomal proliferation. While such diverse substrates as methanol, oleic acid and D‐alanine induce different peroxisomal metabolic pathways, membranes seem to contain common abundant peroxisomal membrane proteins (PMPs). These proteins have been termed PMP31, PMP32 and PMP47. The gene encoding PMP47 has been previously cloned and analysed. We now report the isolation of a second PMP47 gene (or allele) as well as PMP31 and PMP32. PMP47A and PMP47B share 95% sequence identity at the amino acid level. PMP31 and PMP32 each contain 256 amino acids and are highly similar (97% identity) in protein sequence. Both PMP31 and PMP32 are predicted to span the membrane once or twice. All abundant PMPs of C. boidinii are basic in charge; they all have predicted isoelectric points above 10. RNAs corresponding to the PMP47s and to PMPs31‐32 are strongly induced by methanol, oleic acid and D‐alanine. While the PMP47s probably encode substrate carriers, the functions of PMP31 and PMP32 from C. boidinii are still unknown. The GenBank Accession Numbers for PMP47B, PMP31, and PMP32 are L27998, L27999 and L28000, respectively. The 5′ untranslated sequence of PMP47A, accession number J05672, has been corrected.
II. Yeast sequencing reports. The sequence of a 22·4 kb DNA fragment from the left arm of yeast chromosome II reveals homologues to bacterial proline synthetase and murine α‐adaptin, as well as a new permease and a DNA‐binding protein
Authors: de Wergifosse Philippe , Jacques Bernard , Jonniaux Jean‐Luc , Purnelle Bénédicte , Goffeau André , Skala Jacek
Published: May 2005

We report the sequencing of a 22 470 bp DNA fragment from the left arm of Saccharomyces cerevisiae chromosome II. Thirteen open reading frames longer than 300 bp provisionally called YBL0520, YBL0401 to YBL0408 and YBL0410 to YBL0413 have been detected. Five genes were previously sequenced: COR1, encoding a core protein of the mitochondrial coenzyme QH2 cytochrome c reductase complex (Tzagaloff and Crivellone, 1986). PRS3, a proteasome subunit gene (Lee et al., 1992), ERD2, coding for a protein involved in the secretory pathway (Semeza et al., 1990), URA7, which encodes a CTP synthetase (Ozier‐Kalogeropoulos et al., 1991) and the gene for the ribosomal protein L16 (Pan et al., 1993). Among the others, YBL0406 shows striking homologies to FUR4 (Jund et al., 1988) and DAL4 (Yoo et al., 1992), the uracyl and allantoin permeases; YBL0520 is a DNA‐related protein, possibly involved in gene regulation; YBL0412 shares homologies with the mouse α‐adaptins A and C; and YBL0413 is homologous to a protein of Pseudomonas aeruginosa that is likely to be involved in proline biosynthesis. YBL0401, internal to YBL0520, is probably not expressed. The sequence has been deposited in the EMBL data library under accession number X78217
Development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri
Authors: Piredda Sandrine , Gaillardin Claude
Published: May 2005

This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322‐based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per μg of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild‐type URA3 gene by an in vitro generated deletion.

Sequences conferring extrachromosomal replication were isolated from Y. ohmeri DNA. Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 104/μg and were shown to replicate freely in Y. ohmeri at an approximate copy number of 40. Unexpectedly, we observed that BS‐SKR derivatives carrying either YoLEU2 or YoURA3 but no Y. ohmeri ARS also replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four‐fold. The sequences are available for consultation under EMBL accession number Z35101 for YoLEU2 and Z35100 for YOURA3.
Kluyveromyces marxianus small DNA fragments contain both autonomous replicative and centromeric elements that also function in Kluyveromyces lactis
Authors: Iborra François , Ball Maria M.
Published: May 2005

Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences.

Centromeric sequences are organized in a similar way to those of budding yeasts: two well‐conserved elements: CDEI (5′ TCACGTG 3′) and CDEIII (5′ TNTTCCGAAAGTWAAA 3′), are separated by a 165 bp AT‐rich (± 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis.

The ARS‐core consensus sequence (5′ TTTATTGTT 3′) is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae.

A third ARS‐containing fragment with a different organization has been isolated and sequenced.

The nucleotide sequences of DNA fragments reported in this paper will appear in the EMB data library under the accession numbers: Z31562, Z31563, Z31564.
Yeast sequencing reports. CAN1, a gene encoding a permease for basic amino acids in Candida albicans
Authors: Sychrová Hana , Souciet Jean‐Luc
Published: May 2005

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.
IV. Yeast sequencing reports. Sequence of the PHO2‐POL3 (CDC2) region of chromosome IV of Saccharomyces cerevisiae
Authors: Simon Michel , Bénit Paule , Vassal Anne , Dubois Céline , Faye Gérard
Published: May 2005

The nucleotide sequence of a 5 kb EcoRI‐NcoI fragment of chromosome IV, contiguous to gene POL3 (CDC2), has been determined. It contains three open reading frames: QRI1, QRI2 and QRI7. Two of them are essential genes. QRI7 is homologous to the Escherichia coli orfx gene. Accession number to EMBL/Genbank Data Library is X79380.
X. Yeast sequencing reports. Sequence analysis of a 40·2 kb DNA fragment located near the left telomere of yeast chromosome X
Authors: Vandenbol Micheline , Durand Patrick , Bolle Paul‐Andre , Dion Caroline , Portetelle Daniel , Hilger Francois
Published: May 2005

We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae.

The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long. Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y′ repeat region of 65·4 and 12·8 KDa, the cytochrome B pre‐mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein. Of the 16 remaining ORFs, eight show highest homologies with the S. cerevisiae hexose transporters family (two ORFs), the yeast α‐glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O‐acetyltransferase, the S. cerevisiae 137·7 KDa protein located in the Y′ region and a protein of unknown function of Schizosaccharomyces pombe. Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks.

DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends.

Disruption studies have been performed on two ORFs encoding putative proteins of unknown function.

The sequence has been entered in the EMBL Data Library under Accession Number Z34098.
A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae
Authors: Zimmer Thomas , Schunck Wolf‐Hagen
Published: May 2005

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell‐free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG‐containing mRNAs. Moreover, we were able to transfer the non‐universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.
Construction of a set of convenient saccharomyces cerevisiae strains that are isogenic to S288C
Authors: Winston Fred , Dollard Catherine , Ricupero‐Hovasse Stephanie L.
Published: May 2005

A set of GAL2+ yeast strains that are isogenic to strain S288C have been constructed. They contain non‐reverting mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection have been deposited with the ATCC.
Sequence and functional analysis of a 7·2 kb fragment of Saccharomyces cerevisiae chromosome II including GAL7 and GAL10 and a new essential open reading frame
Authors: Schaaff‐Gerstenschläger Ine , Schindwolf Thomas , Lehnert Wolfgang , Rose Matthias , Zimmermann Friedrich K.
Published: May 2005

The nucleotide sequence of a fragment of 7200 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three open reading frames (ORFs). Two genes for galactose metabolism, GAL7 and part of the GAL10 coding region, are localized on the fragment. Comparison to the previously published sequence data showed several differences, leading to changes in the amino acid sequences of GAL7 and GAL10.

One new ORF, YBR0224, was detected, coding for a protein with 918 amino acids. Comparison to the DNA and protein data bases showed no significant homologies. The protein has some interesting features pointing to a function involved in transcription regulation: a leucine zipper motif, a highly acidic region, possibly involved in transcription activation and a putative nuclear localization signal. Deletion analysis showed that the gene is essential when deleted in strain W303. Spores could germinate and form microcolonies, but efforts to propagate the colonies failed. Deletion of this gene in a different genetic background (strain M5) led to very poor‐growing mutant strains with cells showing aberrant cellular morphologies.

The nucleotide sequence of the fragment has been deposited in the EMBL‐database under the Accession Number X81324.
Perfect state of Rhodomyces dendrorhous (Phaffia rhodozyma)
Authors: Golubev Wladyslav I.
Published: May 2005

After mother–daughter cell conjugation, formation of long holobasidia with terminal basidiospores was observed without mycelium production in Rhodomyces dendrorhous (including the type strain of Phaffia rhodozyma) on polyol‐containing media. Basidiospores are not forcibly discharged and germinate by budding. A new genus Xanthophyllomyces (Filobasidiaceae, Tremellales) with a species, X. dendrorhous, is proposed for the telemorphic state of R. dendrorhous.
Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila
Authors: Lages Fernanda , Lucas Cândida
Published: May 2005

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co‐transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose‐ or glycerol‐grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose‐grown cells these were: Km = 0·81 ± 0·11 mM and Vmax = 634·2 ± 164·8 μmol h−1 per g (glycerol); Km = 1·28 ± 0·60 mM and Vmax = 558·6 · 100·6 μmol h−1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M‐NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.
Characterization of Schizosaccharomyces pombe his1 and his5 cDNAs
Authors: Erickson F. Les , Hannig Ernest M.
Published: May 2005

We have isolated Schizosaccharomyces pombe cDNAs corresponding to the genes his1+ and his5+. The his1 cDNA was isolated by functional complementation of the His− phenotype in a his1‐29 gcn3 Saccharomyces cerevisiae strain, while the his5 cDNA was isolated as a suppressor of the 3‐amino‐1,2,4‐triazole (3‐AT) sensitivity in a gcn3 S. cerevisiae strain. his1 and his5 are each present in single copy in haploid S. pombe. As is the case with S. cerevisiae, we have found that the growth of wild‐type strains of S. pombe is sensitive to 3‐AT, an inhibitor of imidazoleglycerol‐phosphate dehydratase. This enzyme is encoded by the HIS3 gene in S. cerevisiae and the his5+ gene in S. pombe. Treatment of S. pombe cells with 3‐AT leads to a small increase in the level of the his5 transcript, but no effect is seen on the level of the his1 transcript. This is in contrast to larger increases in transcription of amino acid biosynthetic genes, regulated by the general amino acid control, seen previously in similarly treated cultures of S. cerevisiae. These results suggest that there are likely to be some differences in the regulation of amino acid biosynthesis between these two yeasts.

Sequences reported here have been deposited with GenBank as U07830 (his1) and U07831 (his5).
Localization of a protein a‐tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes
Authors: Bryant Nia J. , Boyd Alan
Published: May 2005

We have previously reported an immunoisolation procedure which allows purification of Kex2p‐containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity‐tagged at its C‐terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity‐purification of vacuolar membranes.
Genetic and carbon source regulation of phosphorylation of Sip1p, a Snf1p‐associated protein involved in carbon response in Saccharomyces cerevisiae
Authors: Long Roy M. , Hopper James E.
Published: May 2005

The SIP1 gene of Saccharomyces cerevisiae is a carbon‐catabolite‐specific negative regulator of GAL gene transcription and acts as a multicopy suppressor of growth defects associated with impaired Snf1p protein kinase activity. The Sip1 protein is known to undergo phosphorylation when associated in vitro with the Snf1 protein kinase. We have carried out in vivo studies of the genetic and carbon control of Sip1p phosphorylation. Metabolic labeling reveals phosphorylation of Sip1p under both carbon catabolite‐repressing and non‐repressing conditions and in both SNF1 wild‐type and snf1‐deletion cells. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblot assay, we detect apparent changes in Sip1p phosphorylation states in response to changes in carbon source. At least one dephosphorylation of Sip1p occurs with a shift from non‐repressing carbon source to repressing carbon source. The MIG1 gene, acting through SNF1‐dependent and SNF1‐independent pathways, is required for some Sip1p phosphorylations. REG1 appears to be required for at least one dephosphorylation of Sip1p, whereas SSN6 appears to be required for at least one phosphorylation of Sip1p. These results reveal new complexities in carbon response signaling, and may reflect the involvement of the Sip1 protein in the same complex as the Mig1 and Ssn6 proteins.
Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase
Authors: Smith David J. , Proudfoot Amanda E. I. , Detiani Mariastella , Wells Timothy N. C. , Payton Mark A.
Published: May 2005

Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI‐deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64·1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D‐mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells. The nucleotide sequence data reported here will appear in the EMBL database under Accession Number X82024.
Plasmid reorganization during integrative transformation in Hansenula polymorpha
Authors: Bogdanova Aliona I. , Agaphonov Michael O. , Ter‐Avanesyan Michael D.
Published: May 2005

During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non‐selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight‐ to ten‐base AT‐rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.
The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: Regulatory elements and nucleosome positioning motifs
Authors: Erkine Alexander M. , Szent‐Gyorgyi Christopher , Simmons Serena F. , Gross David S.
Published: May 2005

We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat‐shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat‐shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three‐ to seven‐fold enriched within each promoter; a comparable enrichment is seen near the 3′ end of each transcription unit. Located ∼ 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; ∼ 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.
Kluyveromyces lactis killer plasmid pGKL2: Molecular analysis of an essential gene, ORF5
Authors: Schaffrath Raffael , Meacock Peter A.
Published: May 2005

The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One‐step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild‐type plasmids k1 and k2. Northern analysis detected a plasmid‐dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non‐selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over‐expression of an epitope‐tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.