bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of O‐acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts
Authors: Brzywczy Jerzy , Paszewski Andrzej
Published: May 2005

Mutants defective in O‐acetylhomoserine sulfhydrylase (OAH‐SHLase) were obtained in five yeast strains representative of different yeast genera: Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe and Trichosporon cutaneum. In vitro, in all five strains, the enzyme also had O‐acetylserine (OAS) sulfhydrylase activity so it is a ‘bifunctional’ OAH/OAS‐SHLase (Yamagata, 1989). The enzyme was only found to be essential in S. cerevisiae (OAH SHLase‐negative mutants are auxotrophs). Its impairment in K. lactis caused a slower growth rate and a decrease of the sulfur amino acid pool. In T. cutaneum only the pool was affected whereas in Y. lipolytica and S. pombe the lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase‐negative mutants were prototrophs, a monofunctional OAS sulfhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in various yeasts.
Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell in its precursor form
Authors: Scotti Pier A. , Chambert Régis , Petit‐Glatron Marie‐Françoise
Published: May 2005

Levansucrase, a Bacillus subtilis extracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0·3% of total proteins. The absence of any post‐translational modifications, such as signal sequence cleavage or addition of N‐linked sugars, indicated that this protein did not enter the reticulum secretion pathway.

Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted by B. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram scale.
Transformation‐associated recombination between diverged and homologous DNA repeats is induced by strand breaks
Authors: Larionov V. , Kouprina N. , Eldarov M. , Perkins E. , Porter G. , Resnick M. A.
Published: May 2005

Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation‐associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colour marker. Recombination during transformation for covalently closed circular plasmids was over 100‐fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single‐strand breaks that are ligatable, as well as double‐strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation‐associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.
Multipurpose vectors designed for the fast generation of N‐ or C‐terminal epitope‐tagged proteins
Authors: Cullin Christophe , Minvielle‐Sebastia Lionel
Published: May 2005

In this paper are described a set of new high‐copy‐number yeast vectors, which are specially designed for the conditional expression of epitope‐tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction‐amplified open reading frames to be automatically fused in frame with the epitope‐coding sequence, avoiding longer procedures such as site‐directed mutagenesis. This heterologous construction can be realized either at the 5′‐end of the coding sequence, in the pYeF1 vector, or at its 3′‐end, in pYeF2, generating N‐ or C‐terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3‐borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope‐tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.
Characterization of the KIN2 gene product in Saccharomyces cerevisiae and comparison between the kinase activities of p145KIN1 and p145KIN2
Authors: Donovan Mark , Romano Patrick , Tibbetts Michael , Hammond Charlotte I.
Published: May 2005

We have isolated two yeast genes, KIN1 and KIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeast KIN1 gene product (pp145KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine‐specific protein kinase activity. To identify and biochemically characterize the KIN2 gene product, antibodies were raised against a bacterial β‐galactosidase/KIN2 fusion polypeptide. In vivo, the KIN2 gene product is a 145 kDa phosphoprotein, pp145KIN2. In immune complexes, pp145KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [γ‐32P]ATP to either itself or the exogenously added substrates α‐casein, acid‐denatured enolase, or phosvitin. In vitro, kinase activity is dependent on either Mn2+ or Mg2+ ions. Both enzymes, pp145KIN1 and pp145KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine‐specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145KIN1 or pp145KIN2 on tyrosine residues. Both enzymes phosphorylate α‐casein, acid‐denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine‐specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU80TYR20 were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships or substrates in vivo.
Molecular cloning and analysis of the yeast flocculation geneFLO1
Authors: Watari Junji , Takata Yoshihiro , Ogawa Masahiro , Sahara Hirohisa , Koshino Shohei , Onnela Maija‐Leena , Airaksinen Ulla , Jaatinen Risto , Penttilä Merja , Keränen Sirkka
Published: May 2005

The DNA sequence of the flocculation geneFLO1ofSaccharomyces cerevisiae, which is located on chromosome I (Watariet al., 1989) was determined. The sequence contains a large open reading frame (ORF) of 2586 bp and codes for a protein of 862 amino acids. However, further study (genomic Southern and polymerase chain reaction analyses) indicated that the gene we cloned was not the intactFLO1gene but a form with an approximately 2 kb deletion in the ORF region. The intactFLO1gene was then cloned and its nucleotide sequence determined. The sequence revealed that the ORF of the intact gene is composed of 4611 bp which code for a protein of 1537 amino acids. A remarkable feature of the putative Flo1 protein is that it contains four families of repeated sequences composed of 18, 2, 3 and 3 repeats and that it has a large number of serines and threonines. In the deletedFLO1form, a large part of these repeated sequences was missing. The N‐ and C‐terminal regions are hydrophobic and both contain a potential membrane‐spanning region, suggesting that the Flo1 protein is an integral membrane protein and a cell wall component.
XIII. Yeast sequencing reports. SED6 is identical to ERG6, and encodes a putative methyltransferase required for ergosterol synthesis
Authors: Hardwick Kevin G. , Pelham Hugh R. B.
Published: May 2005

Luminal endoplasmic reticulum (ER) proteins carry a sorting signal that allows them to be retrieved from the Golgi apparatus by a specific receptor. In yeast, this receptor is encoded by the ERD2 gene. Although retrieval of ER proteins does not appear to be an essential process, cells lacking ERD2 do not grow. Several multicopy suppressors of this growth defect have been isolated. The sequence of one of these, SED6, is presented here. Its product contains motifs characteristic of methyltransferases, and it is identical to ERG6, the presumed structural gene for S‐adenosylmethionine:Δ24‐sterol‐C‐methyltransferase. The gene is located adjacent to PDR4, near the centromere of chromosome XIII.
Purification of properties of Saccharomyces cerevisiae cystathionine β‐synthase
Authors: Ono Bun‐Ichiro , Kijima Kazuyasu , Inoue Toyomi , Miyoshi Shin‐Ichi , Matsuda Akio , Shinoda Sumio
Published: May 2005

Cystathionine β‐synthase (β‐CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N‐terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation methionine. The purified β‐CTSase catalysed cysteine synthesis from serine (or O‐acetylserine) and H2S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S‐metabolizing enzymes.
Yeast sequencing reports. Genes of the linear mitochondrial DNA of Williopsis mrakii: Coding sequences for a maturase‐like protein, a ribosomal protein VAR1 homologue, cytochrome oxidase subunit 2 and methionyl tRNA
Authors: Drissi Rachid , Sor Frederic , Nosek Jozef , Fukuhara Hiroshi
Published: May 2005

The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single‐stranded loop. These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size. In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase‐like protein. ORF1 genes from W. mrakii and W. suaveolens were mapped and sequenced. Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand. The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes. We identified another open reading frame, ORF2, in W. mrakii mtDNA. It was mapped next to the cytochrome oxidase subunit 3 gene. Rich in adenine‐thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S. cerevisiae mitochondria. Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W. mrakii), X66595 (ORF1, tRNA‐Met and COX2 genes of W. mrakii), X73415 (tRNA‐Met and COX2 genes of W. suaveolens), X73416 (ORF1 gene of W. suaveolens) and X73414 (tRNA‐Met and COX2 genes of P. jadinii).
V. Yeast sequencing reports. Identification of a gene encoding a new Ypt/Rab‐like monomeric G‐protein in Saccharomyces cerevisiae
Authors: Lai Margaret H. , Bard Martin , Kirsch Donald R.
Published: May 2005

A Saccharomyces cerevisiae sequence cloned by serendipity was found to encode a protein that is a new member of the Ypt/Rab monomeric G‐protein family. This sequence shows high homology to the yeast genes SEC4 and YPT1 and, like SEC4 and YPT1, is essential for viability. The sequence was localized to chromosome V based upon hybridization to pulse‐field gel‐separated yeast chromosomes. The sequence has been deposited in the GenBank data library under Accession Number L17070.
XII. Yeast sequencing reports. Sequence, mapping and disruption of CCC1, a gene that cross‐complements the Ca2+‐sensitive phenotype of csg1 mutants
Authors: Fu Dadin , Beeler Troy , Dunn Teresa
Published: May 2005

We have isolated, sequenced, mapped and disrupted a novel gene, CCC1, from Saccharomyces cerevisiae. This gene displays non‐allelic complementation of the Ca2+‐sensitive phenotype conferred by the csg1 mutation. The ability of this gene, in two copies per cell, to reverse the csg1 defect suggests it may have a role in regulating Ca2+ homeostasis. The sequence of CCC1 indicates that it encodes a 322 amino acid, membrane‐associated protein. The CCC1 gene is located on the right arm of chromosome XII. The sequence has been deposited in the GenBank data library under Accession Number L24112.
XIV. Yeast sequencing reports. Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae
Authors: Lalo Dominique , Stettler Sophie , Mariotte Sylvie , Gendreau Emmanuel , Thuriaux Pierre
Published: May 2005

A 15·1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping. It contained six bona fide genes, RPC34, FUN34, CIT1 (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34, RPC34 and RLP7 define strictly essential functions, whereas CIT1, PET8 and MRP7 encode mitochondrial proteins. The PET8 product belongs to a family of mitochondrial carrier proteins. FUN34 encodes a putative transmembraneous protein that is non‐essential as judged from the normal growth of the fun34‐::L̈K18 (URA3) allele, even on respirable substrates. TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins. The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element. This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome. RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome III and XIV. The sequences have been deposited in the EMBL/GenBank data libraries under Accession Numbers L11277, L19167, M11344, M22116, V02536, X00782 and X63746.
Chemical synthesis of the M‐factor mating pheromone from Schizosaccharomyces pombe
Authors: Wang Shu‐Hua , Xue Chu‐Biao , Nielsen Olaf , Davey John , Naider Fred
Published: May 2005

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones. We recently showed that M‐factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C‐terminal cysteine residue is carboxyl‐methylated and S‐alkylated, probably with a farnesyl residue (Davey, 1992): Tyr‐Thr‐Pro‐Lys‐Val‐Pro‐Tyr‐Met‐Cys(S‐farnesyl)‐OCH3. Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone. These results confirm the structure of the M‐factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast.
The frequency of oligopurine. Oligopyrimidine and other two‐base tracts in yeast chromosome III
Authors: Yagil G.
Published: May 2005

The TRACTS program was employed to map the occurrence of base tracts composed of only two bases in Saccharomyces cerevisiae chromosome III. The observed frequencies were compared with those expected in random DNA. A vast excess of long base tracts of the three possible two‐base combinations, namely, purine, pyrimidine (R.Y), keto imino (K.M) and weak;strong (W;S, mainly A,T rich), was documented. The observed excess places yeast in the same category as other eukaryote and organelle genomes analysed. The excess of the two‐base tracts was considerably larger in the 1/3 of the chromosome not coding for a protein, in particular proximal to coding initiation and termination sites, but was observed for coding regions as well. A functional role for the excessive tracts, possibly as unwinding centers of particular genes, is proposed. Multiple occurrence of long two‐base tracts is offered as another diagnostic to determine whether an open reading frame (ORF), or an ORF subregion, is an actually translated gene region.
Transcription regulation of ribosomal protein genes at different growth rates in continuous cultures of Kluyveromyces yeasts
Authors: Hoekstra R. , Groeneveld Ph. , van Verseveld H. W. , Stouthamer A. H. , Planta R. J.
Published: May 2005

We have investigated the relationship between the growth rate of two Kluyveromyces strains that differ in their maximum growth rate, namely K. lactis (μmax = 0·5 h−1) and K. marxianus (μmax = 1·1 h−1), and the transcription rate of ribosomal protein (rp) genes in these strains. The growth rate of either strain was varied by culturing the cells in a chemostat under conditions of glucose limitation at different dilution rates. Although the steady‐state levels of transcription of the rp‐genes of both Kluyveromyces strains were tightly coupled to the cellular growth rate, no clear relationship between the level of rp‐gene transcription and the amount of in vitro binding of the RAP1‐ and ABF1‐like proteins to the promoters of these rp‐genes was observed.

Upon a sudden increase in the growth rate of a steady‐state culture, the transcription of rp‐genes of K. lactis showed a different response from that in K. marxianus. Whereas a substantial overexpression of the K. lactis rp‐genes was found during at least 4–5 h, the level of expression of the K. marxianus rp‐genes was almost immediately adjusted to the new growth rate.
XI. Yeast sequencing reports. The yeast YKL741 gene situated on the left arm of chromosome XI codes for a homologue of the human ALD protein
Authors: Bossier Peter , Fernandes Lisete , Vilela Cristina , Rodrigues‐Pousada Claudina
Published: May 2005

The yeast gene YKL741 is situated on the left arm of chromosome XI, 12 kb closer to the centromere with respect to the previously localized PAS1 gene. The new yeast gene codes for a homologue of the human ALD protein (ALD: adrenoleukodystrophy). The similarity between the YKL741 protein and the ALD protein is very high in the C‐terminal half, which contains an ATP‐binding cassette characteristic of the ABC family of transporters. Additionally the YKL741 protein shows some similarity to the ALD protein in the N‐terminal half in three putative transmembrane spanning domains. The sequence has been deposited in the EMBL data library under Accession Number X76133 SC YKL.
Specific identification of Candida albicans by hybridization with oligonucleotides derived from ribosomal DNA internal spacers
Authors: Botelho Ana R. , Planta Rudi J.
Published: May 2005

In order to develop DNA probes for rapid, sensitive and specific detection of the pathogenic yeast species Candida albicans, we carried out comparative sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) units of C. albicans and the closely related pathogenic species C. tropicalis. While overall sequence similarity between the two species was considerable (65–75%), both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific target sites for the identification of C. albicans. On the basis of these results one ITS1‐derived (ANAB1) and two ITS2‐derived (ANAB2 and ANAB3) oligonucleotides were selected, chemically synthesized, and used as hybridization probes. Their specificity and reliability were evaluated in dot‐blot hybridization experiments with total genomic DNA from 13 strains of medically important Candida species, six strains of other yeast genera associated with man and animals, and ten strains previously identified as C. albicans by phenotypic criteria. Under well‐defined hybridization conditions the three probes hybridized exclusively with DNA derived from strains belonging to the species C. albicans, thus demonstrating their potential clinical usefulness. The failure of four of the (presumed) C. albicans strains to show hybridization to the ITS probes sheds doubt upon their taxonomic classification, which is reinforced by other phenotypic aspects of these strains.
XIII. Yeast mapping reports. MTF1, encoding the yeast mitochondrial RNA polymerase specificity factor, is located on chromosome XIII
Authors: Ulery Terrie L. , Jaehning Judith A.
Published: May 2005

MTF1 is a nuclear gene that encodes the promoter recognition factor of the yeast mitochondrial RNA polymerase. The MTF1 gene was physically mapped to chromosome XIII. Genetic mapping data indicate that the gene is closely linked to RNA1.
Mating in the heterothallic haploid yeast Clavispora opuntiae, with special reference to mating type imbalances in local populations
Authors: Lachance Marc‐André , Nair Pradeep , Lo Patrick
Published: May 2005

Mating was studied in the haploid, heterothallic yeast Clavispora opuntiae to assess the importance of nutritional, genetic, and other factors that may favour mating and recombination. Local populations of this yeast generally exhibit dramatic inequalities in mating type distributions, suggesting that mating is rare in nature even though most isolates mate freely in the laboratory. The absence of assimilable nitrogen is prerequisite to mating competence, presumably by causing G1 arrest. Maximum mating competence is found in cells entering stationary phase in nitrogen‐limited media. Unlike the vast majority of mating yeasts, C. opuntiae does not appear to produce diffusible mating factors (sex pheromones), and mating‐competent cells do not undergo sexual agglutination. Pairwise cell contact appears to be the only signal that triggers the sexual process in this case. In order to determine if mating type imbalances in nature are caused by reduced fertility of ‘consanguine’ crosses, meiotic recombination was measured in pairs of strains that varied in their genetic distances as indicated by restriction mapping. That hypothesis was rejected, as recombination efficiency decreased with increasing genetic distance. We conclude that the rarity of mating in local populations is exacerbated by the stringent physical (pairwise cell contact) and nutritional (nitrogen depletion) conditions that will allow mating to proceed. Parallels are drawn with mating patterns observed in Clavispora lusitaniae.
The peroxisomal targeting signal of 3‐oxoacyl‐coA thiolase from Saccharomyces cerevisiae
Authors: Erdmann Ralf
Published: May 2005

All peroxisomal 3‐oxoacyl‐CoA thiolases identified so far do not contain the previously identified tripeptide peroxisomal targeting signal at their carboxy‐termini. For the two rat thiolases it was shown that their peroxisomal targeting signals are localized within the amino‐terminal region of the proteins and are cleaved upon import. This report demonstrates that the N‐terminal region of the peroxisomal 3‐oxoacyl‐CoA thiolase from Saccharomyces cerevisiae is essential for its peroxisomal targeting, and that the N‐terminal 16 amino acids of yeast thiolase are sufficient to target the otherwise cytosolic small subunit of ribulose‐1,5‐bisphosphate carboxylase to peroxisomes for import.
XIV. Yeast sequencing reports. Nucleotide sequence analysis of an 8887 bp region of the left arm of yeast chromosome XIV, encompassing the centromere sequence
Authors: Verhasselt Peter , Aert Rita , Voet Marleen , Volckaert Guido
Published: May 2005

The nucleotide sequencing of 8887 bp of the left arm of chromsome XIV is described. The sequence includes the centromeric region. Both strands were sequenced with an average redundancy of 5·09 per base pair. The overall G+C content is 37·3% (39·2% for putative coding regions versus 32·5% for non‐coding regions). Six open reading frames (ORFs) greater than 100 amino acids were detected, all of which are completely confined to the 8·9 kbp region. Codon frequencies of the six ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low‐level expressed genes. Comparison of the translated sequences with protein sequences in data bases suggests the presence of two ORFs (N2014 and N2007) encoding ribosomal proteins, the latter of which is the previously sequenced MRP7 gene. Another ORF (N2012) could encode a membrane‐associated protein since it contains secretory signal sequence and two presumed transmembrane helices. This protein might be involved in mitochondrial energy transfer. ORF N2016 is immediately adjacent to the centromere, suggesting that it corresponds to the SPO1 gene, which is very tightly linked to the centromere at the left arm side of chromosome XIV (Mortimer et al., 1989). The sequence has been deposited in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession Number X77114.
In the budding yeast Kluyveromyces marxianus, adenylate cyclase is regulated by Ras protein(s) in vitro
Authors: Verzotti Enrico , Geymonat Marco , Valetti Francesca , Lanzetti Letizia , Giunta Carlo
Published: May 2005

The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn2+‐ and Mg2+‐dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detected a guanyl nucleotide‐dependent activity. Interestingly Y13–259 monoclonal antibody, raised against mammalian p21Ha‐ras, inhibited Mg2+ plus GTP‐γ‐S‐dependent cAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti‐RAS2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.
Predominant localization of non‐specific lipid‐transfer protein of the yeast Candida tropicalis in the matrix of peroxisomes
Authors: Tan Hironobu , Bun‐Ya Masanori , Hirata Aiko , Kamiryo Tatsuyuki
Published: May 2005

PXP‐18 is a 14‐kDa major peroxisomal protein of the yeast Candida tropicalis and a homologue of the non‐specific lipid‐transfer protein (nsLTP) of mammals. Mammalian nsLTP is thought to facilitate the contact of membranes, to stimulate lipid‐transfer between them. If PXP‐18 functions like nsLTP, it must be present on organelle membranes. Immunoelectron microscopy of C. tropicalis cells indicated that gold particles, which visualized PXP‐18, localized exclusively in the matrix of peroxisomes. Subcellular fractionation followed by Western blotting revealed the association of PXP‐18 with peroxisomes in C. tropicalis cells. An enzyme‐linked immunosorbent assay revealed that almost all the PXP‐18 associated with peroxisomes was detectable after the solubilization of the organelle but not before, implying the predominance of PXP‐18 inside peroxisomes. This differential assay was applied to the intracellular import of the intact and truncated PXP‐18s expressed in Saccharomyces cerevisiae cells. Most of the intact PXP‐18 was shown to be imported into the matrix of host‐cell peroxisomes, whereas the truncated PXP‐18, which lacked the C‐terminal tripeptide Pro‐Lys‐Leu, no longer targeted peroxisomes. These results are consistent with the view that PXP‐18 is the matrix protein of peroxisomes and must function in a system other than that of lipid transfer.
II. Yeast sequencing reports. The nucleotide sequence of TTP1, a gene encoding a predicted type II membrane protein
Authors: Romero Pedro A. , Athanassiadis Ariadni , Lussier Marc , Herscovics Annette
Published: May 2005

The DNA sequence of a 2967 bp fragment located near the centromere of chromosome II, between the CEN2 and FUR4 genes, was determined. The segment contains a new open reading frame of 1794 bp. The product encoded by the gene, designated TTP1, is a predicted type II membrane protein of 597 amino acid residues with a short cytoplasmic NH2‐terminus, a membrane‐spanning region and a large COOH‐terminal region containing three potential N‐glycosylation sites. Gene disruption indicated that TTP1 is not essential for cell growth. The sequence has been deposited in the GenBank data library under Accession Number U05211.
Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26S protease complex
Authors: Schnall Ralf , Mannhaupt Gertrud , Stucka Rolf , Tauer Reimund , Ehnle Susanne , Schwarzlose Christa , Vetter Irene , Feldmann Horst
Published: May 2005

There is accumulating evidence for a large, highly conserved gene family of putative ATPases. We have identified 12 different members of this novel gene family (the YTA family) in yeast and determined the nucleotide sequences of nine of these genes. All of the putative gene products are characterized by the presence of a highly conserved domain of 300 amino acids containing specialized forms of the A and B boxes of ATPases. YTA1, YTA2, YTA3 and YTA5 exhibit significant similarity to proteins involved in human immunodeficiency virus Tat‐mediated gene expression but more significantly to subunits of the human 26S proteasome. YTA1 and YTA2 are essential genes in yeast. Remarkably, the cDNA of human TBP‐1 can compensate for the loss of YTA1. Preliminary experiments indicate that YTA1 is a component of the 26S protease complex from yeast. Our findings lead us to propose that YTA1, YTA2, YTA3 and YTA5 function as regulatory subunits of the yeast 26S proteasome. YTA10, YTA11 and YTA12 share significant homology with the Escherichia coli FtsH protein, and together with YTA4 and YTA6 may constitute a separate subclass within this family of putative ATPases.
Purification and immunolocalization of the peroxisomal 3‐oxoacyl‐coA thiolase from Saccharomyces cerevisiae
Authors: Erdmann Ralf , Kunau Wolf‐Hubert
Published: May 2005

A molecular understanding of peroxisome biogenesis depends upon the analysis of peroxisomal proteins. Here we describe the isolation of the 3‐oxoacyl‐CoA thiolase of the peroxisomal β‐oxidation system from Saccharomyces cerevisiae as a dimer of identical subunits, each with a molecular mass of 45 kDa. Monospecific polyclonal antibodies were raised against the purified enzyme, and its peroxisomal origin was demonstrated by immunoblotting of subcellular fractions as well as by immunogold labelling. We also show that these antibodies could be suitable for an immunofluorescence microscopy screening of yeast mutants affected in peroxisome assembly.
The in vivo effects of ethidium bromide on mitochondrial and ribosomal DNA in Candida parapsilosis
Authors: Maleszka R.
Published: May 2005

The ability of Candida parapsilosis to grow in the presence of high levels of ethidium bromide (EB) has been explored to study the effects of this intercalating dye on DNA in vivo. By employing confocal microscopy we have determined that EB penetrates the cellular membranes and binds rapidly to the nucleolus, whereas mitochondrial DNA becomes stained after a longer exposure to this dye. No detectable staining of the nucleus has been detected under these conditions.

Electrophoretic studies of both undigested and restricted DNAs confirm that the nuclear DNA is unaffected by high levels of EB, with the exception of the rDNA‐bearing chromosome that undergoes significant structural alterations in the presence of EB. Moreover, the hybridization signal with the rDNA probe is proportionally reduced in samples obtained from cultures grown in the presence of EB, suggesting that the average copy number of rRNA genes in these cultures may be affected.

In striking contrast to other fungal species, the linear organelle genome in C. parapsilosis retains its structural and functional integrity in the presence of high concentrations of EB.
IV. Yeast sequencing reports. Nucleotide sequence of the SAC2 gene of Saccharomyces cerevisiae
Authors: Kölling Ralf , Lee Angela , Chen Ellson Y. , Botstein David
Published: May 2005

A temperature‐sensitive mutation (act1‐1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC2 gene. A cloned genomic DNA fragment that complements the cold‐sensitive growth phenotype associated with such a suppressor mutation (sac2‐1) was sequenced. The fragment contained an open reading frame that encodes a 641 amino acid predicted hydrophilic protein with a molecular weight of 74 445. No sequences with significant similarity to SAC2 were found in the GenBank and EMBL databases. A SAC2 disruption mutation was constructed which had phenotypes similar to the sac2‐1 point mutation. A haploid SAC2 disruption strain failed to grow at low temperature and the disruption allele suppressed the temperature‐sensitive act1‐1 growth defect. The suppression phenotype was dependent on the strain background. The SAC2 sequence has been submitted to the EMBL data library (Accession Number Z29988).
II. Yeast sequencing reports. The sequence of 12·5 kb from the right arm of chromosome II predicts a new N‐terminal sequence for the IRA1 protein and reveals two new genes, one of which is a DEAD‐box helicase
Authors: Zagulski M. , Bécam A‐M , Grzybowska E. , Lacroute F. , Migdalski A. , Slonimski P. P. , Sokolowska B. , Herbert C. J.
Published: May 2005

We have determined the complete nucleotide sequence of a 12·5 kb segment from the right arm of chromosome II carried by the cosmid α20. The sequence encodes the 5′ end of the IRA1 gene. Two complete new open reading frames and the 3′ non‐coding region of the SUP1 (SUP45) gene. A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N‐terminus of Ira1p compared to the previously predicted sequence. According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids. One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene. The sequence has been deposited in the EMBL data library under Accession Number X78937.
A genetic screen to isolate genes regulated by the yeast CCAAT‐box binding protein Hap2p
Authors: Dang Van‐Dinh , Valens Michèle , Bolotin‐Fukuhara Monique , Daignan‐Fornier Bertrand
Published: May 2005

We have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which the lacZ reporter gene is placed under control of yeast regulatory elements. Two partially representative libraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT‐box binding protein Hap2p. Among 26 fusions shown to be regulated by Hap2p only CYT1 was known to be regulated by this activator. Sequence analysis revealed that most of the remaining regulated fusions are in new yeast genes, while some are in previously characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involved in Krebs cycle, electron transport and heme biosynthesis. Our results suggest that Hap2p could play a more general role by regulating other mitochondrial processes such as protein import and phosphate transport (PTP1) or maturation of mitochondrial tRNAs (RPM2). Among the remaining regulated fusions, two of them correspond to open reading frames (ORFs) on chromosomes III and XI whose nucleotide sequences have been entirely determined. The use of this approach to functionally analyse ORFs of unknown function is discussed.
XIV. Yeast sequencing reports. Sequence of MKT1, needed for propagation of M2 satellite dsRNA of the L‐A virus of Saccharomyces cerevisiae
Authors: Vermut Michael , Widner William R. , Dinman Jonathan D. , Wickner Reed B.
Published: May 2005

MKT1 is required for maintenance of K2 above 30°C in strains with the L‐A‐HN variant of the L‐A double‐stranded RNA virus of Saccharomyces cerevisiae. We report that MKT1 encodes a 92 979 Da protein with serine‐rich regions and the retroviral protease signature, DTG, but with no substantial homology to proteins presently in the databases. This sequence is available from GenBank under Accession Number U09129.
X. Yeast sequencing reports. Sequence and function analysis of a 9·74 kb fragment of Saccharomyces cerevisiae chromosome X including the BCK1 gene
Authors: Miosga T. , Boles E. , Schaaff‐Gerstenschläger I. , Schmitt S. , Zimmermann F. K.
Published: May 2005

In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9743 base pairs of sequence derived from the left arm of chromosome X. This sequence reveals three new open reading frames and includes the published sequence (5′ end and open reading frame) of the gene BCK1/SLK1/SSP31 also identified as ORFAA. Deletion mutants of two earlier unknown open reading frames J0840 and J0904 are viable and the open reading frame J0902 is essential for yeast growth. The sequence has been entered in the EMBL data library under accession number X77923.
V. Yeast sequencing reports. UBP5 encodes a putative yeast ubiquitin‐specific protease that is related to the human Tre‐2 oncogene product
Authors: Xiao Wei , Fontanie Treena , Tang Min
Published: May 2005

A gene from chromosome V of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The deduced amino acid sequence encoded by this gene is similar to several ubiquitin‐specific proteases from yeast, especially at the highly conserved domain. It is thus named UBP5. UBP5 is also closely related to the human Tre‐2 and the mouse Unp oncogene products. This study adds a new member to the ubiquitin protease family and suggests that alteration of ubiquitin protease activity may result in cancer in mammals. However, disruption of the UBP5 gene in a haploid strain did not result in a noticeable phenotypic alteration. The sequence has been deposited in the GenBank data library under Accession Number U10082.
XVI. Yeast sequencing reports. DNA sequence analysis of a 10·4 kbp region on the right arm of yeast chromosome XVI positions GPH1 and SGV1 adjacent to KRE6, and identifies two novel tRNA genes
Authors: Roemer Terry , Fortin Nathalie , Bussey Howard
Published: May 2005

Determination of DNA sequence in the KRE6 region of the Saccharomyces cerevisiae genome completes a 10·4 kbp section on the extreme right arm of chromosome XVI. This segment contains two additional genes, GPH1 and SGV1 (Hwang et al., 1989; Irei et al., 1991) previously assigned physically to chromosome XVI, as well as a new tRNAGly gene, and a novel tRNAAla gene which is flanked by a sigma element. The complete 10·4 kbp DNA sequence has been entered in GenBank with accession number L33835.
IIX. Yeast mapping reports. ATP1 and ATP2, the F1F0‐ATPase α and β subunit genes of Saccharomyces cerevisiae, are respectively located on chromosomes II and X
Authors: Takeda Masaharu , Okushiba Takahito , Hayashida Takeya , Gunge Norio
Published: May 2005

Southern blot analysis showed that ATP1 and ATP2 map on chromosomes II and X, respectively. Physical mapping of ATP1 and ATP2 by chromosome fragmentation showed that ATP1 is at the left end of chromosome II and ATP2 is at the right end of chromosome X. Both are located close to telomere sequences of each chromosome; ATP1 and ATP2 being approximately 30 kb and 85 kb from the respective telomeres.
Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin
Authors: Gilbert S. C. , van Urk H. , Greenfield A. J. , McAvoy M. J. , Denton K. A. , Coghlan D. , Jones G. D. , Mead D. J.
Published: May 2005

Recombinant human haemoglobin A (rHbA) was produced by a leucine‐requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of α and β globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed‐batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co‐operative tetramer.
Effect of site‐directed mutagenesis of conserved lysine residues upon Pas1 protein function in peroxisome biogenesis
Authors: Krause Thomas , Kunau Wolf‐H. , Erdmann Ralf
Published: May 2005

The Pas1 protein (Pas1p) is required for peroxisome biogenesis in Saccharomyces cerevisiae and contains two putative ATP‐binding sites, each within a domain which is conserved among members of the recently characterized AAA‐family. To elucidate whether both putative ATP‐binding sites are essential for Pas1p function, lysine467 of the first and lysine744 of the second putative ATP‐binding site were each changed to glutamate by site‐directed mutagenesis. While replacement of lysine744 abolished the function of the Pas1 protein in peroxisome biogenesis, replacement of lysine467 had no obvious effect.
XIV. Yeast sequencing reports. A 21·7 kb DNA segment on the left arm of yeast chromosome XIV carries WHI3, GCR2, SPX18, SPX19, an homologue to the heat shock gene SSB1 and 8 new open reading frames of unknown function
Authors: Jonniaux Jean Luc , Coster Francoise , Purnelle Benedicte , Goffeau Andre
Published: May 2005

We report the amino acid sequence of 13 open reading frames (ORF > 299 bp) located on a 21·7 kb DNA segment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Five open reading frames had been entirely or partially sequenced previously: WHI3, GCR2, SPX19, SPX18 and a heat shock gene similar to SSB1. The products of 8 other ORFs are new putative proteins among which N1394 is probably a membrane protein. N1346 contains a leucine zipper pattern and the corresponding ORF presents an HAP (global regulator of respiratory genes) upstream activating sequence in the promoting region. N1386 shares homologies with the DNA structure‐specific recognition protein family SSRPs and the corresponding ORF is preceded by an MCB (MluI cell cycle box) upstream activating factor. The sequence has been deposited in the EMBL data library under Accession Number X78898.
II. Yeast sequencing reports. Analysis of a 17·4 kb DNA segment of yeast chromosome II encompassing the ribosomal protein L19 as well as proteins with homologies to components of the hnRNP and snRNP complexes and to the human proliferation‐associated p120 antigen
Authors: van Dyck Luc , Jonniaux Jean‐Luc , de Melo Barreiros Tania , Goffeau Andre , Kleine Karl
Published: May 2005

We report the nucleotide sequence of a 17·4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains 12 open reading frames (ORFs) longer than 300 bp and a putative autonomously replicating sequence (ARS). The ORF YBL0418 contains the KH motif present in several nucleic acid‐binding proteins and shares homologies with the mouse X protein of the heterogeneous nuclear ribonucleo‐protein (hnRNP) complexes involved in pre‐mRNA processing. YBL0424 is the yeast member of the ribosomal protein L19 (YL14) family. YBL0425 is related to the D1 core polypeptide of the small nuclear ribonucleoprotein (snRNP) particles involved in the splicing of introns. YBL0437 is a putative homologue of the human protein p120, one of the major antigens associated with malignant tumours. Mcm2, a protein important for ARS activity, as well as Aac2, one of the three isoforms of the mitochondrial ATP/ADP carrier, were previously described (Yan et al., 1991; Lawson and Douglas, 1988). Four ORFs show no homology or particular features that could help to assess their functions. The last ORFs are not likely to be expressed for they are localized on the complementary strand of longer ORFs. The sequence has been submitted to the EMBL data library under Accession Number X77291.
The IFH1 gene product interacts with a fork head protein in Saccharomyces cerevisiae
Authors: Cherel Isabelle , Thuriaux Pierre
Published: May 2005

FHL1 encodes a polypeptide closely related to the fork head protein family of transcriptional activators. Deleting this gene leads to a slow‐growth phenotype with impaired rRNA maturation. IFH1 (located on chromosome IV) was isolated as a dosage‐dependent suppressor partially correcting the growth defect of the fhl1 deletion. It codes for a highly hydrophilic protein with a predicted molecular weight of 122 kDa and a pI of 4·8, that is very rich in charged residues (mostly acidic) but otherwise unrelated to any known protein. Carboxy‐terminal deletions removing the last third of the protein lead to a leaky growth phenotype with impaired rRNA maturation, as in the case of the fhl1 deletion. A full deletion of IFH1 is lethal, but growth was restored in a strain deleted for both IFH1 and FHL1. Thus, Ifh1p is essential for growth, but only in the presence of a functional Fhp1p protein. Conversely, its overexpression by increased gene dosage partially compensates for the genetic inactivation of FHl1p. These data suggest a direct interaction between the Fhl1p and Ifh1p proteins, and are consistent with a model where Fhl1p is converted from a transcriptional repressor to an activator on binding of Ifh1p. The sequence has been deposited in the EMBL data library under Accession Number Z29488.
Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure
Authors: Gietz R. Daniel , Schiestl Robert H. , Willems Andrew R. , Woods Robin A.
Published: May 2005

An improved lithium acetate (LiAc)/single‐stranded DNA (SS‐DNA)/polyethylene glycol (PEG) protocol which yields >1 × 106 transformants/μg plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS‐DNA/PEG transformation. The highest transformation efficiency was observed when 1 × 108 cells were transformed with 100 ng plasmid DNA in the presence of 50 μg SS carrier DNA. The yield of transformants increased linearly up to 5 μg plasmid per transformation. A 20‐min heat shock at 42°C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P‐labelled plasmid DNA than did double‐stranded (DS) carrier. The LiAc/SS‐DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS‐DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS‐DNA/PEG method is discussed.