AFG1, a new member of the SEC18-NSF, PAS1, CDC48-VCP, TBP family of ATPases.

We have sequenced a gene that encodes a 377 amino acid putative protein with an ATPase motif typical of the protein family including SEC18p (NSF = N-ethyl maleimide-sensitive fusion protein; vesicle-mediated endoplasmic reticulum to Golgi protein transfer), PAS1p (peroxisome assembly), CDC48p (VCP = valosin-containing protein; cell cycle) and TBP1 (Tat-binding protein). This gene, AFG1 for ATPase family gene, also has substantial homology to these proteins outside the ATPase domain. AFG1 is located on chromosome V immediately centromere-proximal to MAK10.     

XIII. Yeast mapping reports. MTF1, encoding the yeast mitochondrial RNA polymerase specificity factor,is located on chromosome XIII

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.     

Large-scale phenotypic analysis—the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. © 1997 John Wiley & Sons, Ltd.     

XIII. Yeast sequencing reports. Cloning and sequencing of the NES24 gene of Saccharomyces cerevisiae

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.     

Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).     

Sequence of a segment of yeast chromosome XI identifies a new mitochondrial carrier, a new member of the G protein family, and a protein with the PAAKK motif of the H1 histones.

We have entirely sequenced an 8.3 kb segment localized on the left arm of chromosome XI of Saccharomyces cerevisiae. Five new open reading frames have been uncovered. One of them encodes a new mitochondrial carrier protein which is dispensable for growth on glycerol medium. Another could be a new member of the G protein family. A third possesses the PAAKK motif common to H1 histones.     

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.     

Functional analysis of three adjacent open reading frames from the right arm of yeast chromosome XVI

A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process. © 1998 John Wiley & Sons, Ltd.     

Sequence and analysis of a 26·9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048. The analysis of the 26 857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A). Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work. Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable). O5248 is a probable folylpolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome. O5273 shows homology with a yeast protein required for vanadate resistance. O5268 shows homology with putative oxidoreductases of different organisms. O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast. The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function. The present sequence corresponds to coordinates 772 331 to 799 187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp. mips. embnet. org).     

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374–376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33·4%, 26·7%, 23·4% and 29·2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68·4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L37111.     

VI. Yeast sequencing reports. The sequence and potential regulatory elements of the HEM2 promoter of Saccharomyces cerevisiae

This paper reports the 1890-bp sequence located upstream of the HEM2 gene of Saccharomyces cerevisiae. The following potential regulatory protein-binding motifs were found: ABF1-binding site, yAP1-binding site, two REB1-binding sites, a cyclic AMP-responsive element, RAP1-binding site, and several HAP2-HAP3-HAP4 binding sites, implicating a complex regulatory mechanism governing expression for the HEM2 gene.     

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

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.     

II. Yeast sequencing reports. Sequence of a segment of yeast chromosome II shows two novel genes,one almost entirely hydrophobic and the other extremely asparagine-serine rich

A 3·2 kb EcoRI fragment of yeast Saccharomyces cerevisiae was entirely sequenced. Two new open reading frames were identified. The first is extremely hydrophobic, and would likely be an integral membrane protein. It has significant similarity to only one reported gene, a gene of unknown function from Drosophila melanogaster. The second ORF is asparagine-rich and very serine-rich, with a remarkable stretch of nearly 26 consecutive asparagine residues comprised of the same codon. It has no significant similarity to any reported gene. The fragment maps to chromosome II on the left arm between the CDC27 and ILS1 loci. The nucleotide sequence reported in this paper has been deposited in the GenBank database with the Accession Number M89908.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

Yeast sequencing reports. APL1, a yeast gene encoding a putative permease for basic amino acids

A Saccharomyces cerevisiae gene (1722 bp), encoding a protein (574 aa) highly homologous to the basic-amino-acid permeases LYP1 and CAN1, was sequenced. The gene, which was named APL1 (Amino-acid Permase Like), is located 881 bp upstream from LYP1 (lysine-specific permease), and in head-to-head orientation to it. These sequence data have been deposited in the EMBL/GenBank/DDBJ nucleotide sequence data libraries under Accession Number X74069.     

Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan^™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10–90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.