Mak5p, which is required for the maintenance of the M1 dsRNA virus, is encoded by the yeast ORF YBR142w and is involved in the biogenesis of the 60S subunit of the ribosome.

In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.

Ria1p (Ynl163c), a protein similar to elongation factors 2, is involved in the biogenesis of the 60S subunit of the ribosome in Saccharomyces cerevisiae.

RIA1 (YNL163c) is a quasi-essential gene that encodes a protein with strong similarities to elongation factors 2. Small C-terminal deletions in the protein lead to a severe growth defect. In the case of a 22-residue C-terminal deletion this can be suppressed by intragenic mutations in the RIA1 gene or dominant extragenic mutations in TIF6, which is thought to be involved in the biogenesis of the 60S subunit of the ribosome. The dominant TIF6 alleles can also suppress the phenotype associated with a complete deletion of the RIA1 gene. Depletion of Ria1p has a dramatic effect on the polysome profile: there is a severe reduction in the level of the 80S monosomes, an imbalance in the 40S/60S ratio, and halfmers appear. Dissociation of the monosomes and polysomes in the Ria1p depletion mutant revealed a specific reduction in the amount of 60S subunits. Localization experiments with HA-tagged derivatives of Ria1p did not detect any stable association of Ria1p with ribosome subunits, 80S monosomes or polysomes. Cell fractionation experiments show that Ria1p is found in both the cytoplasmic fraction and the nuclear fraction. Taken together, these data suggest that Ria1p is involved in the biogenesis of the 60S subunit of the ribosome.

Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae.

We have studied the CBK1 gene of Saccharomyces cerevisiae, which encodes a conserved protein kinase similar to the human myotonic dystrophy kinase. We have shown that the subcellular localization of the protein, Cbk1p, varies in a cell cycle-dependent manner. Three phenotypes are associated with the inactivation of the CBK1 gene: large aggregates of cells, round rather than ellipsoidal cells and a change from a bipolar to a random budding pattern. Two-hybrid and extragenic suppressor studies have linked Cbk1p with the transcription factor Ace2p, which is responsible for the transcription of chitinase. Cbk1p is necessary for the activation of Ace2p and we have shown that the aggregation phenotype is due to a lack of chitinase expression. The random budding pattern and the round cell phenotype of the CBK1 deletion strain show that in addition to its role in regulating chitinase expression via Ace2p, Cbk1p is essential for a wild-type morphological development of the cell.

A 'natural' mutation in Saccharomyces cerevisiae strains derived from S288c affects the complex regulatory gene HAP1 (CYP1).

The HAP1 gene encodes a complex transcriptional regulator of many genes involved in electron-transfer reactions and is essential in anaerobic or heme-depleted conditions. We show here that strains derived from S288c carry a defective Ty1 element inserted in the 3' region of the HAP1 ORF. This mutant allele acts as a HAP1 null allele in terms of cytochrome c expression and CYC1 UAS1-dependent transcription, but is able to sustain limited growth in heme-depleted conditions.

The yeast gene YJR025c encodes a 3-hydroxyanthranilic acid dioxygenase and is involved in nicotinic acid biosynthesis.

We have deleted the yeast gene YJR025c and shown that this leads to an auxotrophy for nicotinic acid. The deduced protein sequence of the gene product is homologous to the human 3-hydroxyanthranilic acid dioxygenase (EC 1.13.11.6) which is part of the kynurenine pathway for the degradation of tryptophan and the biosynthesis of nicotinic acid. In cell-free extracts the 3-hydroxyanthranilic acid dioxygenase activity is proportional to the copy number of the YJR025c gene. As YJR025c encodes the yeast 3-hydroxyanthranilic acid dioxygenase, we have named this gene BNA1 for biosynthesis of nicotinic acid.

Sequencing and functional analysis of the yeast genome.

The genome of the yeast Saccharomyces cerevisiae was sequenced by an international consortium of laboratories from Europe, Canada, the U.S.A. and Japan. This project is now finished and the complete sequence of the first eukaryotic genome was released to the public data bases in April 1996. An overview and preliminary analysis of the entire genome sequence was presented in a special issue of Nature in May 1997, entitled "The yeast genome directory". At its origin the Yeast Genome Sequencing Project provoked much debate and controversy; however, the final results obtained and the insights this has given us into the organisation and content of a eukaryotic genome have more than justified the expectations of the supporters of the project. The importance of genomic sequencing and analysis, especially of model organisms, is now widely accepted and this has resulted in the birth of the new science of genomics (Botstein & Cherry, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 5506). The information from gene and protein sequences ultimately lead to functional description of all genes. The main strategies describing possible ways to analyse the function of new genes that have been identified by systematic sequencing of Saccharomyces cerevisiae genome are described.

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase.

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3. Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ::CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1, which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a delta cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a delta cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase of Saccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein.

The NAM2 gene of Saccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrial cytb gene (bI4) and the fourth intron of the mitochondrial coxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.

Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.

The sequence of 12.8 kb from the left arm of chromosome XIV reveals a sigma element, a pro-tRNA and six complete open reading frames, one of which encodes a protein similar to the human leukotriene A4 hydrolase.

We have determined the nucleotide sequence of a 12.8 kb fragment from the left arm of chromosome XIV carried by the cosmid 14-16d. An analysis of the sequence reveals the presence of a sigma element, a pro-tRNA gene and eight open reading frames, six of which are complete. All of the eight open reading frames correspond to new genes. Of the eight new genes, two show strong similarities to a pair of new genes from chromosome IX, suggesting an ancestral duplication, and one gene encodes a protein similar to mammalian leukotriene A4 hydrolase.

The sequence of 36.8 kb from the left arm of chromosome XIV reveals 24 complete open reading frames: 18 correspond to new genes, one of which encodes a protein similar to the human myotonic dystrophy kinase.

We have determined the complete nucleotide sequence of a 36.8 kb segment from the left arm of chromosome XIV carried by the cosmid 14-11. The sequence encodes the 5' coding region of the PSD1 gene, the 3' coding region of an unknown gene and 24 complete open reading frames, of which 18 correspond to new genes and six (SKO1, SCL41A, YGP1, YCK2, RPC31 and MFA2) have been sequenced previously. Of the 24 new genes, five show significant similarities to sequences present in the databanks. These include elongation factors 2 and the human myotonic dystrophy kinase.

The CBP2 gene from Saccharomyces douglasii is a functional homologue of the Saccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome.

In Saccharomyces cerevisiae the only known role of the CBP2 gene is the excision of the fifth intron of the mitochondrial cyt b gene (bI5). We have cloned the CBP2 gene from Saccharomyces douglasii (a close relative of S. cerevisiae). A comparison of the S. douglasii and S. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that the S. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome, but not in the presence of an intronless S. cerevisiae mitochondrial genome. Also the S. douglasii and S. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from the S. douglasii mitochondrial genome.

Artificial antisense RNA regulation of YBR1012 (YBR136w), an essential gene from Saccharomyces cerevisiae which is important for progression through G1/S.

YBR1012 (YBR136w) is an essential gene from Saccharomyces cerevisiae identified during the systematic sequencing of part of the right arm of chromosome II. We previously constructed a conditional allele of YBR1012 based on antisense RNA, by inserting a small fragment of this gene downstream from the inducible UASGAL10-CYC1 promoter. Several other antisense RNA constructions have since been made and their activity tested. The response of the system appears to be very delicate, as the presence or absence of 13 nucleotides of polylinker in the 300 nucleotide antisense transcript can dramatically modify its effectiveness. The most effective antisense RNA construction was used in flow cytometry studies to investigate the role of ybr1012p. The results show that during the antisense RNA block some 80% of the cells are arrested with their DNA unreplicated, suggesting that Ybr1012p is needed for progression through G1 or early S phase.

The sequence of 24.3 kb from chromosome X reveals five complete open reading frames, all of which correspond to new genes, and a tandem insertion of a Ty1 transposon.

We have determined the complete nucleotide sequence of a 24.3 kb segment from chromosome X carried by the cosmid pEJ103. The sequence encodes five complete open reading frames (ORFs), none of which correspond to previously described genes; however, four of these ORFs display interesting similarities with sequences present in the databanks. The sequence also contains a tandem insertion of a Ty1 element. An investigation of the Ty1 polymorphism in other strains has revealed that the original insertion occurred within an ORF. Finally, the structure of the Ty1 repeat suggests a mechanism by which it may have been generated.

Heat shock protein HSP60 can alleviate the phenotype of mitochondrial RNA-deficient temperature-sensitive mna2 pet mutants.

mna2, which belongs to the class I temperature-sensitive pet mutants that lose mitochondrial (mt)RNA at restrictive temperature, was shown by complementation and sequence determination to correspond to the gene coding for HSP60. Both mna2-1 and mna2-2, the two available alleles of mna2, have conservative single amino acid substitutions in the HSP60 gene. Valine substitutes for an alanine (position 47) in mna2-1, and an isoleucine substitutes for a valine (position 77) in mna2-2. These substitutions result in defects in respiration and in steady-state mtRNA accumulation. Wild-type hsp60 alleviates the mtRNA phenotype completely, while partially relieving the respiratory deficiency.

Subcellular relocalization of a long-chain fatty acid CoA ligase by a suppressor mutation alleviates a respiration deficiency in Saccharomyces cerevisiae.

We have isolated an extragenic suppressor, FAM1-1, which is able to restore respiratory growth to a deletion of the CEM1 gene (mitochondrial beta-keto-acyl synthase). The sequence of the suppressor strongly suggests that it encodes a long-chain fatty acid CoA ligase (fatty-acyl-CoA synthetase). We have also cloned and sequenced the wild-type FAM1 gene, which is devoid of suppressor activity. The comparison of the two sequences shows that the suppressor mutation is an A-->T transversion, which creates a new initiation codon and adds 18 amino acids to the N-terminus of the protein. This extension has all the characteristics of a mitochondrial targeting sequence, whilst the N-terminus of the wild-type protein has none of these characteristics. In vitro mitochondrial import experiments show that the N-terminal half of the suppressor protein, but not of the wild-type, is transported into mitochondria. Thus, we hypothesize that the suppressor acts by changing the subcellular localization of the protein and relocating at least some of the enzyme from the cytosol to the mitochondria. These results support the hypothesis that some form of fatty acid synthesis, specific for the mitochondria, is essential for the function of the organelle.

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

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.

We have determined the complete nucleotide sequence of a 12.5 kb segment from the right arm of chromosome II carried by the cosmid alpha 20. 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.

YBR1012 an essential gene from S. cerevisiae: construction of an RNA antisense conditional allele and isolation of a multicopy suppressor.

The gene YBR1012 was identified during the systematic sequencing of chromosome II of the yeast Saccharomyces cerevisiae. We have inactivated the gene and shown that it is essential for cellular viability. Using antisense RNA technology we have constructed a conditional allele, expression of the antisense RNA strongly inhibits growth. To our knowledge this is the first successful use of antisense RNA technology in S. cerevisiae. Comparison of the deduced ybr1012p sequence with the data banks revealed the presence of a putative phosphatidylinositol kinase domain and a strong homology to the Schizosaccharomyces pombe rad3p. These results suggest that ybr1012p may be involved in signal transduction, possibly related to the control of replication and/or DNA damage repair. The link with DNA damage repair was reinforced by the isolation of the DUN1 gene as a multicopy suppressor of the YBR1012 deletion.

An analysis of the sequence of part of the right arm of chromosome II of S. cerevisiae reveals new genes encoding an amino-acid permease and a carboxypeptidase.

We have analysed two new genes, YBR1007 and YBR1015, discovered during the systematic sequencing of chromosome II of S. cerevisiae. YBR1007 shows strong similarities to amino-acid permeases, in particular the high-affinity proline permeases of S. cerevisiae and A. nidulans. The number and position of the predicted membrane-spanning domains suggest a conserved structure for these proteins, with 12 trans-membrane domains. YBR1015 shows strong similarities to serine carboxypeptidases; all three residues of the "catalytic triad" typical of this family of enzymes are conserved in the YBR1015 protein. In a preliminary functional analysis we have created a null allele of the YBR1015 gene, and shown that it is not essential for cellular viability.