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.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

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.

Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human.

LAG1 is a longevity gene, the first such gene to be identified and cloned from the yeast Saccharomyces cerevisiae. A close homolog of this gene, which we call LAC1, has been found in the yeast genome. We have cloned the human homolog of LAG1 with the ultimate goal of examining its possible function in human aging. In the process, we have also cloned a homolog from the nematode worm Caenorhabditis elegans. Both of these homologs, LAG1Hs and LAG1Ce-1, functionally complemented the lethality of a lag1delta lac1delta double deletion, despite low overall sequence similarity to the yeast proteins. The proteins shared a short sequence, the Lag1 motif, and a similar transmembrane domain profile. Another, more distant human homolog, TRAM, which lacks this motif, did not complement. LAG1Hs also restored the life span of the double deletion, demonstrating that it functions in establishing the longevity phenotype in yeast. LAG1Hs mapped to 19p12, and it was expressed in only three tissues: brain, skeletal muscle, and testis. This gene possesses a trinucleotide (CTG) repeat within exon 1. This and its expression profile raise the possibility that it may be involved in neurodegenerative disease. This possibility suggests at least one way in which LAG1Hs might be involved in human aging.

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 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.

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.

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.

The sequence of 29.7 kb from the right arm of chromosome II reveals 13 complete open reading frames, of which ten correspond to new genes.

We have determined the complete nucleotide sequence of a 29.7 kb segment from the right arm of chromosome II carried by the cosmid alpha 61. The sequence encodes the 3' region of the IRA1 gene and 13 complete open reading frames, of which ten correspond to new genes and three (CIF1, ATPsv and CKS1) have been sequenced previously. The density of protein coding sequences is particularly high and corresponds to 84% of the total length. Two new genes encode membrane proteins, one of which is particularly large, 273 kDa. In one case (ATPsv), the comparison of our sequence and the published sequence reveals significant differences.

The ogd1 and kgd1 mutants lacking 2-oxoglutarate dehydrogenase activity in yeast are allelic and can be differentiated by the cloned amber suppressor.

The activity of mitochondrial 2-oxoglutarate dehydrogenase in S. cerevisiae can be impaired either by the ogd1 or the kgd1 mutation. The OGD1 gene and two suppressor genes were isolated by complementation of the ogd1 mutant. The complementation of the kdg1 mutant by the OGD1 gene, an allelism test, and meiotic mapping, revealed that the ogd1 and kgd1 mutations are allelic. The two mutations were differentiated by the cloned suppressor gene which was able to partially complement ogd1, but not kgd1. The molecular analysis of the suppressor gene revealed its identity with the natural tRNA(GlnCAG) gene found in the upstream region of URA10.