Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.

Differential evolution of the Saccharomyces cerevisiae DUP240 paralogs and implication of recombination in phylogeny.

Multigene families are observed in all genomes sequenced so far and are the reflection of key evolutionary mechanisms. The DUP240 family, identified in Saccharomyces cerevisiae strain S288C, is composed of 10 paralogs: seven are organized as two tandem repeats and three are solo ORFs. To investigate the evolution of the three solo paralogs, YAR023c, YCR007c and YHL044w, we performed a comparative analysis between 15 S.cerevisiae strains. These three ORFs are present in all strains and the conservation of synteny indicates that they are not frequently involved in chromosomal reshaping, in contrast to the DUP240 ORFs organized in tandem repeats. Our analysis of nucleotide and amino acid variations indicates that YAR023c and YHL044w fix mutations more easily than YCR007c, although they all belong to the same multigene family. This comparative analysis was also conducted with five arbitrarily chosen Ascomycetes-specific genes and five arbitrarily chosen common genes (genes that have a homolog in at least one non-Ascomycetes organism). Ascomycetes-specific genes appear to be diverging faster than common genes in the S.cerevisiae species, a situation that was previously described between different yeast species. Our results point to the strong contribution, during DNA sequence evolution, of allelic recombination besides nucleotide substitution.

The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship.

Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope-exchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the aminoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gln and of the the deacylation of glutaminyl-tRNA Gln. At acidic pH, glutaminyl-tRNA synthetase catalyzes the synthesis of the glutaminyl-tRNA Gln and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating than glutaminyl-tRNA Gln cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.

Structural studies on aminoacyl-tRNA synthetases. A tentative correlation between the subunit size and the occurrence of repeated sequences.

Recent studies have shown that those synthetases with subunits greater than 85,000 daltons contain extensive repeated sequences, whilst those with small subunits (40,000 daltons) do not. We have undertaken a comparative study of four aminoacyl-tRNA synthetases (glutamyl-, arginyl-, valyl-, and phenylalanyl-tRNA synthetases) with subunit sizes ranging from 56,000 to 130,000 daltons in an attempt to correlate the occurrence and extent of the repeats with the length of the polypeptide chain. Our results show that monomeric glutamyl-tRNA synthetase from Escherichia coli (56,000 daltons) contains few repeated sequences, whereas both subunits of yeast phenylalanyl-tRNA synthetase (alpha, 73,000 daltons; beta, 62,000 daltons) and yeast arginyl-tRNA synthetase (74,000 daltons) do have a significant amount of repeats. Thus 56,000 dalton appears to be the minimum size compatible with the existence of such repeats.

Allosteric regulation of carbamoylphosphate synthetase-aspartate transcarbamylase multifunctional protein of Saccharomyces cerevisiae: selection, mapping and identification of missense mutations define three regions involved in feedback inhibition by UTP.

The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase). The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb). All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain. In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms. Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain. They are clustered in two regions of this domain which must be directly involved in the feedback process.

Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae.

Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura(+) revertants of a particular ura2 mutant. Among 133 Ura(+) revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura(+) reversion rate was increased 4.2-fold in a rad52Delta strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Delta context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.

Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex.

The 6636 bp of the yeast URA2 gene encoding the carbamoylphosphate synthetase-aspartate transcarbamylase complex have been sequenced. The protein is organized into four regions, three of which are functional domains as indicated previously by genetic analysis. The fourth domain corresponds to a defective dihydroorotase called DHOase-like. The URA2 gene complex with the same organization as the equivalent genes in higher eukaryotes suggests an evolution from a common ancestral gene.

The Candida albicans Na(+)/H(+) antiporter exports potassium and rubidium.

The Candida albicans Cnh1p belongs to the family of Na(+)/H(+) antiporters (TC 2.A.36) but it transports besides toxic sodium and lithium also rubidium and potassium. Upon heterologous expression in a Saccharomyces cerevisiae salt-sensitive strain, the Cnh1p is targeted to the plasma membrane and its transport activity results in increased tolerance of cells to external alkali metal cations. The cation efflux activity of Cnh1p in S. cerevisiae depends on the gradient of protons across the plasma membrane, and a Cnh1p-mediated K(+) efflux is involved in a cell response to sudden rise of cytoplasmic pH.

Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae.

The NHA1 gene (2958 nt) encoding a putative Na(+)/H(+) antiporter (986 aa) in Saccharomyces cerevisiae was cloned by selection based on increased NaCl tolerance. The putative protein is highly similar to sodium/proton antiporters from Schizosaccharomyces pombe (gene sod2), and Zygosaccharomyces rouxii (gene Z-SOD2). Overexpression of the NHA1 gene results in higher and partially pH-dependent tolerance to sodium and lithium; its disruption leads to an increased sensitivity towards these ions.

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast.

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.

Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii.

This paper reports the genomic analysis of strain CBS732 of Zygosaccharomyces rouxii, a homothallic diploid yeast. We explored the sequences of 4934 random sequencing tags of about 1 kb in size and compared them to the Saccharomyces cerevisiae gene products. Approximately 2250 nuclear genes, 57 tRNAs, the rDNA locus, the endogenous pSR1 plasmid and 15 mitochondrial genes were identified. According to 18S and 25S rRNA cladograms and to synteny analysis, Z. rouxii could be placed among the S. cerevisiae sensu lato yeasts.

Genomic exploration of the hemiascomycetous yeasts: 15. Pichia sorbitophila.

This paper reports the genomic analysis of the strain CBS7064 of Pichia sorbitophila, a homothallic diploid yeast. We sequenced 4829 random sequence tags of about 1 kb and compared them to the Saccharomyces cerevisiae gene products. Approximately 1300 nuclear genes, 22 tRNAs, the rDNA locus, and six mitochondrial genes have been identified. The analysis of the rDNA genes has permitted to classify this organism close to the Candida species. Accession numbers from AL414896 to AL419724 at EMBL databank.

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.

Genomic exploration of the hemiascomycetous yeasts: 19. Ascomycetes-specific genes.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific'. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes. Half of the 'Ascomycetes-specific' genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

Genomic exploration of the hemiascomycetous yeasts: 21. Comparative functional classification of genes.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

Studies on transcription of the yeast URA2 gene.

The multifunctional protein carbamoylphosphate synthetase (CPSase)-aspartate transcarbamylase (ATCase) encoded by the URA2 gene catalyses the first two steps of the yeast pyrimidine pathway. An excess of the final product, the intracellular UTP (uridine triphosphate), inhibits both the transcription of the URA2 gene and the enzymatic activities. Results presented in this paper suggest that transcription of URA2 is negatively regulated (repression-derepression) and establish that this regulation is less efficient in the flow of the pyrimidine pathway than feedback inhibition.