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.     

Mass-murdering: deletion of twenty-three ORFs from Saccharomyces cerevisiae chromosome XI reveals five genes essential for growth and three genes conferring detectable mutant phenotype

In the frame of the European Network for Functional Analysis (EUROFAN), two regions from chromosome XI covering 54kb have been subjected to 'mass-murder'. Ten deletions covering 23 novel open reading frames (ORFs) were constructed in haploid and diploid strains. Six deletions were lethal in haploid strains. One deletion caused slow germination of spores and slow cellular growth, and another one was associated with both cellular growth thermosensitivity and poor growth on glycerol. These two defects were assigned to two different genes. All mutant phenotypes were complemented by a single gene, enabling us to identify five genes essential for vegetative growth, three genes with detectable phenotype and 15 dispensable genes under standard physiological conditions.     

CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae.

By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.     

Evidence for a new chromosome in Saccharomyces cerevisiae.

The current yeast map has 16 chromosomes, each originally defined by a centromere-linked gene unlinked to previously defined centromere markers. We examined four genes, cly2, KRB1, AMY2, and tsm0115, each centromere linked, but previously thought to be not on chromosomes I to XVI. We found that AMY2 is linked to cly2, and both are on chromosome II. tsm0115 is on the left arm of chromosome XVI. We confirm the earlier evidence that KRB1 is not on chromosomes I through XVI. This gene thus defines a new chromosome XVII. We also report meiotic linkage of met4 and pet8 (on chromosome XIV), confirming the connection between the petx-kex2 fragment of XIV and the centromere of XIV.     

Role of essential genes in mitochondrial morphogenesis in Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.     

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes.

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.     

SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae.

We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum. These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides. The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins. Their sequences, however, are sufficiently different to indicate that they are not true ras proteins. More extensive sequence identity (approximately 55%) is shared with the YPT1 and SEC4 proteins from Saccharomyces cerevisiae. These yeast proteins are essential for growth and are believed to be involved in intracellular signaling associated with membrane function. SAS1 and SAS2 exhibit distinct patterns of genomic organization and developmentally regulated gene expression. SAS1 contains introns and is associated with a developmentally regulated repetitive element. SAS2 is colinear with its mRNA and does not appear to be closely linked with this repetitive element. Both genes are expressed during growth and throughout development. SAS1 is maximally expressed during cytodifferentiation, when two sizes of SAS1 mRNA are detectable. SAS2 mRNA levels are maximal during culmination. On the basis of the expression patterns of the SAS genes and their relationship to the YPT1 and SEC4 genes, we discuss possible functions of the SAS proteins.     

Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.