REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase

We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.     

Schistosoma mansoni: the IMP4 gene is involved in DNA repair/tolerance after treatment with alkylating agent methyl methane sulfonate

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.     

Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae.

The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START'. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200-nucleotides mRNA. We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain. Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein.     

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae

Abstract We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae . The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues ( M _r 76.4 kDa) with strong sequence similarites to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae .     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.     

Identification and analysis of a DNA fragment from Saccharomyces kluyveri that can complement the loss of CDC25 function in Saccharomyces cerevisiae.

In the budding yeast, Saccharomyces cerevisiae, the function of wild-type Ras proteins is dependent on the CDC25 protein, which promotes the exchange of guanine nucleotides bound to Ras. To facilitate the identification of proteins which similarly regulate Ras function in higher eukaryotes, we have identified the CDC25 gene from another budding yeast, Saccharomyces kluyveri, by low-stringency hybridization to an S. cerevisiae CDC25 restriction fragment. This protein, SKCDC25, shares significant amino acid homology with CDC25, SCD25, and Ste6 of Schizosaccharomyces pombe in the C-terminal portion of the protein. The expression of SKCDC25 in a temperature-sensitive cdc25 strain of S. cerevisiae complements the loss of endogenous CDC25 activity. The identification of the highly conserved C-terminal sequences, which direct bona fide CDC25 activity within these proteins, will aid in the isolation of CDC25 genes from higher eukaryotes.     

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.     

Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene

The function of the cell division cycle gene, CDC4, is required in Saccharomyces cerevisiae for progression beyond the G1 phase of the cell cycle. The wild-type gene was isolated from a plasmid library by selection for complementation of a recessive, temperature-sensitive allele. Hybridization of genomic sequences with the cloned gene revealed the presence of a duplicated sequence. Both CDC4 and the duplicated sequence were subjected to DNA sequence analysis. These analyses revealed (1) that CDC4 contains a large open reading frame encoding a protein of 779 amino acids, and (2) that the duplicated sequence bears strong homology with the carboxy-terminal segment of this open reading frame. Presence of a nonsense codon within the duplicated sequence suggested that it does not encode a functional product. Disruption of the duplicated sequence within the yeast genome provided a more critical test for function. The absence of any detectable phenotype for this disruption confirms that the sequence should be considered a pseudogene. The marker inserted to disrupt the sequence also served to map the duplication and to establish that it is not genetically linked to CDC4. The structural features determined suggest evolutionary relationships between these genes as well as between the CDC4 product and other proteins.     

Characterization of a yeast mitochondrial ribosomal protein structurally related to the mammalian 68-kDa high affinity laminin receptor.

We have cloned the nuclear gene MRP4 coding for a mitochondrial ribosomal protein of the yeast, Saccharomyces cerevisiae. The gene was isolated by complementation of a respiratory-deficient mutant with a pleiotropic defect in mitochondrial gene expression. The nucleotide sequence of MRP4 revealed that it has sequence similarity with Escherichia coli ribosomal protein S2 and related proteins of chloroplast ribosomes from different plants. Further characterization of the MRP4 protein revealed that it is a component of the 37 S subunit of mitochondrial ribosomes. Moreover, the phenotype of cells carrying a disrupted copy of MRP4 is consistent with the MRP4 protein being an essential component of the mitochondrial protein synthetic machinery. Finally, we note that the MRP4 protein and other members of the S2 family of ribosomal proteins have regions of sequence similarity with the mammalian 68-kDa high affinity laminin receptor.     

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310. The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.     

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation.

Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S. cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thus, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.     

Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.