Cloning and integrative deletion of the RAD6 gene of Saccharomyces cerevisiae

Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad⁺ levels of resistance to U.V., MMS and -rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids.Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae

Summary A DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 m DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

Cloning of maltase regulatory genes in Saccharomyces cerevisiae

Summary By hybridization with a putative MAL2p regulatory sequence we have identified a 19 kb long BamH1 DNA fragment to contain the MALp sequence in a MAL4 strain. A mixture of recombinant plasmids was prepared by ligation of purified 19 kb BamH1 fragments partially digested with Sau3A into the multicopy vector YEp1357. The source of DNA was a strain carrying the MAL4 locus. Yeast maltose non-fermenting strains were transformed with the plasmid mixture. A recombinant plasmid, pRM-4, containing the MAL4p regulatory gene was isolated that complements the maltose-negative phenotype. The plasmid was shown to confer the ability to synthesize maltase to recipient strains grown under inducing as well as under repressing conditions.The MAL4p regulatory sequence cloned was used as a probe in hybridization experiments to study the degrees of homology between the different MAL regulatory genes. The results showed that the sequence from MAL4 strains is strongly homologous to that of MAL3 strains whereas it shows significant differences to the ones of MAL1 and MAL2 strains.Southern analysis of the segregants of crosses between maltose-positive strains and ma10 strains allowed us to localize the maltase regulatory sequence of each MAL locus within a characteristic BamH1 fragment of genomic DNA hybridizing to the isolated sequence.     

Cloning and characterization of the SKI3 gene of Saccharomyces cerevisiae demonstrates allelism to SKI5

Summary We have identified a mutant strain of the yeast Saccharomyces cerevisiae which overproduces killer toxin. This strain contains a single mutation which fails to complement defects in both the SKI3 and SKI5 genes, while a cloned copy of this gene complements both the ski3 and ski5 defects. The level of secreted toxin from a cDNA based plasmid is not increased in a ski3 strain, showing that the overproduction phenotype is dependent upon an increased level of M1 dsRNA.     

Phenotypic identification of amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae

Primary gene amplification, i.e., mutation from one gene copy to multiple gene copies per genome, is important in genomic evolution, as a means of producing anti-cancer drug resistance, and is associated with the progression of tumor malignancy. Primary amplification has not been studied in normal eukaryotic cells because amplifications are extremely rare in these cells. A system has been developed to phenotypically identify co-amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae and 21 independent spontaneous amplifications have been isolated.     

Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae

5-Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6-hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1-overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.     

Sequence analysis of a Papaver somniferum L. mitochondrial DNA fragment promoting autonomous plasmid replication in Saccharomyces cerevisiae and Kluyveromyces lactis

The minimal fragment of mitochondrial DNA from Papaver somniferum L. (poppy) able to promote autonomous plasmid replication in the yeast Saccharomyces cerevisiae was sequenced. Sequence analysis of the 917-bp MK4/8 DNA fragment revealed a high AT content, and the presence of two 12-bp sequences differing from the ARS core consensus of S. cerevisiae only by a T and C insertion, respectively. The mitochondrial insert contains a further six 11-bp sequences with one mismatch to the S. cerevisiae core consensus, more then 20 related sequences with two base pair exchanges, numerous direct and inverted repeats, and many copies of a sequence motif called the ARS box. The original 4.2-kb mitochondrial DNA fragment, as well as the minimal 917-bp subfragment in vector pFL1-E (a variant of YIP5, lacking an origin of replication in yeast), were then tested for their ability to replicate autonomously in another fungus, Kluyveromyces lactis.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

A recessive mutant allele of the HNM1 gene of Saccharomyces cerevisiae is responsible for hyper-resistance to nitrogen mustard

Summary A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementations group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.     

Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae

The ICL1 gene encoding the isocitrate lyase from Saccharomyces cerevisiae was cloned and sequenced. A reading frame of 557 amino acids showing significant similarity to isocitrate lyases from seven other species could be identified. Construction of icl1 null mutants led to growth defects on C₂ carbon sources while utilization of sugars or C₃ substrates remained unaffected. Using an ICL1-lacZ fusion integrated at the ICL1 locus, a more than 200-fold induction of -galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions. A preliminary analysis of the ICL1 upstream region identified a 364-bp fragment necessary and sufficient for this regulatory phenotype. Sequence motifs also present in the upstream regions of co-regulated genes were found within this region.     

Cloning and expression on a multicopy vector of five invertase genes of Saccharomyces cerevisiae

Summary Six unlinked loci for invertase structural genes are known in the yeast Saccharomyces cerevisiae: SUC1-SUC5 and SUC7. These genes are similar in structure and expression but not identical. Different yeast strains possess none, one or several of these genes.We have isolated the genes SUC1-SUC5, subcloned them into the multicopy vector YEp24 and compared the expression of the five SUC genes in one recipient strain. SUC2 was isolated by transformation of a suc0 strain with a gene pool and complementation to sucrose fermentation. SUC4 was cloned from a minipool of chromosomal fragments which were shown to contain SUC4 by Southern hybridization. SUC1, SUC3 and SUC5 were isolated using the method of plasmid eviction. A plasmid containing regions flanking SUC4 was integrated next to these SUC genes. The plasmid together with the SUC genes were then cut out of the chromosome using an appropriate restriction endonuclease.The length of chromosomal DNA fragments containing the different SUC genes were 4.8 kb for SUC1, 5.2 kb for SUC2, 4.8 kb for SUC3, 12.8 kb for SUC4 and 17.2 kb for SUC5.Fragments containing the complete SUC genes and the sequences controlling their expression were subcloned into YEp24 and transformed into a strain without any active invertase gene. Invertase activity of transformants was measured after growth repressing (8% glucose) and derepressing (2% raffinose) conditions. As expected from results with strains carrying the individual SUC genes in a chromosomal location, the SUC genes were expressed to a different extent.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthdayThis work was supported by Deutsche Forschungsgemeinschaft     

Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase

Summary The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48 500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.     

Chromosomal mapping of the uracil permease gene of Saccharomyces cerevisiae

Summary The gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.     

Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae

A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37–48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.     

Physical mapping of the MEL gene family in Saccharomyces cerevisiae

Nine members, MEL2–MEL10, of the MEL gene family coding for -galactosidase were physically mapped to the ends of the chromosomes by chromosome fragmentation. Genetic mapping of the genes supported the location of all the MEL genes in the left arm of their resident chromosomes.     

A method to sharply delimit a yeast nuclear gene in a cloned DNA fragment

Summary We have developped a procedure to delimit the boundaries of a cloned gene carried on a DNA fragment as large as 4 to 5 kilobases. The method consists in the following. Two series of limit digest products generated with a tetranucleotide recognition sequence endonuclease and originating from either of the two ends of this DNA segment are tested for their complementing capacity by yeast transformation. The gene is then delimited by the overlap of the two shortest complementing fragments.