Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, -aminoadipate reductase. The LYS5 gene of S. cerevisiae was cloned by functional complementation of a lys5 mutant, X4004-3A, using a YEp24 plasmid library. The cloned LYS5 gene was contained within a 7.5 kb DNA insert of the recombinant plasmid pSC5. Cloning of LYS5 gene was confirmed by second cycle transformation of a lys5 mutant with the pSC5 plasmid, growth response studies, and plasmid loss experiments with Lys5 ⁺ transformants. Analysis of restriction digests of the pSC5 plasmid revealed 3 EcoRI, 5 PvuII, 1 PstI, 1 BglII and 2 HpaI sites in the 7.5 kb insert. A 3.9 kb internal pSC5 fragment hybridized only to the plasmid pSC5, but no homology was observed with LYS2 DNA or the YEp24 vector. The pSC5 transformed Lys5 ⁺ cells and the wild-type strain exhibited same level of -aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2 ⁺ transformants consistently had five times greater -aminoadipate reductase activity when compared with the wildtype and the Lys5 ⁺ transformant. The -aminoadipate reductase activity was repressed in lysine-grown wildtype and Lys5 ⁺ transformed cells but not in Lys2 ⁺ transformed cells. A Lys2 ⁺ and Lys5 ⁺ double transformant exhibited higher a-aminoadipate reductase activity than lys2 ⁺ or lys5 ⁺ transformant.     

Physical and functional characterization of the cloned lysl + gene of Schizosaccharomyces pombe

The α-aminoadipate pathway for the biosynthesis of lysine is present in yeast and other higher fungi. The lys2 and lys5 mutants of Saccharomyces cerevisiae as well as the lys1– and lys7 –mutants of Schizosacharomyces pombe are blocked at the α-aminoadipate reductase step of this pathway. The cloned lys1 + gene in the plasmid pLYS1 isolated from a S. pombe genomic library complemented lys1– mutant of S. pombe. The cloned LYS2 gene in the plasmid YEp620 and the LYS5 gene in the plasmid pSC5 of S. cerevisiae exhibited heterologous complementation of lys1– and lys7– mutants, respectively, of S. pombe. The homologous lys1 + transformed cells exhibited five fold higher α-aminoadipate reductase activity while the heterologous lys1 + and lys7 + transformed cells exhibited much less activity than the the wild type cells. The DNA insert of the plasmid pLYS1 was determined to be 16.7 kb long and the lys1 + gene has been subcloned within a 9.1 kb Clal-Clal DNA insert of the recombinant plasmids pLYS1B and pLYS1C. The restriction pattern for 12 enzymes of the 9.1 kb DNA insert, (Apal, Aval, BamHI, Clal, EcoRI, EcoRV, HindIII, Hpal, Pstl, Pvull, Sphl, and Xbal), exbibited no obvious similarity to that of the LYS2 gene of S. cerevisiae. A 1.7 kb EcoRI-HindIII DNA fragment of pLYS1B and pLYS1C complemented the lys1-131 mutation in an integrative transformation. Although the lys1 + gene of S.pombe is isofunctional to the LYS2 gene of S. cerevisiae, the restriction sites, and expression of these two genes exhibited considerable divergence.     

A yeast strain producing lys2 deletions at a high frequency after sporulation

Summary A diploid yeast strain with extensive sequence dissimilarity in homologous regions near the LYS2 locus was sporulated, and spontaneous lys2 and lys5 mutant spores, selected on -amino adipate, were analyzed. As many as 50% of the mutant spores contained a deletion in LYS2. These deletions occurred at a frequency of 5.0 × 10^–7. While deletions of various sizes and endpoints were obtained, all the deletions recovered in this study included the border between homologous and non-homologous sequences located 4 kb upstream of LYS2. Large lys2 deletions that extended into an adjacent CYH2 duplication occurred at a frequency of 2.0 × 10^–7, more than 1,000 times the frequency of the CYH2-LYS2 deletions found in a related haploid strain. This high frequency of CYH2-LYS2 deletions was observed only after sporulation of the diploid strain, and was dependent upon extensive sequence dissimilarity near the LYS2 locus.     

The Saccharomyces cerevisiae HIS3 and LYS2 genes complement the Schizosaccharomyces pombe his5-303 and lys1-131 mutations,respectively: new selectable markers and new multi-purpose multicopy shuttle vectors,pSP3 and pSP4

Three new S. pombe plasmids are described. Plasmids pSP3 and pSP4 are two Schizosaccharomyces pombe ars1 multicopy vectors with the Saccharomyces cerevisiae HIS3 or LYS2 genes as selectable markers. They complement the S. pombe his5-303 or lys1-131 mutations, respectively. Plasmid pSPars1 is a vector carrying the S. pombe ars1 and a unique NdeI site which allows the introduction of any selectable marker therefore bringing a unified vector backbone for the construction of new S. pombe/S. cerevisiae/E. coli shuttle vectors. These plasmids permit classical molecular genetic techniques to be performed directly.     

Molecular cloning of the ARG7 gene of Schizosaccharomyces pombe encoding argininosuccinate lyase

Summary A gene bank of Sau3A partially restricted Schizosaccharomyces pombe DNA in YEp13 was used to transform an arg4 mutant of Saccharomyces cerevisiae. One colony was recovered which contained the YEp13 plasmid bearing a large insert complementing the argininosuccinate lyase (ASL) mutation. As shown by restriction mapping and subcloning experiments, the DNA sequence required for complementation is localized on a 2 kb BamHI-BamHI fragment. The plasmid complemented several S. cerevisiae arg4 mutants of independent origin and a S. pombe arg7 mutant lacking ASL. Low but significant ASL activities were detected in crude extracts of these transformants. No complementation of the E. coli argH mutant was observed. Southern blot hybridizations showed that the insert originates from the S. pombe genome. No cross-hybridization was found between this sequence and S. cerevisiae DNA. It can be concluded that the cloned DNA fragment bears the S. pombe ARG7 gene coding for ASL.Abbreviations ASL argininosuccinate lyase - bp base pair - kb kilobase pair     

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa⁺) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa^- diploid cells yield -Aa⁺ mutants at a rate of 7.8±3.6×10^-9. As in haploid strains, approximately 97% (30/31) of -Aa⁺ mutants are spontaneous lys2-x recessive mutations. -Aa⁺ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10^-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10^-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement.The research of the authors was supported by the Director, Office of Energy Research, Biological Research Division of the U.S. Department of Energy under Contract No. DE-ACO3-76SF00098, grants to M. S. E. and C. V. B. from the National Institutes of Health and the National Aeronautics and Space Administration, and a National Science Foundation postdoctoral research fellowship award to R. M. R.     

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 ⁺ gene, which is homologous to Saccharomyces cerevisiae LYS4 ⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 ⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7 ⁺ gene with a lys2 ⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2 ⁺ gene with a his7 ⁺-marked PCR product. Strains carrying these deletions of lys2 ⁺ or his7 ⁺ serve as relatively efficient hosts for the deletion of the ade6 ⁺ gene by lys2 ⁺- or his7 ⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 ⁺ as a selectable marker, along with improved strains for the use of his7 ⁺ to mark gene deletions.     

Biosynthetic and regulatory role of lys9 mutants of Saccharomyces cerevisiae

Summary Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, -aminoadipate aminotransferase, -aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 S mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

A gene (Cargl) that regulates tissue resistance toCandida albicansmaps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeastCandida albicansis genetically regulated. Analysis of the strain distribution pattern of theC. albicansresistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant forCarg1,TcraandRib1. Therefore,Carg1is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes.     

Isolation of a DNA fragment which complements glutamine synthetase deficient strains ofS. pombe

Summary From a gene bank ofS. pombe DNA, a 5.6 kb clone was isolated which complemented mutants defective in glutamine synthetase (GS) activity. Sub-cloning fragments of this 5.6 kb clone showed that the complementing activity was localised in a 1.6 kb HindIII-Aval fragment and a partial DNA sequence revealed an open reading frame preceded by TATA sequences and a TGACTA sequence. Plasmid constructs carrying up to 3.4 kb of DNA used to transformgln ⁻ strains gave transformants which showed a wide range of GS activity, in some cases 100 times the wild-type level. These constructs identify DNA sequences lying downstream from the putative coding sequence which have effects on the total amount of enzyme activity, but do not affect the control imposed by the nitrogen source on which the cells are grown.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.     

Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii

Summary In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment. This fragment was cloned and sequenced. In this report, the galK expression plasmid, pK01 was used to screen for the presence of any prokaryotic promoter within the cloned fragment. The insertion of 2 AluI fragments yielded galK⁺ colonies. Sequence analyses of these Alul inserts revealed prokaryotic promoter consensus regions. Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert. Two back-to-back prokaryotic promoters were mapped on a 79 by Alul fragment located within the displacement loop region.     

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.     

Chromium sensitive mutants of the yeast Saccharomyces cerevisiae

Summary Ten mutants of Saccharomyces cerevisiae sensitive to CrO₃ were obtained and genetically analyzed. All of them were recessive. Segregation of the sensitivity after crossing them with a wild type strain indicated that the sensitivity of some mutants was determined by single mutations and that of others was caused by multiple mutations. Among 6 mutations found in mutants having single mutations responsible for the sensitivity, 6 loci were distinguished by complementation. One of them was identified as LYS7, the gene previously known as the structure gene for homocitric dehydrase, in terms of co-segregation and co-reversion of chromium sensitivity and lysine dependency which did not complement an authentic lys7 mutation.     

Sequences of three dsRNAs associated with La France disease of the cultivated mushroom (Agaricus bisporus)

Summary La France disease of the cultivated mushroom, Agaricus bisporus, is known to be associated with the presence of a number of dsRNA segments. The nucleotide sequences of the dsRNAs M2 (1.3 kb), M1 (1.55 kb) and L3 (2.8 kb), invariably associated with the disease, were determined. Putative coding sequences for proteins with molecular weights of 38, 40 and 87 kDa were found for M2, M1 and L3 dsRNAs, respectively. The average G+C content of these dsRNAs was 43%, close to that of A. bisporus nuclear DNA. The nucleotide sequences, as well as the amino acid sequences, appear to be unique, as no matching sequences could be found among databases. S3 dsRNA (0.39 kb), which is occasionally found in large amounts in diseased mushrooms, is an internally deleted variant of M2 dsRNA and is largely composed of the non-coding ends of that dsRNA.     

Identification of a lys2 mutant of Candida maltosa by means of transformation

Summary We have isolated five mutants of Candida maltos, which lack the 2-aminoadipate reductase activity, an enzyme involved in the lysine biosynthesis. By means of complementation analysis using protoplast fusion, the isolated mutants were divided into two complementation groups. Thereof the C. maltosa strain G457 could be transformed by the plasmids pDP12 and pDP13, which contain the L YS2-coding gene of Saccharomyces cerevisiae. On the basis of our presented results obtained by studies on hybridization, stability, and recovery of plasmids from C. maltosa transformants, we suggest that transformation does proceed integratively.Abbreviations AA 2-aminoadipate - PRA phosphoribosylanthranilate