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.     

Adenine and pyrimidine genes of Aspergillus niger and evidence for a seventh linkage group

Summary Mutants of Aspergillus niger requiring adenine and one mutant requiring cytosine were isolated after low-dose mutagenesis and enrichment. In addition we had mutants of two genes involved in the pyrimidine biosynthesis isolated as 5-fluoro-orotic acid-resistant mutants. The fifteen adenine-less mutants could be placed in seven complementation groups. From each group a representative mutant was analyzed in order to determine the linkage group by analysis of the mutants in a heterozygous diploid carrying markers in six linkage groups. AdeF could not be assigned to any one of these linkage groups and proved to be linked to nicB, oliC and cnxC, none of which could be placed in a linkage group. Thus, conclusive evidence was obtained for a seventh linkage group. As pyrA was used as selection marker for transformation, we constructed a pyrA strain with a linked marker which can be used in the genetic analysis of transformants.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

Isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae by functional complementation in yeast

Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment.By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.     

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.     

Isolation,characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.     

MAL63 codes for a positive regulator of maltose fermentation in Saccharomyces cerevisiae

Summary Genetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63 Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a ma163 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

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.     

Isolation of a selective marker from Cephalosporium acremonium by complementation of an auxotrophic mutant of E. coli

Summary A Cephalosoporium acremonium gene library has been constructed using cosmid vector pYcl. After transformation of E. coli leuB strain JA221 with cosmids isolated from this gene library a cosmid (pCeleuB43) was isolated which complements the leucine auxotrophy of JA221. The same cosmid is also able to complement a leu2 strain of S. cerevisiae. The leuB and leu2 genes both code for -isopropylmalate dehydrogenase. The complementation of a leuB E. coli strain and a leu2 S. cerevisiae strain strongly suggests that the analogous gene from C. acremonium has been isolated.     

Photoperception mutants in Trichoderma: mutants that sporulate in response to stress but not light

Summary In an effort to find photoreceptor mutants, we isolated mutants of the fungus Trichoderma defective in blue-light induced conidiation, but still inducible by stress, an alternate stimulus. This selection criterion was based on a physiological model in which both stimuli share a common pathway for expression (output).Complementation in heterokaryons was used to estimate the number of different lesions responsible for the selected phenotypes. As a working hypothesis, each pair that did not complement when tested at a standard light level was assumed to represent a complementation group. When medium depletion, the least stringent stress condition was used, an average of 17 complementation groups was estimated using different mathematical techniques. These groups would represent about 17 gene products; all required for only the light-specific portion of the sporulation pathway. The number of candidates for mutants in the photoreceptor pathway was further reduced by choosing only those strains that responded to a transient stress by a brief incubation in water, which seemed more stringent. The mutants in this subclass mapped into four complementation groups. Two of these groups had both defective absorption and modified action spectra, and are probably defective in the photoreceptor, cryptochrome.     

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.     

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.     

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function

Summary This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.     

Identification of mutations preventing n-hexadecane uptake among 26 n-alkane non-utilizing mutants of Yarrowia (Saccharomycopsis) lipolytica

Summary Genetic analyses of n-alkane non-utilizing mutants of the yeast Yarrowia (Saccharomycopsis) lipolytica were continued. By analyses of inter-mutant complementation and recombination a total of 26 genetic loci have been identified. Mutations representing these loci have phenotypes characteristic of defects in substrate uptake or in one or more of the enzymatic activities making up the hydroxylase complex. Tests of ¹⁴C n-hexadecane uptake by a set of alkane-negative mutants representing the 26 loci show that 16 of the mutations cause a significant reduction in n-alkane uptake. N-alkane uptake by Y. lipolytica is shown to be inducible and to be inhibited by the metabolic poisons 2–4 dinitrophenol and KCN. The latter observation indicates that n-alkane uptake of Y. lipolytica is due to active transport.     

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     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.     

Transformation of the mycoparasite Parasitella simplex to neomycin resistance

Summary The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.     

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae

Summary We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MAT rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36°C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36°C for both mitotic recombination and sporulation.     

Isolation ofDrosophila melanogaster mutants with a wavelength-specific alteration in their phototactic response

A method is described for the isolation ofDrosophila mutants whose positive spontaneous phototactic reaction in repeated choices between UV light (368 nm) and blue light (481 nm) tended less to the UV than that of the wild type. From those 32 strains which have been isolated up till now and which fulfill this criterion, most showed only a relatively slight deviation from normal behavior. Among those exhibiting a marked change in the choice tendency was one allele of the mutantrdgB (receptor degeneration B) and seven alleles of the mutantsevenless (receptor cell 7 missing); four other mutants belong to two complementation groups which have not been identified as yet. The method described should be suitable for detecting further mutants with defects in receptor-cell function. In addition, it should be possible to isolate mutants in which the mechanisms of wavelength-specific processing are altered.