The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine.

Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.     

Isolation and characterization of Saccharomyces cerevisiae glycolytic pathway mutants.

Yeast strains carrying recessive mutations representing four different loci that cause defects in pyruvate kinase, pyruvate decarboxylase, 3-phosphoglycerate kinase, and 3-phosphoglycerate mutase were isolated and partially characterized. Cells carrying these mutations were unable to use glucose as a carbon source as measured in turbidimetric growth experiments. Tetrad analysis indicated that these mutations were not linked to each other; one of the mutations, that affecting phosphoglycerate kinase, was located on chromosome III.     

Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae were isolated which have decreased ability to hydrolyze leucine beta-naphthylamide, a chromogenic substrate for amino-peptidases. The mutations were shown by starch gel electrophoresis to affect one of four different aminopeptidases. Mutations affecting a given enzyme belong to a single complementation group. The four genes were symbolized lap1, lap2, lap3, and lap4, and the corresponding enzymes LAPI, LAPII, LAPIII, and LAPIV. Both lap1 and lap4 were mapped to the left arm of chromosome XI, and lap3 was mapped to the left arm of chromosome XIV. Strains which possessed only one of the four leucine aminopeptidases (LAPs) were constructed. Crude extracts from these strains were used to study the properties of the individual enzymes. Dialysis against EDTA greatly reduced the activity of all the LAPs except for LAPIII. Of the cations tested, Co2+ was the most effective in restoring activity. LAPIV was the only LAP reactivated by Zn2+. LAPI was purified 331-fold and LAPII was purified 126-fold from cell homogenates. Both of the purified enzymes had strong activity on dipeptides and tripeptides. The activity levels of the LAPs are strongly dependent on growth stage in batch culture, with the highest levels in early-stationary phase. Strains lacking all four LAPs have slightly lower growth rates than wild-type strains. The ability of leucine auxotrophs to grow on dipeptides and tripeptides containing leucine is not impaired in strains lacking all four LAPs.     

Isolation and characterization of temperature-sensitive mak mutants of Saccharomyces cerevisiae.

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton linear double-stranded ribonucleic acid molecule. Using simplified screening and complementation procedures, we have isolated mutants in three chromosomal genes that are temperature sensitive for killer plasmid maintenance or replication. One of these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during spore germination and outgrowth at nonpermissive temperatures, but during vegetative growth, they only lowered the plasmid copy number. These two mutants did not lose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killer plasmids in laboratory yeast strains.     

Isolation and characterization of Metolachlor-resistant mutants of Saccharomyces cerevisiae

The herbicide Metolachlor (-chloroacetamide group) inhibits the growth of Saccharomyces cerevisiae on complete, minimal, and non-fermentative media. Spontaneous and induced resistant mutants showed monogenic segregation patterns. Among the resistant clones, 70% were recessives, 16.4% were partially dominants and 13.4% were dominants. The spontaneous partially dominant mutation Mtc1 was mapped on linkage group XV at 33.3cM from ade2 and 31.7cM from his3, in a region that is characterized by the presence of several resistant genes. The recessive mutation mtc2 was located on chromosome IV. Although all the mutants had the ability to grow in the presence of the herbicide, they remained affected in their respiration efficiency, indicating two different mechanism of action of Metholachor on yeast cells.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.     

Escherichia coli mutants completely deficient in adenosylmethionine decarboxylase and in spermidine biosynthesis.

Mutants of Escherichia coli deficient in adenosylmethionine decarboxylase, an enzyme in the biosynthetic pathway for spermidine, were isolated after mutagenesis of E. coli K 12 with N-methyl-N-nitro-N-nitrosoguanidine or with the bacteriophage Mu. The mutated gene, designated speD, is at 2.7 min on the E. coli chromosome map. In several of the mutants resulting from Mu insertion both adenosylmethionine decarboxylase activity and spermidine were undetectable. The absence of spermidine from speD strains proves the essential role of adenosylmethionine decarboxylase in the biosynthetic pathway for spermidine. Despite the complete absence of spermidine, these mutants grew at 75% of the wild type rate.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae

The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.     

Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.

S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Catalase T deficient mutants of Saccharomyces cerevisiae

A method for the isolation of catalase T deficient mutants of Saccharomyces cerevisiae is described. Ten mutants lacking catalase T and belonging to 5 complementation groups were isolated. CTT1 locus was identified as the structural gene for catalase T. It is under the control of CTT2, CTT3 and CTT4 loci.     

Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.     

S-adenosylmethionine decarboxylase and spermidine synthase from chinese cabbage

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virus-infected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO₂ and of decarboxylated S-adenosylmethionine (dSAM). The K_m for SAM is 38 micromolar. The reaction is not stimulated by Mg⁺⁺ or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5.1.16), present in leaf or protoplast extracts in many fold excess over SAM decarboxylase, has been purified approximately 1900-fold in steps involving ammonium sulfate precipitation, affinity chromatography, and gel filtration on Sephacryl S-300. Standardization of the Sephacryl column by proteins of known molecular weight yielded values of 35,000 and 81,000 for the decarboxylase and synthase, respectively.     

Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae

Thirty Ca2+-sensitive (cls: calcium sensitive) mutants of Saccharomyces cerevisiae were isolated by replica-plating. These mutants, which each had a single recessive chromosomal mutation, were divided into 18 complementation groups. Some cls mutants showed a phenotype of specific sensitivity to Ca2+, while others showed phenotypes of sensitivities to several divalent cations. From measurements of the calcium contents and initial rates of Ca2+ uptake of the cls mutants, 16 of the 18 cls complementation groups were classified into four types: type I mutants (cls5, cls6, cls13, cls14, cls15, cls16, cls17, and cls18) had both elevated calcium contents and increased uptake activities. A type II mutant (cls4) had a normal calcium content and normal uptake activity; type III mutants (cls1, cls2 and cls3) had elevated calcium contents but normal initial rates of Ca2+ uptake; type IV mutants (cls8, cls9, cls10 and cls11) had normal calcium contents but increased initial rates of Ca2+ uptake. Two of the mutants (cls7 and cls12) had intermediate biochemical properties. The primary defects of these four types of cls mutants were considered in terms of the Ca2+ transport system(s). Both type I and type III mutants, which had elevated calcium contents, simultaneously showed a trifluoperazine-sensitive phenotype, suggesting a close correlation of this phenotype with elevated calcium content. In addition, all type IV mutants were unable to utilize nonfermentable sugars. One CLS gene, CLS7, was located on the left arm of chromosome V.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

Summary The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit ^- mutants. These mutants were isolated in an op1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1—cytochrome c oxidase subunit 2, OXI2 — subunit 3, OXI3 — subunit 1, and COB — cytochrome b.The ability of diploid mit ^- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit ⁺ tetrads, both mit ⁺ and mit ^- tetrads, or only mit ^- tetrads. Mutants forming mit ^- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit ^- mutants have retained the ability to perform meiosis and sporulation.