Biochemical and genetic classification of riboflavine deficient mutants of Saccharomyces cerevisiae

Summary Twenty six riboflavine deficient mutants of Saccharomyces cerevisiae were isolated. They can be divided by biochemical methods into four classes, accumulating (i) no specific product (S), (ii) 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine (AP), (iii) 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine (HP), (iv) 6,7-dimethyl-8-ribityllumazine (LU).In genetic experiments six complementation groups were found. Complementation group I is congruent with class S, group II with AP, III and IV with HP and V and VI with LU. In the crosses tested so far, no linkage between complementation groups could be detected by tetrad analysis. Intragenic complementation was found within complementation group V (mutants accumulating LU).Genetic experiments were performed at the Forstbotanisches Institut der Universität Freiburg. The senior author (O.O.) wishes to thank Prof. Dr. Dr. Hans Marquardt for generously providing the facilities of the Forstbotanisches Institut. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to O.O.     

Nature of riboflavin precursors in Pichia guilliermondi yeasts]

Thirty-nine riboflavin-deficient mutants have been isolated from three yeast strains of Pichia guilliermondii (ATSS 9058, VKM Y-1256, VKM Y-1257) and F5-121 mutant which is capable of production of large amounts of riboflavin in the presence of iron in the medium. All mutants were divided into five groups according to the nature of precursors accumulated in the medium and growth reaction in media with 6,7-dimethyl-8-ribityllumasine and diacetyl. The mutants of the first group did not accumulate specific precursors of riboflavin either in the cells or in the medium. The mutants of the second, third and fourth groups accumulated, after the incubation with diacetyl, 2-amino-4-hydroxy-6,7-dimethylpteridine, 2-amino-4-hydroxy-6,7-dimethyl-8-ribitylpteridine and 6,7-dimethyl-8-ribityllumasine; therefore, they synthesized the following precursors of riboflavin: 2,4,5-triamino-6-hydroxy-pyrimidine, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine. The mutants of the fifth group accumulated 6,7-dimethyl-8-ribityllumasine in the medium and lacked riboflavin synthetase activity, as was confirmed by enzymatic studies.     

Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii

The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively. It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein). No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant. Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene).     

Mechanism of retroinhibition in e regulation of flavinogenesis in yeasts of genus Pichia]

The kinetics of the synthesis of a riboflavin (RF) precursor, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine (DHARAP), was studied using the washed cells of RF-deficient mutants of Pichia guilliermondii R7G and Pichia ohmeri R32 with blocked lumasine synthetase. RF inhibited the synthesis of DHARAP while cycloheximide in the absence of RF had no effect on this process. The data suggest that flavins regulate the biosynthesis of RF in P. guillier mondii and P. ohmeri b7 means of feed-back inhibition mechanism.     

Origin of the ribityl side-chain of riboflavin from the ribose moiety of guanosine triphosphate in Pichia guilliermondii yeast.

In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.     

Operon of riboflavin biosynthesis in Bacillus subtilis. XVII. A study of the regulatory functions of the intermediate products and their derivatives

Repression of synthesis of GTP-cyclohydrolase and riboflavinsynthetase was studied in different regulatory mutants of Bacillus subtilis. The results of experiments with some riboflavin precursors and their derivatives revealed that 5-amino-2,6-dioxo-4-ribitylaminopyrimidine and 6-methyl-7-(1',2'-dioxyethyl)-8-ribityllumazine can serve as effectors in riboflavin biosynthesis.     

Genetics of somatic mammalian cells. XIV. Genetic analysis in vitro of auxotrophic mutants

Genetic analysis has been carried out on auxotrophic mutants produced by treatment of Chinese hamster ovary and the Chinese hamster lung cells with mutagenic agents in vitro. Thirty-six different mutants were subjected to complementation analysis and biochemical tests. The different mutations studied result in growth requirements for proline; glycine; glycine or folinic acid; adenine or several of its precursors; inositol; adenine plus thymidine; and glycine plus adenine plus thymidine. The mutants which require glycine fall into four different complementation classes while those requiring adenine or hypoxanthine form two different complementation classes. The biochemical blocks of the latter two classes both occur somewhere in the steps involved in conversion of 5-phosphoribosyl-1-pyrophosphate to 5-aminoimidazole 4-carboxylic acid ribonucleotide. The auxotrophic mutants described exhibit all-or-none responses to their specific nutrilite supplements and are stable with respect to reversion. They involve alterations in ten different genes, and hence form a useful set of mutants for a variety of genetic studies. All the auxotrophies produced in a single exposure to a mutagen are due to single gene mutations, even when multiple nutritional requirements were produced. All the mutations studied are recessive.     

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units.     

Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracil, 5-Azacytidine, and Ethyl Methane Sulfonate

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.     

Menaquinone biosynthesis in Bacillus subtilis: isolation of men mutants and evidence for clustering of men genes

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis were selected by simultaneous resistance to two aminoglycoside antibiotics. These men mutants fell into two groups: group I, in which the nutritional requirement was satisfied either by o-succinylbenzoic acid or by 1,4-dihydroxy-2-naphthoic acid; and group II, comprising those capable of growing only when supplemented with 1,4-dihydroxy-2-naphthoic acid. The latter group could be further subdivided into two classes on the basis of syntrophy experiments, fine-structure genetic mapping, and in vitro complementation by cell-free extracts (Meganathan et al., J. Bacteriol., 145:328-332, 1981). These subclasses of group II defined the menB and menE genes, whereas group I appeared to comprise mutations in the menC and menD genes. All of the men mutations tested, whether occurring in menB, menE, or menC,D, could be placed by genetic mapping with bacteriophage PBS1 between bioB and ald on the B. subtilis genome.     

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.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.     

The nature of the pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase participating in riboflavin biosynthesis in yeasts

2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine-5'-phosphate are studied for their effect on the activity of 6,7-dimethyl-8-ribityllumazine synthase of Pichia guilliermondii yeasts. It is shown that when nonphosphorylated form of pyrimidine and ribose-5-phosphate (donor C-4--a fragment) is used as a substrate, the specific activity of 6,7-dimethyl-8-ribityllumazine synthase is high and Be2+ and F- ions, inhibitors of alkaline phosphatases, do not inhibit it. The value of Km for this pyrimidine is 1.1 X 10(-5) M. Phosphorylated pyrimidine being used as a substrate in the presence of Be2+ and F-, the reaction practically does not proceed. Therefore, only 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine is a pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase of yeast.     

Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants.

Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%).     

Isolation, complementation and partial characterization of mutants of the methanol autotroph Xanthobacter H4-14 defective in methanol dissimilation

Seven mutants of Xanthobacter H4-14, unable to grow on methanol but capable of growth on formate, were isolated and complemented with a chromosomal clone bank constructed in the broad-host-range cosmid pVK100. One mutant could not be complemented but the others fell into four distinct complementation groups that involved three different recombinant clones. All of the complementing regions were separated by at least 10 kbp. The five complementation classes had different phenotypic characteristics and were defective in different aspects of methanol and formaldehyde oxidation. Class I mutants were defective in methanol oxidation, class II mutants were impaired in formaldehyde oxidation, class III mutants appeared to be defective in a regulatory element involving the methanol oxidation system, and class IV mutants appeared to be defective in a regulatory element involving formaldehyde oxidation. Class V mutants exhibited a methanol-sensitive phenotype, which was correlated with an imbalance between methanol and formaldehyde dehydrogenase activities. Analysis of this class suggested it was defective in a repressor that regulated methanol dissimilation functions.     

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.     

Genetics of aggregation pattern mutations in the cellular slime mould Dictyostelium discoideum.

A class of aggregation pattern mutants called 'streamers' have been isolated from Dictyostelium discoideum and analysed genetically. The streamer phenotype is the formation of very large streams of centripetally moving amoebae which are collected from abnormally large territories during the aggregation phase of this organism. Such mutants do not show the pleiotropic developmental defects seen with most other classes of aggregation mutants and after the abnormal aggregation phase they develop into normally differentiated stalk cells and spores. Twenty-four haploid streamers were isolated and assigned to seven complementation groups, stmA to stmG, after selecting diploids formed between pairs of the mutants. The complementation loci were assigned to the following linkage groups using parasexual genetic techniques: stmA and stmF, linkage group VII; stmB, stmD and stmG, linkage group II; stmC and stmE, linkage group III. Use was made of a new temperature sensitive for growth marker, tsgK21, which was assigned to linkage group VII. The total number of complementation groups giving the streamer phenotype is estimated from statistical calculation, based on the frequency of allelism, to be between seven and nine.     

Mutants of S. CEREVISIAE Defective in the Maintenance of Minichromosomes

We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.