The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.     

Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi

The colchicine-binding activity of a high speed supernatant from fourteen colchicine- and/or vinblastine-resislant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activity produce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type.     

Formate hydrogenlyase system in Salmonella typhimurium LT2.

Isolation from Salmonella typhimurium of mutants unable to reduce benzyl viologen under anaerobic conditions has allowed the study of the factors involved in the multienzymic formate hydrogenylase system. 1. Depending on the affected activities, different classes of mutants were found: FHL-A mutants have lost formate dehydrogenase 1 and formate dehydrogenase 2 activities; mutations in fdhA (117 min) or fdhB (33 min) lead to such a phenotype. FHL-B and FHL-C mutants have lost formate dehydrogenase 2 activity and part or all of hydrogenase activity, respectively; both types correspond to mutations in the hyd gene (approximately 90 min). FHL-D mutants have lost only formate dehydrogenase 2 activity; fhlD gene maps at 120 min. 2. In some cases, mixtures of extracts from two mutants display formate dehydrogenase 2 and formate hydrogenylase activities. Restoration studies suggest the existence of one factor sensitive to growth conditions and inactivated by oxygen or heating. This factor which is present and active in FHL-C mutants, is probably the one missing in FHL-D mutants. 3. A new scheme for the formate hydrogenylase system is proposed, in which hydrogenase transfers electrons directly to benzyl viologen.     

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.     

Photomorphogenic mutants of tomato

Photomorphogenesis of tomato (Lycopersicon esculentum Mill.) is being studied with the aid of mutants which are modified either in their photoreceptor composition or in their signal transduction chain(s). Phytochrome chromophore mutants, presumably deficient in all phytochromes, and mutants specifically deficient in phytochrome A (phy A) or B1 (phyB1) have been used to study the roles played by phytochromes in photomorphogenesis. In addition, other mutants, including transgenic lines overproducing phyA, exhibit exaggerated photomorphogenesis. Studies using these mutants are reviewed, with emphasis being placed on anthocyanin biosynthesis and plastid development as model systems for the dissection of the complex interactions between photoreceptors and to elucidate the nature of photoreceptor transduction chains. Recently, new mutants have been isolated by screening in a phyA, phyB1-deficient background. The novel phenotypes selected are candidates for mutants in additional photoreceptors or their transduction chains.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.     

Independent action of ELF3 and phyB to control hypocotyl elongation and flowering time

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those of phyB mutants, we isolated two new elf3 mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants, elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA or hy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.     

Genetic and enzymic studies on the recombination process in Bacillus subtilis

We have isolated recombination deficient mutants of Bacillus subtilis on the basis of their sensitivity to methyl-methane-sulfonate or ultraviolet light, or of their inability to be transformed on solid medium. We have analyzed the mutants for several recombination and repair properties; we have grouped them in 5 classes on the basis of their phenotype and tested them for the activity of several enzymes acting on DNA, ie. DNA polymerase, polynucleotide ligase, ATP dependent DNase, and a DNase acting on single-stranded DNA. One mutant was found reduced in the latter DNase. Some of the mutants have been mapped, and they correspond to three different genes denominated rec D, rec F and rec G. All the recombination deficient mutants of B. subtilis described in the literature have been grouped in 7 classes; the mutations belong to 13 (and possibly 15) different genes distributed along the map. A coherent nomenclature and the criteria for a standard study of the rec mutants are proposed.     

Isolation of pleiotropic yeast mutants requiring ergosterol for growth

Mutant strains of Saccharomyces cerevisiae which require ergosterol for growth have been isolated. These mutants are all petite and require a fatty acid. Several mutants require methionine in addition. These mutants have been classified into 6 complementation groups. For one of the mutants the enzymatic block has been localized after lanosterol. These mutants do not show a stringent requirement for ergosterol, as sitosterol, stigmasterol or cholesterol also support growth. Mutants of this type will be of value not only in studies of sterol biosynthesis, but also in assessing the biological role of sterols in the cytoplasmic yeast membrane. Similar mutants but without a stringent requirement for a sterol have been previously isolated by Resnick and Mortimer (8).     

Growth studies of three phi chi 174 mutants in tsDNA mutants Escherichia coli.

Three mutants of phi chi 174 were examined for their abilities to grow in temperature-sensitive dna,A, dnaC, dnaE, or dnaG mutants of Escherichia coli. The results indicate that the phage mutants have acquired the ability to grow in some tsDNA mutants that normally block the replication of wild-type phi chi 174. Evidence is presented indicating that the phage mutants contain one or more altered structural proteins. Several models are presented to explain how altered phage structural proteins could affect phi chi 174 replication.     

Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development.

The ascomycete Aspergillus nidulans produces green conidia (asexual spores). Recessive mutants which produce yellow conidia have been previously isolated from haploid strains and have been shown to be deficient in laccase (diphenol oxidase), an enzyme that requires copper for activity. Using a diploid parent strain, we isolated dominant yellow conidial mutants which, in the haploid state, produced even less laccase activity than a recessive mutant. Three isolates of such mutants behaved similarly and define a single complementation group (yB) on chromosome VIII distinct from the yA locus on chromosome I defined by recessive mutants. Unlike yA mutants, whose only discernable phenotype is their conidial color, yB mutants are pleiotropic: conidial germination was delayed relative to the wild type, and sexual development was blocked at an early stage. The three phenotypes of yB mutants were expressed on yeast extract-glucose medium containing 1.6 microM of added copper. When copper was added to above 5 microM, all three phenotypes were remediated, and near wild-type levels of laccase were produced. We conclude that yB mutants have a reduced availability of copper. The dominance of yB mutants could result, for example, from an alteration in transport or storage of copper. Using an immunological assay, we detected no laccase antigenic cross-reacting material in yB mutants grown on medium of low copper content. We conclude that either the synthesis or the stability of laccase is copper dependent.     

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.     

Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.     

alpha-Amanithin-resistant mutants of Chinese hamster ovary (CHO) cells

Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

8-Azaguanine Resistance in Mammalian Cells I. Hypoxanthine-Guanine Phosphoribosyltransferase

Chinese hamster cells were treated with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, and mutants resistant to 8-azaguanine were selected and characterized. Hypoxanthine-guanine phosphoribosyltransferase activity of sixteen mutants is extremely negative, making them suitable for reversion to HGPRTase(+). Ten of the extremely negative mutants revert at a frequency higher than 10(-7) suggesting their point mutational character. The remaining mutants have demonstrable HGPRTase activity and are not useful for reversion analysis. Five of these mutants have < 2% HGPRTase and are presumably also HGPRTase point mutants. The remaining 14 mutants utilize exogenous hypoxanthine for nucleic acid synthesis poorly, and possess 20-150% of wild-type HGPRTase activity in in vitro. Their mechanism of 8-azaguanine resistance is not yet defined.     

Mutations in Francisella tularensis, decreasing the virulence of these bacteria and leading to a change in the immune response upon infection of experimental animals with them]

The research was aimed at isolation of Francisella tularensis mutants possessing the decreased virulence for experimental animals and mediating the changes in the animal immune response. A number of spontaneous and induced mutants of the American and European subtypes of Francisella tularensis were selected for antibiotics resistance or detergent sensitivity. All the obtained mutants have the decreased virulence and differ in their ability to induce the protective antitularemia immunity or ability to induce the humoral immune response in the laboratory animals. The dimeric immunoprecipitation in gel as well as immunoblotting have shown the mutations decreasing the virulence to cause the loss by bacteria of a number of antigenic structures (in case the virulence is completely lost) or changes in antigenic structure resulting in inability of bacteria to induce the humoral immune response when immunizing the laboratory animals. The latter occurs in partially virulent mutants of the vaccine mutant type. The concomitant changes in virulence, ability to cause protective immunity or humoral immune response of the mutants is discussed.     

Isolation and characterization of mutants at the APRT locus in the L-5178Y TK+/TK- mouse lymphoma cell line

2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.