Kynurenine hydroxylase mutants of the nematode Caenorhabditis elegans

Summary The relation of intestinal autofluorescence to tryptophan catabolism in the free-living nematode Caenorhabditis elegans has been investigated. L-Kynurenine hydroxylase (EC. 1.14.13.9) activity has been detected in normal (wild-type) individuals. Mutants in the gene flu-1 which are characterized by an altered autofluorescence of the intestine cells, i.e., more intense than wild type and bluish purple instead of light blue have also been examined. They show a markedly reduced activity of kynurenine hydroxylase. The finding supports the previously proposed model for altered fluorescence based on chromatographic identification of tryptophan catabolites present.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 M, whereas the IC₅₀ value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Age-associated and tissue-specific decline in autophagic activity in the nematode C. elegans

Macroautophagy/autophagy is a cellular recycling process that is required for the extended life span observed in many longevity paradigms, including in the nematode C. elegans. However, little is known regarding the spatiotemporal changes in autophagic activity in such long-lived mutants as well as in wild-type animals during normal aging. In a recent study, we report that autophagic activity decreases with age in several major tissues of wild-type C. elegans, including the intestine, body-wall muscle, pharynx, and nerve-ring neurons. Moreover, long-lived daf-2/insulin-signaling mutants and glp-1/Notch receptor mutants display increased autophagic activity, yet with different time- and tissue-specific differences. Notably, the intestine appears to be a critical tissue in which autophagy contributes to longevity in glp-1, but not in daf-2 mutants. Our findings indicate that autophagic degradation is reduced with age, possibly with distinct kinetics in different tissues, and that long-lived mutants increase autophagy in a tissue-specific manner, resulting in increased life span.     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: Consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-1-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5 end nucleotides of the mt-s-rRNA and mt-1-rRNA genes were determined to be adjacent to the 3 end nucleotides of the tRNA^Glu and tRNA^His genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3 64% of mt-1-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3 64% of the mt-1-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-1-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-1-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived from more classical, morphological, and developmental data.Correspondence to: D.R. Wolstenholme     

C-banding and DNA content in three species of Belostoma (Heteroptera) with large differences in chromosome size and number

C-banding was carried out on Belostoma elegans (2n=26+X₁X₂Y) (), B. micantulum (2n=14+XY) () and B. oxyurum (2n=6+XY) () (Belostomatidae, Heteroptera). The C-bands always have a telomeric localization and no interstitial bands were detected. An inverse relationship between chromosome size and chromosome number exists, and besides, an inverse relationship between chromosome size and the size of the C-bands was observed. The DNA content was determined in all three species. B. elegans has a C content of 1.55±0.06 pg, B. micantulum has 0.88±0.04 pg and B. oxyurum had 0.53±0.04 pg.Considering the male meiotic characteristics, the chromosome complement and the results of C-banding and DNA content, the karyotype of B. oxyurum probably originated through autosomal fusions. The karyotype of B. micantulum and B. elegans could have originated through autosomal fusions or fragmentations respectively; with the information available up to now it is not possible to discard any of the two pathways.     

Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation

A screening method for mutants of Arabidopsis thaliana hypersensitive to -radiation has been devised. Plants grown from ethyl methanesulfonate (EMS)-treated seeds were irradiated at the seedling stage, which is highly radiosensitive due to extensive cell division. Severe growth inhibition of mutant plants by a -ray dose which only slightly affects wild-type plants was the selective criterion. Twelve true-breeding hyper-sensitive lines were isolated from a total of 3394 screened plants. Genetic analysis of five of the lines revealed five new genes, designated RAD1-RAD5. These Arabidopsis RAD mutants are phenotypically similar to mutants in the RAD52 epistasis group of Saccharomyces cerevisiae, which are highly sensitive to ionizing radiation but not hypersensitive to UV light. One possibility is that the Arabidopsis mutants are defective in a nonhomologous or illegitimate recombination mechanism used by plants for repair of chromosome breaks.     

The soluble citric acid cycle enzymes of Drosophila melanogaster. I. Genetics and ontogeny of NADP-linked isocitrate dehydrogenase

Fifty-five wild-type stocks of Drosophila melanogaster have been screened for electrophoretic variants of NADP-dependent isocitrate dehydrogenase. Three stocks are polymorphic, and one is homozygous for a fast (toward the anode at pH 8.7) electrophoretic variant. Using the variants and the mapping stock rucuca, the enzyme's structural locus has been mapped at 3–27.1±0.4. The symbols IDH-NADP and Idh-NADP are proposed for the enzyme and its genetic locus, respectively. Enzyme activity per organism increases throughout larval development, decreases during the pupal period, and increases again in anticipation of adult emergence. The activity profile is thus similar to that of a number of other Drosophila enzymes which have been studied.This project was supported by NIH training grant HD-139 and by NSF research grant GB-7803. The data have been taken from a dissertation presented to the Department of Biology of the Johns Hopkins University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Analysis of mutations affecting Ty-mediated gene expression in Saccharomyces cerevisiae

Summary Yeast translocatable, Ty, elements can cause constitutive synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when inserted upstream from the 5 end of the structural gene, ADR2. These insertion mutations, ADR3 ^c, are unstable and give rise to secondary ADHII^– mutations. The majority of such mutants, adr3, can be attributed to excision of the insertion sequence, leaving behind a single copy of the -sequence which occurs as a direct repeat at the ends of the Ty elements. A few adr3 mutants appear to be generated by DNA-rearrangements in the vicinity of the Ty insertion. The occurrence of recessive mutants, tye, which are unlinked to ADR2 indicates that the constitutive expression of ADR2 caused by the Ty insertions requires the function of trans-acting genes. These results support the idea that regulation of Ty-linked ADR2 is actively mediated by the insertion sequence and is probably not due to a mere disruption of the wild-type controlling site.     

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae

Summary A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way.The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F-factor carrying the relA ⁺ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent.The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA ^- allele, showed the wild-type level of nitrogenase activity under the same conditions.Fellow of the 6th International Training Course jointly sponsored by UNDP/UNESCO Hungarian Academy of Sciences. Present address: Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und Experimentelle Therapie Jena, Beuthenberg Str. 11, DDR-69 Jena     

Mutants of Salmonella typhimurium deficient in DNA polymerase. I. Detection by their failure to produce colicin E1

Summary A colicinogenic strain of Salmonella typhimurium was treated with nitrosoguanidine, and the survivors were tested for spontaneous production of colicin E1. Among about 10000 clones tested, two were found which appeared to have lost the ColE1 factor and had become sensitive to methyl methanesulphonate (MMS). These two isolates also proved to be more sensitive to ultraviolet (UV) irradiation and ionizing () radiation than their parent strain, and to be at least partly deficient in ability to host-cell reactivate bacteriophages damaged by UV-irradiation, -irradiation or MMS treatment. A third mutant with these properties has previously been described. Revertants of all three mutants selected on the basis of resistance to MMS were found to have regained wild-type resistance to UV, , or MMS treatment, suggesting that each of the original mutants carries a single mutation responsible for increased radiation sensitivity and reduced HCR capacity. All three mutants were of approximately normal fertility in transduction, and released temperate phages spontaneously at a significantly higher frequency than did their parent strain. Assays performed on crude extracts obtained by ultrasonic treatment established that the various mutants were deficient in an enzyme with DNA polymerase activity, and that their MMS-resistant derivates had regained almost 100% of the enzyme activity found in extracts of the wild-type parent strain. Preliminary mapping by conjugation indicated that the mutation conferring radiation sensitivity in one of the three strains lies between cysI and rha on the S. typhimurium chromosome, but attempts to determine its location more precisely by P22-mediated transduction were unsuccessful.     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Mutants of carotene production in Blakeslea trispora

The filamentous fungus Blakeslea trispora, an industrial carotene source, contains -carotene and precursors of its synthesis — phytoene, phytofluene, lycopene, and -carotene. Strain improvement through mutagenesis is difficult because all life stages are multinucleate. Mutants have been obtained following exposure of wild-type spores to N-methyl-N-nitro-N-nitrosoguanidine. Changes in the colour of the mycelia reflect variations in the accumulation of various precursors and the final product. Quantitative analysis of the mutants leads to the conclusion that the biosynthetic pathway is similar to that of the related fungus Phycomyces blakesleeanus, but the regulation is completely different. In particular, interruption of the pathway does not lead to overacummulation of precursors.     

Genetic analysis of mutants producing defective beta-galactosidase which can be activated by specific antibodie

Summary Eleven lac ^- mutants have been isolated producing -galactosidase mutant proteins, which can be activated to enzyme activity upon addition of anti -galactosidase antibodies (lac _aba ^- -mutants). The mutants have been mapped using P1 transduction and deletion mapping. Seven of them fall into one group (1), two others into another group (2). Two mutants map at sites different from the two groups (Fig. 2). Lac _aba ^- -mutant sites farthest apart correspond to a distance of about 3/4 of the z gene.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Physiological consequences of mitochondrial antibiotic-resistant mutations in Paramecium: growth-rates, cytochromic defects and cyanide-insensitive respiration of mutant and erythromycin-treated wild-type strains.

Summary A set of mitochondrial antibiotic-resistant mutants of Paramecium have been analyzed with respect to their growth-rates, cytochromic content and respiratory properties. The mutants could be arranged in a continuous series ranging from strains equivalent to wild-type to severely affected ones; affected strains display longer generation times, reduced amount of cytochrome oxidase and very high levels of cyanideinsensitive respiration. Perfect phenocopies of the mutants were obtained by treating wild-type cells with low concentrations of erythromycin suggesting that the mutations exert their pleiotrophic effect by perturbating mitochondrial protein synthesis in agreement with the idea that these mutations affect the mitochondrial ribosomes. In the mitochondria of some of the mutants, electrons can be channelled with equal efficiency into the classical cyanide-sensitive pathway and the alternate cyanide insensitive (and SHAM_sensitive) one, providing direct demonstration of the branching of these two respiratory pathways. In the absence of any added inhibitor, however, electrons tend to be channelled in the cyanide-sensitive pathway.All the physiological data fit perfectly the genetic data concerning the stability of the various mutations in mixed mitochondrial populations, i.e., markers that were known to be strongly counter-selected with respect to wild-type in such populations correspond to severely affected strains, while markers that were known to be stable correspond to healthy strains. A more quantitative analysis of the data shows that that there is little or no complementation between wild-type and mutated mitochondria in mixed cells indicating a high extent of functional autonomy of mitochondria in Paramecium.     

The target of the negative regulator of pMB1 replication overlaps with part of the repressor coding sequence

Summary Plasmid mutants (svir), insensitive to inhibition by the repressor of initiation of pMB1 replication, have been selected by exploiting their ability to support growth in the presence of repressor and inhibitor of plasmid replication. The alteration in the mechanism that controls plasmid replication causes a change in the plasmid copy number. svir mutants are dominant, as expected for mutants in the target of a repressor, but at the same time they are unable to synthesise a repressor active on the wild-type target. This lack of cross interaction between svir mutants and a co-resident wild-type plasmid results in their compatibility. These findings are explained by postulating that the target of the inhibitor of pMB1 replication coincides with part of the DNA segment that codes for the inhibitor itself. As a consequence single base pair changes in the target result in altered repressor molecules.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

Characterization of site-directed mutants in manganese-stabilizing protein (MSP) of Synechocystis sp. PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550

To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c-550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant psO strain lacking MSP as well as a double-deletion strain psbO:psbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (psbV), the two single amino acid substitution mutants (psbV:MSP-D159N and psbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O₂-evolving activity of 68–77% compared to the wild-type control using CO₂ as an electron acceptor and maximal uncoupled O₂-evolution rates of 42–57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (psbV:MSP-D159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-D159N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.