The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

A technique for targeted mutagenesis of the EF-Tu chromosomal gene by M13-mediated gene replacement

A generally applicable system for targeted mutagenesis of a chromosomal sequence is described. The Escherichia coli tufA gene was mutated using a recombinant M13mp9 phage vector carrying a tuf gene. Integration via crossing over with the chromosomal tufA target gene produced an M13 lysogen. These lysogens were screened for resistance to kirromycin. The M13 phage carrying tufA mutations were efficiently retrieved by a genetic procedure. Genetic mapping was performed with the M13 vectors. The same recombinant M13 phage was used for mutagenesis, lysogen formation, gene replacement, retrieval, mapping and sequencing of kirromycin mutants. Three different mutations yielding resistance to kirromycin were found: two of these have previously been found and characterised, while the third mutation, Gly316 Asp, is a new mutant. We also report the identification of a fourth kirromycin-resistant mutant, Gln124 Lys.     

A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene

Summary An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.     

Interaction of RecBCD enzyme with DNA damaged by gamma radiation

Summary The DNA of a gene 2 mutant (T4 2 ^–) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD ⁺ cells are therefore incapable of supporting the growth of phage T4 2 ^–. Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2 ^–-infected bacteria able to form plaques. We found that recBCD ⁺ cells can form plaques if, before infection with T4 2 ^–, they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2 ^– DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of RecBCD enzyme on T4 2 ^– DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Whole Genome Phage Display Selects for Proline-rich Boi Polypeptides against Bem1p

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second ``src homology region 3' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1_{[Asn142-Ile551]}, which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained ~7.7 × 10⁷ independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.     

Overexpression of an alkaline lipase gene from Proteus vulgaris K80 in Escherichia coli BL21/pKLE

Over-expression of Proteus vulgaris K80 lipase gene in Escherichia coli BL21 (DE3)/pKLE was achieved, with the enzyme being produced in an as active soluble form, using the T7 RNA polymerase system in a modified M9 salt or M9ZB medium. d-Lactose (55 mM) was used to induce gene expression and gave twice the lipase activity achieved with 0.4 mM IPTG. The expression of the lipase gene depended on the feeding rate of glucose being optimal at 12 g l^–1 h^–1.     

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd ⁺ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.     

Effect of DNA sequence changes on UV mutability of a purine anticodon triplet of glyU cloned on M13 phage

Summary Mutant forms of the glyU (glycyl tRNA) gene cloned in M13mp8 were subjected to uninduced targeted UV mutagenesis; i.e. phage particles were irradiated and used to infect unirradiated umuC ⁺ or irradiated umuC mutant cells. The irradiated phage carried GAG at the anticodon triplet and transitions to GAA were scored. The uninduced targeted mutation rate was reduced by altering the sequence of the gene in the vicinity of the target purine (Pu) residue. In particular a triplet of pyrimidines (PyPyPy) 5 to the target G was changed to PyPuPy in order to prevent formation of cyclcobutane and 6-4 pyrimidine dimers close to the target. On this basis we suggest a mechanism for one type of uninduced regionally targeted UV mutagenesis.     

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa andO. glaberrima) contain more than 18% amylose in endosperm starch, whilewaxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even inwaxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with aMr of 60 kd, whereaswaxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is thewaxy protein encoded by theWx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands withMr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum againstWx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous toWx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from theWx gene expressed in the endosperm.     

Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene

It is shown here that a plasmid (p29) derived from the transducing phage aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by comigration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene.Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5-to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.Non-Standard Abbreviation MOPS 4-morpholine-propane sulfonic     

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn²⁺ significantly inhibited activity. The K _m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

Analyses of the acetate-producing pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen-deprived conditions

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions. An erratum to this article can be found at     

<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V _max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD⁺ than with NADP⁺. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench

The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.Abbreviations CAD cinnamyl-alcohol dehydrogenase - HPLC high-performance liquid chromatography - m/z mass number - OMT caffeic acid O-methyltransferase