Genetic Map Position of the pdxH Gene in Escherichia coli

The gene for pyridoxine phosphate oxidase, pdxH, is located 1.2 min beyond aroD, proximal to trp.     

植物6-磷酸山梨醇脱氢酶

6-磷酸山梨醇脱氢酶是植物合成山梨醇的关键酶.文章从酶学特性、山梨醇转化、碳水化合物代谢等方面对6-磷酸山梨醇脱氢酶的研究进展以及此酶的某些应用研究情况作介绍.     

Gene Order and Co-Transduction in the leu-ara-fol-pyrA Region of the Salmonella typhimurium Linkage Map

The gene order and orientation in the leu-pyrA region of the Salmonella typhimurium linkage map was established by phage P22-mediated transductions. The gene order, in counterclockwise orientation, is leuO-leuA-leuB-leuC-leuD-ara-fol-pyrA. The fol locus is co-transducible with either the ara and leu loci or the pyrA locus, whereas no co-transduction for the ara and pyrA loci can be found.     

Atypical Genetic Locus Associated with the zwf Gene Encoding the Glucose 6-Phosphate Dehydrogenase from Enterococcus mundtii CRL35

The zwf gene encoding glucose 6-phosphate dehydrogenase (G6PD, EC.1.1.1.49) from Enterococcus mundtii CRL35 was cloned as a 4921 bp EcoRI fragment and analyzed. The predicted zwf gene product consists of 506 residues with a molecular mass of 58.4 kDa, and is fully active in Escherichia coli as demonstrated by its heterologous expression in the zwf-negative mutant E. coli Su294. It shows a high degree of sequence identity (40–60%) to G6PDs described in other bacteria. Upstream of the zwf gene, a homolog of the DtxR family was identified (ORF D). Analysis of the 5′ sequence of ORF D revealed a potential promoter sequence, which would suggest the presence of an operon-like structure between ORF D and the zwf gene. Finally, it was found that Fe²⁺ levels have an important role as a modulator of G6PD activity. This is the first report of this type of regulation of G6PD activity. A possible involvement in oxidative stress is discussed.     

Glucose-6-Phosphate Dehydrogenase/6-Phosphogluconate Dehydrogenase Ratio and the Glucose-6-Phosphate, 6-Phosphogluconate and Fructose-6-Phosphate Contents in Tobacco Plants Infected with Potato Virus Y

The ratio of activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6P DH/6PG DH), and the contents of glucose-6-phosphate (G6P), 6-phosphogluconate (6PG) and fructose-6-phosphate (F6P) were studied at various stages of potato virus Y (PVY) multiplication in Nicotiana tabacum cv. Samsun. G6P DH/6PG DH increased through the experiment from 0.42 to 0.53 in leaves of healthy tobacco, and up to 0.59 in PVY systemically infected leaves. However, these ratios in the ruptured protoplast preparations, and the chloroplast and cytosol fractions of healthy protoplasts were similar to that from infected ones. The ratio lower than 1, found in the healthy and/or PVY- infected leaf tissues and in the infected protoplasts as well, confirms the assumption that G6P DH is the control enzyme of oxidative pentosephosphate pathway not only in the healthy but also in the infected plants. The contents of G6P, 6PG and F6P in the period of the highest PVY multiplication were strongly decreased (to 30 – 50 % when compared with control healthy leaves) and were negatively correlated with the G6P DH and 6PG DH activities.     

Genetic Locus of a Gene Affecting Leucine Transport in Salmonella typhimurium

By using a series of deletion mutations in the region of the tryptophan operon, it has been shown that a gene governing the transport of leucine maps on the side of the chr locus distal to the trp operon.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

Evidence for a Methionine-controlled Homoserine Dehydrogenase in Salmonella typhimurium

Evidence is presented for the existence of a second homoserine dehydrogenase in Salmonella typhimurium. The formation, but not the activity, of this enzyme is controlled by methionine. Two distinct homoserine dehydrogenases were separated from wild-type cells by diethylaminoethyl (cellulose) column chromatography. Sucrose gradient ultracentrifugation gave molecular weight estimates for the threonine-regulated enzyme (HSD I) of 220,000 to 240,000 and for the methionine controlled enzyme (HSD II) of 130,000 to 140,000. Approximately 12% of the total HSD activity in wild-type cells was accounted for by HSD II. A threonine-requiring strain of S. typhimurium was found to lack HSD I but not HSD II. Under certain conditions, this mutant grew rapidly in minimal medium. Rapid growth in minimal medium was correlated with the appearance of an enzyme with similar characteristics to HSD I. The possible origins of this HSD I-like enzyme are presented.     

Histidinol Dehydrogenase (hisD) Mutants of Salmonella typhimurium

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F' factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F' factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide.     

Genetic Regulation of SN-Glycerol-3-Phosphate Dehydrogenase in Brown Adipose Tissue

The levels of sn-glycerol-3-phosphate dehydrogenase (GPDH) were determined in the brown adipose tissue (BAT) of different inbred strains of mice. The BAT of the BALB/cJ strain contains twice as much enzyme activity per milligram protein as do other strains. The appearance of this difference is developmentally dependent, since it is not detected in BAT until 25-30 days postpartum. Genetic analysis of this strain difference has shown that the mechanism of inheritance involves at least two genes, one of which is linked to the Gdc-1 structural locus on chromosome 15. Determinations of GPDH synthesis by immunoprecipitation of GPDH protein labeled in vivo with [3H]leucine, and of GPDH mRNA by Northern blot analysis, establish that in BALB/cJ mice higher rates of enzyme synthesis are determined by elevated levels of GPDH mRNA. It was also found that cold stress increases GPDH mRNA levels in all the strains examined.     

人G6PD T279A和T279S两种突变子的体外构建

目的:构建葡萄糖6磷酸脱氢酶(Glucose 6-phosphate dehydrogenase,G6PD)T279A和T279S两种突变子.方法:以Genbank No X03674为参考序列设计并合成引物、以含G6PD基因的质粒(Philip JMason博士惠赠)为模板,PCR扩增获得G6PD野生型基因片段,琼脂糖凝胶电泳后回收PCR产物,连接、转化构建克隆质粒pMD18T-G6PD;酶切pMD18T-G6PD质粒、电泳后回收目的基因片段,连接、转化构建含G6PD野生型基因的重组质粒pAL-G6PD;设计并合成含有突变序列的引物,以pAL-G6PD为模板,体外扩增获得G6PD835-海口(835A→G,T279A)和835-中国-1(835 A→T,T279S)突变子.结果:酶切后经电泳鉴定表明获得与预期大小相符的pMD18T-G6PD质粒,EcoRI和Hind Ⅲ双酶切获得与预期大小相符的pAL-G6PD,测序结果与参考序列完全一致.0.8%的琼脂糖凝胶电泳鉴定,并定量pAL-G6PD单链DNA浓度约为200ng/uL.经测序鉴定并与参考序列比对结果表明获得了G6PD的T279A和T279S两种突变子.结论:成功构建了G6PD的T279A和T279S两种突变子,为下一步原核表达、生化性质以及酶动力学等研究奠定了基础.     

New Methionine Structural Gene in Salmonella typhimurium

Eight metH mutants in Salmonella typhimurium with closely linked sites of mutation which could grow only on methionine were isolated from a metE mutant deficient in N(5)-methyltetrahydropteroyltriglutamate-homocysteine transmethylase; their deficiency in cobalamin-dependent N(5)-methyltetrahydrofolate-homocysteine transmethylase was supported by the results of enzyme studies of one of them. Cotransduction of metH and metA (homoserine O-transsuccinylase) mutants was obtained, thus revealing linkage between a second pair of the six known methionine structural genes. One metH mutant clearly differed from the rest in that it reverted at a higher frequency, was temperature sensitive, complemented all other metH mutants, and was located farthest from the metA gene.     

Regulation of the Expression of the sn-Glycerol-3-Phosphate Dehydrogenase Gene in Drosophila melanogaster

P element-mediated transformation has been usedto investigate the regulation of expression of thesn-glycerol-3-phosphate dehydrogenase gene ofDrosophila melanogaster. A 13-kb constructcontaining the eight exons and associated introns, 5 kb of the5′ region, and 3 kb downstream from the structuralgene produced normal levels of enzyme activity andrescued the poor viability of flies lacking the enzyme. All the regulatory elements essential fornormal enzyme expression were located in a fragment thatincluded the exons and introns and 1-kb upstreamnoncoding sequence. Deletions of the 1.6-kb secondintron reduced activity to 25%. Transformants withfusion constructs between the sn-glycerol-3-phosphatedehydrogenase gene and the beta-galactosidase gene fromE. coli revealed three elements that affectedexpression. A (CT)₉ repeat element at the5′ end of the second intron increased expressionin both larvae and adults, particularly at emergence. Asecond regulatory element, which includes a(CT)₇ repeat, was located 5′ to the TATA box and had similareffects on the gene's expression. A third, undefined,enhancer was located in the second intron, between 0.5and 1.8 kb downstream of the translation initiationcodon. This element increases enzyme activity to asimilar extent in larvae and adults but has littleeffect when the enhancer at the 5′ end of theintron is present.     

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf).     

Postnatal Expression of Glucose-6-Phosphate Dehydrogenase in Different Brain Areas

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differred from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.     

Characterization of Enterobacteria by Starch-Gel Electrophoresis of Glucose-6-Phosphate Dehydrogenase and Phosphogluconate Dehydrogenase

Specific activities and electrophoretic mobilities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase were determined in 38 isolates of the family Enterobacteriaceae and in 10 isolates of the related Pasteurella. The deficiency of glucose-6-phosphate dehydrogenase in P. pestis was verified. Enzymes obtained from different strains of the same species exhibited an unexpected degree of heterogeneity. For example, 8 and 11 apparent variants of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase, respectively, were found in 14 strains of Escherichia coli. Although similar frequencies of heterogeneity were noted in 7 strains of P. pseudotuberculosis, 5 species of Shigella, and 8 species of Salmonella, differences in mobility were generally small in comparison with those observed between strains of E. coli. Values obtained for the pasteurellae, shigellae, and salmonellae, thus fell within narrow ranges that may prove typical for the genera. However, most of these ranges, as well as many values observed for single species of other genera, were overlapped by the wide range recorded for E. coli. The significance of this observation was discussed with respect to the relative age and taxonomic position of the organisms in question. The method could be used to distinguish between most wild-type strains of the same species and should thus facilitate investigations of genetic transfer and epidemiology.