Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.     

Dephosphorylation of rabbit skeletal muscle glycogen synthase by phosphoprotein phosphatase and human placental alkaline phosphatase

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase results in the incorporation of ³²P into two major tryptic peptides (P-1 and P-2) which are identified by isoelectric focusing on polyacrylamide gel. When ³²P-labeled synthase is incubated with rabbit muscle phosphoprotein phosphatase both P-1 and P-2 are hydrolyzed. Incubation of ³²P-labeled synthase with human placental alkaline phosphatase results in a specific hydrolysis of P-1. Measurement of the increase in synthase activity ratio accompanied by the dephosphorylation of P-1 with human placental alkaline phosphatase and, subsequently, of P-2 with phosphoprotein phosphatase shows that both P-1 and P-2 affect the glucose-6-P dependency of the synthase.     

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.     

Cloned truncated recA genes in E. coli

Summary Plasmids pMH1 and pDR1461, possessing the control region and 22% or 73% of the E. coli recA gene, conferred UV sensitivity to wild-type uvrA, and umuC bacteria. Sensitization was less in recA441 (tif-1) mutants and absent in lexA cells. Radiosensitization correlated with inhibition of recombinational repair, even through induced recA protein synthesis and recombination in Hfr matings were normal. Plasmids pMH1 and pDR1461 also prevented induction of some, but not all, SOS functions. Mutagenic reversion to tryptophan prototrophy and induced reactivation of UV-irradiated phage were eliminated, and the efficiency of lysogenic induction reduced. However, naladixic acid induced filamentous growth, mitomycin-C induced uvrA gene expression and post UV-irradiation DNA degradation control were little changed. Explanations of these effects are discussed which involve the presence of either truncated recA protein or multiple copies of the recA gene control sequence.A preliminary account of this work is presented in Chromosome Damage and Repair, edited by E. Seeberg and K. Klepper, to be published by Plenum Press     

Replication of E. coli duplex DNA in vitro

Summary An E. coli lysate after being gently washed to remove soluble components, supports replicative DNA synthesis, if soluble proteins and the deoxyribonucleotide triphosphates are added. This DNA synthesis is dependent on ATP and on the presence of the gene products of the dnaB, dnaG, and polC (DNA polymerase III) genes. It continues at the replication forks preformed in vivo and Okazaki fragments are intermediate products of the reaction.Two different methods were used to prepare the washed DNA containing fraction. The one method involves washing of a cell lysate situated on a dialysis membrane. The other method involves DNAase treatment of a lysate and sedimentation of the degraded DNA through a glycerol gradient. Both washed preparations contain not only the DNA and the replication forks but also functional amounts of DNA polymerase III and of the dnaB gene product. Other factors, that are essential for replicative DNA synthesis, including the dnaG gene product, are washed out of the DNA containing preparations and the system is reconstituted by readdition of the soluble proteins.     

大肠杆菌的蛋白分泌机制

大肠杆菌的分泌蛋白定位于内膜、外膜、周质空间和胞外环境,它们在N端或C端带有一定的结构包含着分泌信号,这两类分泌蛋白在各自特定的一组蛋白因子的协助下跨越内膜,再通过目前尚不清楚的方式实现其最终定位.N端带有信号肽的分子在跨越内膜时得到Sec家族蛋白因子协助,信号肽在跨膜过程中可能被切除,该过程由ATP和电化学势提供能量.C端带分泌信号的分子主要受到Hly家族分子协助,一次穿过内膜和外膜而不经过周质空间.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

基于卷积神经网络的大肠杆菌启动子预测

目的 基于位点特异性打分矩阵（position-specific scoring matrices,PSSM）的预测模型已经取得了良好的效果,基于PSSM的各种优化方法也在不断发展,但准确率相对较低,为了进一步提高预测准确率,本文基于卷积神经网络（convolutional neural networks,CNN）算法做了进一步研究。方法 采用PSSM将启动子序列处理成数值矩阵,通过CNN算法进行分类。大肠杆菌K-12（Escherichia coli K-12,E.coli K-12,下文简称大肠杆菌）的Sigma38、Sigma54和Sigma70 3种启动子序列被作为正集,编码（Coding）区和非编码（Non-coding）区的序列为负集。结果 在预测大肠杆菌启动子的二分类中,准确率达到99%,启动子预测的成功率接近100%;在对Sigma38、Sigma54、Sigma70 3种启动子的三分类中,预测准确率为98%,并且针对每一种序列的预测准确率均可以达到98%以上。最后,本文以Sigma38、Sigma54、Sigma70 3种启动子分别和Coding区或者Non-coding区序列做四分类,预测得到的准确性为0.98,对3种Sigma启动子均衡样本的十交叉检验预测精度均可以达到0.95以上,海明距离为0.016,Kappa系数为0.97。结论 相较于支持向量机（support vector machine,SVM）等其他分类算法,CNN分类算法更具优势,并且基于CNN的分类优势,编码方式亦可以得到简化。     

On the nature of sbcA mutations in E. coli K12

Summary We have recently shown (Kaiser and Murray 1979) that many E. coli K12 strains carry a defective prophage (Rac) located a few minutes clockwise of the trp operon on the genetic map. The Rac genome contains recE, the determinant for the ATP-independent exonuclease, ExoVIII. E. coli K12 strains which carry sbcA mutations express recE constitutively. This paper describes an investigation of several such strains. We show that the SbcA phenotype may arise from more than one type of mutational change. The most readily explained SbcA phenotype is that of sbcA8 strains in which a large section of the Rac genome (including one hybrid attachment site and probably the prophage repressor gene) is deleted. Three sbcA ^- strains carry multiple (and probably tandemly repeated) copies of the Rac genome while two others carry a single Rac prophage that is indistinguishable in its hybridisation behaviour from that carried by sbcA ⁺ strains.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.