Hyper expression ofBacillus stearothermophilus α-amylase gene inBacillus subtilis

Summary Utilizing plasmids pUB110, pBR322 and pRG71, we have constructed a number of vectors which could be utilized for expression of cloned genes both in gram positive and gram negative bacteria. This report discusses the hyperexpression of -amylase gene ofB. stearothermophilus BR135 using these vectors.     

The 25-kilodalton hemolytic protein affinity-purified from parasporal inclusions ofBacillus thuringiensis strain PG-14 (serotype 8a∶8b)

A simple method using an antibody-mediated affinity chromatography was developed for rapid and specific purification of the 25-kilodalton protein from alkali-solubilized and silkworm (Bombyx mori) larval gut juice-digested parasporal inclusions of theBacillus thuringiensis strain PG-14 (serotype 8a8b). Affinity-purified 25-kilodalton protein was highly hemolytic to red blood cells (RBCs) of two avian (chicken and goose) and six mammalian (horse, mouse, cow, rabbit, guinea pig, and sheep) species. The concentration of the 25-kolodalton protein required for 100% hemolysis was in the range of 2–16 g/ml, and an apparent RBC species-dependent variation was observed in hemolytic activity of this protein. Of the RBCs tested, chicken and house RBCs were the most susceptible to hemolysis by this protein; sheep RBCs wre 4–8 times less susceptible than the others.     

Denaturation ofBacillus stearothermophilus α-amylase by urea and detergents

Denaturation ofBacillus stearothermophilus -amylase by urea and detergents was investigated for the purpose of understanding the mechanism of denaturation of this enzyme. The enzyme was extremely resistant to denaturation by detergents at 60° C, either in the presence or absence of added calcium. Addition of EDTA was necessary to obtain denaturation by detergents. The rate of denaturation of the -amylase by urea was strongly dependent on the incubation pH and presence or absence of calcium ions. Calcium-binding groups were shown to have pKa values of 5.5 for exogenous calcium and 4.7 to 4.8 for endogenous calcium. A mechanism is proposed for the denaturation ofBacillus stearothermophilus -amylase.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

Effect of additives on the thermostability ofBacillus stearothermophilus α-amylase

The effect of additives on the thermostability ofBacillus stearothermophilus -amylase was determined. Polyols, dimethyl formamide, and dimethyl sulfoxide all increased the half life of the enzyme approximately 2-fold when tested at a 10% (w/v) addition. These results suggest that the enzyme's structure is stabilized against thermal denaturation through ionic interactions. Addition of dextran or polyvinyl alcohol (hydrophilic polymers which increase the viscosity of the solution) had a slight positive effect on enzyme stability while addition of polyethylene glycol or polyvinylpyrrolidone (hydrophobic polymers which increase the viscosity of the solution) resulted in a 2-fold decrease in enzyme half life.     

Resolution of the extracellular α-glucosidase system ofBacillus sp. NCIB 11203

Summary Two extracellular -glucosidases (EC 3.2.1.20, -D-glucoside glucohydrolase) of the alkalophilic bacterium,Bacillus sp. NCIB 11203, were separated, purified and partially characterised. Resolution of the system into two separate enzymes was achieved by fractionation with (NH₄)₂SO₄ and chromatography on DEAE-Biogel A. The first of these activities, an -glucosidase, hydrolysed p-nitrophenyl--D-glucopyranoside preferentially and had minor activity on isomaltose and isomaltotriose. The second enzyme was a maltase and displayed highest activity on maltose and maltotriose and some activity on p-nitrophenyl--D-glucopyranoside.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Transfer ofBacillus subtilis endo-β-1,4-glucanase gene intoZymomonas anaerobia

Summary The endo--1,4-glucanase gene ofBacillus subtilis origin cloned previously in a plasmid pBS1 was subcloned in a new plasmid pSCR815, and with the new plasmidZymomonas anaerobia was transformed. TheBacillus glucanase gene expressed in theZymomonas cells with efficiency much lower than inEscherichia coli.     

Nonspecific ionic effects on the cytolytic and hemolytic properties ofBacillus thuringiensis δ-endotoxins

We investigated the in vitro effects of ions, carbohydrates, lectins, and charged compounds on the cytolytic and hemolytic action of the purified -endotoxin ofBacillus thuringiensis var.darmstadiensis 73-E10-2, and other -endotoxins. Cytotoxicity was inhibited by preincubating the toxin with N-acetylneuraminic, glucuronic, galacturonic, and N-acetylglutamic acids and ATP. Lectins were unable to inhibit toxicity. Pretreatment of sheep erythrocytes with neuraminidase enhanced hemolysis. These results suggested a nonspecific inhibition of cytotoxicity based on the presence of a negative charge. Supraphysiological concentrations of divalent cations such as Ca²⁺, Mg²⁺, and Zn²⁺ in the medium yielded a reduced toxicity, whereas EDTA and EGTA enhanced cytotoxicity.     

Investigation of the membrane lesion induced in vitro by two mosquitocidal δ-endotoxins ofBacillus thuringiensis

Bacillus thuringiensis produces insecticidal crystalline -endotoxins during sporulation. Leakage of radiolabeled markers from insect tissue culture cells and erythrocytes supports a mechanism of colloid osmotic lysis for the 23K and 27K polypeptides from the -endotoxins ofBacillus thuringiensis var.darmstadiensis 73-E10-2 and var.israelensis, respectively. Both toxins produce a plasma membrane pore 0.6–1.0 nm in radius.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

C-Terminal processing ofBacillus subtilis BSE616 endo-β-1, 4-glucanase inBacillus megaterium

Summary A large form of endo--1, 4-glucanase (endoglucanase) encoded byBacillus subtilis BSE616 gene inB. megaterium (pCK98) was exocellularly produced at early log phase. The C-terminal portion of the secreted enzyme of 52 kilodaltons (Kd) was gradually cleaved and then converted to the smallest product of 33 Kd. N-terminal amino acid residues of both 52 Kd and 33 Kd enzyme were determined to be the same, Ala, the 30th residue of the primary translation product. Cleavage of the C-terminal portion increased specific and molecular activity of the enzyme.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

The effect of glucose on the expression of a clonedBacillus amyloliquefaciens α-amylase gene in strains ofBacillus subtilis

Bacillus subtilis strain 1A297 was shown to relieve the glucose repression of a clonedB. amyloliquefaciens -amylase gene carried on the hybrid plasmid pVC102 without affecting its temporal activation. However, glucose repression of -amylase occurred when pVC102, was introduced intoB. subtilis strain 1A289. Glucose repression was relieved by -methyl-d-glucoside, an analog of glucose that blocks its uptake. The relief of glucose repression in 1A297 did not act at the level of plasmid copy number. As 1A297 was capable of exerting glucose repression on a homologous chromosomally encoded gene, it is postulated that the putativetrans-acting product involved in glucose repression inB. subtilis (Nicholson and Chambliss, 1986, J. Bacteriol. 165:663–670) is altered in strain 1A297 and does not recognize theB. amyloliquefaciens -amylase gene.     

生理卫生(下册)教学参考资料(续完)

第三节祖国在防治疾病上的伟大成就这节课是在学生已掌握了传染病的基本知識的基础上来进行講授的,首先在教材里提出以血吸虫病、天花、瘧疾、黑热病、痳疯等传染病說明了新旧社会的对比;同时也说明了社会主义社会制度的优越性。我們的党和政府对于人民健康的关怀是无微不至的,并且对于各种疾病的积极防治工作加强了党的领导,及时提出了一系列的方針、政策和措施;建立了专业防治机构,培养了专业干部;結合生产发动了广大羣众来参加防治。因此在防治各种传染病的工作中取得了很大的成績。     

生理卫生(下册)教学参考资料(续)

第五节大脑皮层的机能——高级神經活动本节是本章也是本書的高峯,是把过去学习过有关反射作用的知識进行总結和提高,使学生能从理論上理解神经对人的意义——保持內部各器官的协調,同外界环境的统一。     

Structure of Poly (dT)·Poly (dA)·Poly (dT)

Abstract

The molecular structure of poly (dT)·poly (dA)·poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right- handed triple-helix of pitch 38.4 Å and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2′-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

稀有内养囊霉(Entrophosporainfrequens(Hall)AmesetSchneider)的研究(英)

Hall（1977）首次在新西兰的土壤中发现GlomusinjtequensHall。Ames和Schneider（979）在美国的加利福尼亚再次发现了这个种。他们认为该种与无梗囊霉（Acauloryora）有相似之处,抱子都是从泡囊发育而成,但无梗囊霉的抱于是在产抱囊的侧面形成,而该种的抱子则是在产抱囊柄的内部发育而成的。为此,Ames和Schneider（197）为这个种建立起一个新属——内养囊霉属（Entrophospora）,把这个种改名为稀有内养囊霉Entrophoworam斤equens（HallAmesetSchnelder。SChellGk和Spain（1984）报道了这个属的另一个种哥伦比亚内养囊霉（Entro…     

Domain organization ofBacillus thuringiensis CryIIIA δ-endotoxin studied by denaturation in guanidine hydrochloride solutions and limited proteolysis

Denaturation ofBacillus thuringiensis CryIIIAδ-endotoxin—an insecticidal protein, active againstColeoptera larvae—in concentrated guanidine hydrochloride solutions was pursued by fluorescence and circular dichroism spectroscopy and limited proteolysis. It was found that the protein consists of two fragments that differ by their stability to denaturation by guanidine hydrochloride atpH 3. The less stable fragment corresponds to the N-terminalα-helical domain limited by Leu-279; the more stable one starts with Ile-280, contains about 330 amino acid residues, and corresponds to the molecule C-terminal moiety that consist of its twoβ-structural domains forming a superdomain.     

Robert (Robin) Hill (1899–1991)

Obituary

Robert (Robin) Hill (1899–1991)