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101.
Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum 总被引:7,自引:1,他引:6
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A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg2+ in the γ-glutamyl transferase assay, but there was no evidence that the GS was adenylylated. 相似文献
102.
Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability. 总被引:5,自引:4,他引:1
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Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains. 相似文献
103.
Hydration force and bilayer deformation: a reevaluation 总被引:20,自引:0,他引:20
The hydration repulsive force between lipid bilayers and the deformability of both gel and liquid-crystalline bilayers have been quantitated by an X-ray diffraction analysis of osmotically stressed liposomes. Both sampling theorem reconstructions and electron density distributions were calculated from diffraction data obtained from multilayers with applied osmotic pressures of 0-50 atm. The bilayer thickness and area per lipid molecule remain nearly constant (to within about 4%) in this pressure range, as adjacent bilayers move from their equilibrium separation in excess water to within 2-4 A of each other. This analysis indicates that the bilayers are relatively incompressible. This results differs from previously published X-ray diffraction studies of bilayer compressibility but agrees with direct mechanical measurements of the bilayer compressibility modulus. It is also found that the hydration repulsive force decays exponentially with separation between bilayers with a decay constant of 1.4 A for gel-state dipalmitoylphosphatidylcholine and 1.7 A for liquid-crystalline egg phosphatidylcholine bilayers. This implies that the exponential decay constant is not necessarily equal to the diameter of a water molecule, as has been previously suggested on experimental and theoretical grounds. 相似文献
104.
M D Kramer L Binninger V Schirrmacher H Moll M Prester G Nerz M M Simon 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(12):4644-4651
We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed. 相似文献
105.
M M Simon U Hochgeschwender U Brugger S Landolfo 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(8):2755-2762
In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor. 相似文献
106.
John H. Caldwell Jennifer Van Brunt Franklin M. Harold 《The Journal of membrane biology》1986,89(1):85-97
Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr2+ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr2+. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor. 相似文献
107.
M Caillol M Meunier M Mondain-Monval P Simon 《Journal of reproduction and fertility》1986,78(2):479-486
In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 micrograms LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0.23 +/- 0.14 ng/ml from February to a maximum of 1.44 +/- 0.57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47.0 +/- 8.0 ng/ml in January to 106 +/- 33 ng/ml in July and decreased in August (69.4 +/- 10.6 ng/ml). The values of the second peak rose from 11.0 +/- 2.2 ng/ml in January to 90.6 +/- 12.4 ng/ml between March and July and decreased in August (24.5 +/- 5.1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season. 相似文献
108.
Nigel L. Brown Tapan K. Misra Joseph N. Winnie Annette Schmidt Michael Seiff Simon Silver 《Molecular & general genetics : MGG》1986,202(1):143-151
Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes. 相似文献
109.
Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes 总被引:17,自引:13,他引:4
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Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis. 相似文献
110.
The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study. 总被引:1,自引:0,他引:1
Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion. 相似文献