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1.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.   总被引:3200,自引:408,他引:2792       下载免费PDF全文
A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.  相似文献
2.
Regulation of the expression of IL-6 in human monocytes   总被引:15,自引:0,他引:15  
IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.  相似文献
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The primary structure of bovine satellite 1.715.   总被引:12,自引:5,他引:7       下载免费PDF全文
The primary structure of the 1402 bp repeat unit of bovine satellite 1.715 has been determined using a dimer inserted at the SalI site of plasmid pBR322 and cloned in E. coli. In contrast with bovine satellites 1.706, 1.720b and 1.711a, the 1.715 satellite has a complex sequence with no obvious internal short prototype repeat. The sequence consists however of repeats ranging in length from 6 to 13 nucleotides. In addition, the hexanucleotide, AGATGA, present in the prototype sequences of satellites 1.706, 1.720b and 1.711a, is found in satellite 1.715 in repeats as long as, or longer than, 8 nucleotides, establishing a homology link among those satellites on one hand and satellite 1.715 (and the related satellite 1.711b) on the other. In turn, this suggests a common evolutionary origin. A comparison of the maps for 15 restriction enzymes of cloned and uncloned satellite indicates very little sequence divergence among the repeat units of the latter, most of the differences being due to methylation.  相似文献
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Soluble DNA dependent RNA polymerase from prostate nuclei   总被引:4,自引:0,他引:4  
J Doly  M Ramuz  P Mandel  P Chambon 《Life sciences》1965,4(20):1961-1966
7.
Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.  相似文献
8.
A Civas  M Dion  G Vodjdani    J Doly 《Nucleic acids research》1991,19(16):4497-4502
The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.  相似文献
9.
The capacity of IL-6 to mediate the antiparasitic activity of IL-1 on intrahepatic development of malaria parasite was demonstrated. The comparisons of IL-6 levels in infected and noninfected hepatocyte cultures, either purified or enriched with nonparenchymal cells and stimulated by IL-1 or IL-6, indicate that subtle interactions exist between intrahepatocytic development of Plasmodium yoelii and liver synthesis of IL-6. During its intrahepatic multiplication, the parasite causes a decline in IL-6 production. IL-6 mRNA was not detected in the livers of infected mice during development of either hepatic or blood stage parasites although IL-6 activity was found in the sera during both stages.  相似文献
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