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Rapid isolation of high molecular weight plant DNA.   总被引:215,自引:12,他引:203       下载免费PDF全文
A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.  相似文献
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The poliovirus polyprotein is cleaved at three different amino acid pairs. Viral polypeptide 3C is responsible for processing at the most common pair (glutamineglycine). We have found that a cDNA fragment encoding parts of the capsid protein region (P1) and the nonstructural protein region (P2), and including the P1-P2 processing site (tyrosine-glycine), can be expressed in E. coli. The translation product was correctly processed. Disruption of the coding sequence of 2A, a nonstructural polypeptide mapping carboxy-terminal to the tyrosine-glycine cleavage site, by linker mutagenesis or deletion, prevented processing. Deletion of the adjacent polypeptide 2B had no such effect. Antibodies against 2A specifically inhibited processing at the 3C'-3D' processing site (tyrosine-glycine) in vitro. We conclude that poliovirus encodes the second proteinase 2A, which processes the polyprotein at tyrosine-glycine cleavage sites.  相似文献
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Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.  相似文献
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Nucleic acid quantitation by continuous flow fluorometry   总被引:1,自引:0,他引:1  
A system for rapid and sensitive fluorometric measurement of nucleic acids is described. Samples can be analyzed virtually as fast as they can be injected into the analyzer (three to five per minute). 5-microliter samples ranging in concentration from 0.05 to 40 micrograms/ml can be measured accurately. The sample can easily be recovered.  相似文献
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Home range, dispersal and the clan system of impala   总被引:1,自引:0,他引:1  
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