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The circular dichroism of membrane-free folded chromosomes from E. coli was measured and analyzed. The spectrum can be explained as a simple linear combination of the individual spectra of E. coli RNA, and E. Coli DNA in the B form. No contribution from A form or C form DNA was detected. There was evidently some real variation in the ratio of the two nucleic acids from preparation to preparation, but the average value was 24% RNA and 76% DNA. No significant light scattering was observed and the analyses indicated no contribution to the circular dichroism from scattering artifacts. Apparently, combining DNA, RNA, and protein into membrane-free folded chromosomes does not change the secondary structure of the DNA or RNA from that found for the free nucleic acid in the same solvent system. 相似文献
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Rapid electroelution of nucleic acids from agarose and acrylamide gels 总被引:19,自引:0,他引:19
The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [3H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., 14C-labeled DNA) relative to the elution of “reference” DNA (i.e., 3H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ~0.190 and ~0.017 min?1. 相似文献
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Y Oda S Iwai E Ohtsuka M Ishikawa M Ikehara H Nakamura 《Nucleic acids research》1993,21(20):4690-4695
To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding. 相似文献
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《Reviews in Molecular Biotechnology》2000,74(1):15-25
In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as ‘universal receptors’ that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes. 相似文献
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A simple and efficient method was devised to permit the isolation of total nucleic acids (poly(A+) and poly(A-) RNAs and DNA) from cells and tissues (e.g. testis) enriched in DNA. Chaotropic reagents (guanidine thiocyanate and LiBr) were utilized to inactivate nucleases rapidly, minimize the viscosity of the homogenization solution and to precipitate RNA selectively (LiBr). Both total and poly(A+)-enriched mRNAs were recovered in a biologically active form as demonstrated by their ability to programme an in vitro translation reaction. High molecular weight DNA (greater than 22 kilobases) was recovered from the LiBr-soluble supernatants by selective ethanol precipitation, subsequently purified and was in a form suitable for further biochemical analysis. 相似文献
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S Bauer Y Milner E Ziv J Shiloach N de Groot Y Lapidot 《Biotechnology and bioengineering》1973,15(6):1081-1088
A procedure for the preparation of about 50 g batches of tRNA from 25 kg E. coli W is described. The method involves phenolic extraction of the cells, batch absorption of the tRNA on DEAE-cellulose, washing the DEAE-cellulose and packing it into a column, elution of the tRNA from the column and precipitation of the tRNA with ethanol. The method is less time and labor consuming than the methods described in the literature and can be carried out with relatively simple equipment. 相似文献
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B D Stollar 《CRC critical reviews in biochemistry》1975,3(1):45-69
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Chandar S. Thakur Margaret E. Brown Jacob N. Sama Melantha E. Jackson T. Kwaku Dayie 《Applied microbiology and biotechnology》2010,88(3):771-779
Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from
stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR)
spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria
grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies,
it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such
information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol
combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media
and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous
gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts
to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs. 相似文献
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