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Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization 总被引:2,自引:0,他引:2
Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions. 相似文献
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R M Schimpff M Bozzola P Chatelain A M Repellin 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1986,302(19):683-686
After serum Sephacryl 300 gel filtration, the major circulating forms of TA in serum are macromolecular. However different patterns are found in cord blood and in the blood of newborns at 24 hr of life. The low values found in cord blood are not due to inhibiting factor(s). TA in serum fractions does not parallel immunoreactive Sm-C levels. The pattern of immunoreactive Sm-C are similar in both cord blood and newborn blood. 相似文献
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In vitro conditions and kinetics of 14C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls. 相似文献
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Lin N Yan J Huang Z Altier C Li M Carrasco N Suyemoto M Johnston L Wang S Wang Q Fang H Caton-Williams J Wang B 《Nucleic acids research》2007,35(4):1222-1229
The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection. 相似文献
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The uptake and incorporation of adenosine and thymidine by infective larvae, 10-day-old juvenile, and adult stages of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. No evidence of thymidine incorporation by the worm was obtained in this study. Scintillation counting methods demonstrated that 14C-labeled adenosine was incorporated by all three stages of this filarial worm. Autoradiography, performed on worms incubated in [3H] adenosine from 5 min through 2 hr, revealed that following 5–15 min incubation the greatest degree of adenosine incorporation occurred in the hypodermis and somatic cords. Adenosine incorporation into the deeper body tissues, including the gut, increased significantly with longer periods of incubation. The results obtained further support the concept that nutrient uptake in B. pahangi occurs by a transcuticular route. 相似文献
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P A Straat H Wolochow R L Dimmick M A Chatigny 《Applied and environmental microbiology》1977,34(3):292-296
As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells. 相似文献
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R M Schimpff M Bozzola 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1984,299(7):235-237
Using thymidine uptake by activated human lymphocytes to measure the growth-stimulating activity of human serum, the authors demonstrate that thymidine activity was significantly correlated (p less than 0.001) with GH levels. r = 0.729 when GH values are below 3.5 ng/ml, r = 0.509 when GH values are below 8 ng/ml and r = 0.337 for all of the patients. Low levels of GH are sufficient for the generation of this activity. 相似文献
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Membrane anchors of vesicular stomatitis virus: characterization and incorporation into virions. 总被引:1,自引:5,他引:1
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Wild-type vesicular stomatitis virus-infected cells contained multiple carboxy-terminal fragments of the envelope glycoprotein G. They migrated in 16% polyacrylamide gels with two dominant apparent molecular weights, 14,000 and 9,000. Both fragments were immunoprecipitated by two antibodies, anti-G(COOH) and anti-G(stem), made against the last 15 amino acids at the carboxy terminus and against the first 22 amino acids of the ectodomain adjacent to the transmembrane region of G, respectively. Pulse-chase experiments in the presence and absence of tunicamycin indicated that the higher-molecular-weight fragment, Gal, was generated first, presumably in the rough endoplasmic reticulum, and then apparently chased into the faster-migrating, stable fragment, Ga2. Exposure of infected cells to radioactive palmitic acid labeled Ga2. Ga2 was detected in purified virions. These results show that a polypeptide approximately 71 amino acids long is transported and incorporated into budding virions. What signals are operative and whether this C-terminal fragment of G protein is transported as a complex with other viral or host cell proteins are presently unknown. 相似文献
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Gasparutto D Cognet S Roussel S Cadet J 《Nucleosides, nucleotides & nucleic acids》2005,24(10-12):1831-1842
An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment. 相似文献
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DF 31, a sperm decondensation factor from Drosophila melanogaster: purification and characterization. 总被引:3,自引:0,他引:3
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We have purified to homogeneity a Drosophila protein which is able to decondense Xenopus sperm chromatin. This protein, which we have called DF 31, is a heat-stable phosphoprotein which displays a molecular weight of 31 kDa on SDS-PAGE, but which has an apparent molecular weight of > 200 kDa on gel filtration. We show that DF 31 decondenses sperm DNA by displacement of sperm-specific proteins. In addition to its sperm decondensation activity, DF 31 is also able to facilitate nucleosome loading on both decondensed sperm DNA and on naked DNA template. The reaction as catalysed by DF 31 is quite efficient; however, the nucleosomes appear to be loaded randomly onto the DNA, not in regular arrays. Although the mechanism by which DF 31 aids nucleosome loading is not yet clear, it most probably occurs through binding of DF 31 to core histones. 相似文献
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C Sorg 《European journal of biochemistry》1975,55(2):423-430
For chemical characterization of the products of activated lymphocytes a radioactive double-label technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum-free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [3H]leucine or [14C]leucine were fractionated on Sephadex G-75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0--.35 (approx. mol. wt 75000-20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5-10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture. 相似文献