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Electrophoretic separation of histones and high-mobility-group proteins on acid-urea-Triton gels 总被引:3,自引:0,他引:3
T Boulikas 《Analytical biochemistry》1985,149(2):379-386
The electrophoretic mobilities of calf thymus histones and high-mobility-group (HMG) nonhistone proteins were studied on a newly modified polyacrylamide gel containing acetic acid, urea, and the nonionic detergent Triton X-100 in combination with glycine in the electrode buffer. This gel system avoids stacking gel, photopolymerization of acrylamide, and preelectrophoresis. Under extremely low Triton concentrations some H3 variant forms (H3.1) were preferentially separated by their slower migration from bulk H3. Under increasing concentrations of Triton in the gel in the presence of 3 or 6 M urea, the mobilities of H2A.1, H3.2, H2A.2, H4, and H2B were sequentially retarded. The mobilities of H1 and HMGs remained virtually unchanged under all conditions. This gel system is able to resolve charge-modified histones. 相似文献
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Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation 总被引:1,自引:0,他引:1
T Boulikas 《Biochimie et biologie cellulaire》1986,64(5):474-484
Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure. 相似文献
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T Boulikas 《Journal of cellular biochemistry》1992,50(2):111-123
Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis. 相似文献
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Present studies deal with the role of inhibin in proliferation and growth. The effect of inhibin on incorporation of 3H-thymidine in prostatic DNA in vivo as well as by NRK-49F and Balb/c3T3 cell lines in vitro, was investigated. Also studied the immunocytochemical localization of inhibin in normally proliferating and differentiated tissues of human prostate and endometrium. The in vivo studies revealed a suppression of 3H-thymidine uptake both in ventral (33%) and dorsolateral (26%) lobes of rat prostate. Interestingly, the histology of inhibin treated rat prostate manifested amidst the epithelial lining, an appearance of apoptotic bodies which are considered to be indicative of cell death. Further, the immunocytochemical studies for localization of inhibin showed intense staining in the differentiated human prostate and endometrium as compared to the respective proliferative tissues. Is inhibin kept suppressed in these proliferating tissues, because it is antiproliferative? The present in vitro experiments demonstrated that, at low inhibin concentrations, the incorporation of 3H-thymidine is stimulated while at higher doses it is suppressed. Thus, it is clear that prostatic inhibin seems to have a concentration-dependent dual role in the regulation of DNA synthesis. 相似文献
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Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid-urea-Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis. 相似文献
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