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41.
42.
Heterogeneous binding of high mobility group chromosomal proteins to nuclei 总被引:7,自引:5,他引:2
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A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs. 相似文献
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A 55-residue protein containing the DNA binding domain of Hin recombinase, residues 139-190, with the tripeptide Gly-Gly-His (GGH) at the NH2 terminus was synthesized by stepwise solid-phase methods. GGH(Hin139-190) binds sequence specifically to DNA at four 13 base pair sites (termed hixL and secondary) and, in the presence of Ni(OAc)2 and monoperoxyphthalic acid, reacts predominantly at a single deoxyribose position on one strand of each binding site [Mack, D.P., & Dervan, P.B. (1990) J. Am. Chem. Soc. 112, 4604]. We find that, upon treatment with n-butylamine, the DNA termini at the cleavage site are 3'- and 5'-phosphate, consistent with oxidative degradation of the deoxyribose backbone. The nickel-mediated oxidation can be activated with peracid, iodosylbenzene, or hydrogen peroxide. The sequence specificity of the reaction is not dependent on oxidant, but the rates of cleavage differ, decreasing in the order peracid greater than iodosylbenzene greater than hydrogen peroxide. Optimal cleavage conditions for a 1 microM concentration of protein are 50 microM peracid, pH 8.0, and 1 equiv of Ni(OAc)2. The preferential cleavage at a single base pair position on one strand of the minor groove indicates a nondiffusible oxidizing species. A change of absolute configuration in the GGH metal binding domain from L-His to D-His [Ni(II).GG-(-D-)H(Hin139-190)] affords cleavage at similar base pair locations but opposite with regard to strand specificity. 相似文献
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46.
Molecular evolution of rodent insulins 总被引:1,自引:0,他引:1
Several trees of amino acid sequences of rodent insulins were derived with
the maximum-parsimony procedure. Possible orthologous and paralogous
relationships were investigated. Except for a recent gene duplication in
the ancestor of rat and mouse, there are no strong arguments for other
paralogous relationships. Therefore, a tree in agreement with other
biological data is the most reasonable one. According to this tree, the
capacity to form zinc-binding hexamers was lost once in the ancestor of the
hystricomorph rodents, followed by moderately increased evolutionary rates
in the lineages to African porcupine and chinchilla but highly increased
rates in at least three independent lines to other taxa of this suborder:
guinea pig, cuis, and Octodontoidea (coypu and casiragua).
相似文献
47.
Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses 总被引:57,自引:0,他引:57
The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical. 相似文献
48.
Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase 总被引:6,自引:0,他引:6
Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein. 相似文献
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50.
The vascular endothelial growth factor (VEGF) and its receptors have been implicated as key-factors in tumor angiogenesis and are major targets in cancer therapy. New oligomers which mimic the architecture of DNA-binding polyamides have been designed to target the hypoxia inducible factor (HIF-1) binding site on the promoter of VEGF gene. These oligomers incorporate an increasing number of six–five fused rings such as hydroxybenzimidazole–imidazole, benzimidazole–pyrrole, benzimidazole–chlorothiophene, and imidazopyridine–pyrrole, and bind the VEGF hypoxia response element (HRE) 5′-TACGT-3′ with high affinity and selectivity. 相似文献