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Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date. 相似文献
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HMG-17 is a highly conserved and ubiquitous nonhistone chromosomal protein that binds to nucleosome core particles. HMG-17 and HMG-14 form a family of chromosomal proteins that have been reported to bind preferentially to regions of active chromatin structure. To study the functional role of the single-copy chicken HMG-17 gene, null mutants were generated by targeted gene disruption in a chicken lymphoid cell line, DT40. Heterozygous and homozygous null mutant cell lines were generated by two independent selection strategies. Heterozygous null mutant lines produced about half the normal level of HMG-17 protein, and homozygous null lines produced no detectable HMG-17. No significant changes in cell phenotype were observed in cells harboring either singly or doubly disrupted HMG-17 genes, and no compensatory changes in HMG-14 or histone protein levels were observed. It is concluded that HMG-17 protein is not required for normal growth of avian cell lines in vitro, nor does the absence of HMG-17 protein lead to any major changes in cellular phenotype, at least in lymphoid cells. 相似文献
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Neither HMG-14a nor HMG-17 gene function is required for growth of chicken DT40 cells or maintenance of DNaseI-hypersensitive sites. 总被引:1,自引:1,他引:0
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HMG-14 and HMG-17 form a family of ubiquitous non-histone chromosomal proteins and have been reported to bind preferentially to regions of active chromatin structure. Our previous studies demonstrated that the chicken HMG-17 gene is dispensable for normal growth of the DT40 chicken lymphoid cell line. Here it is shown that the major chicken HMG-14 gene,HMG-14a, is also dispensable and, moreover, that DT40-derived cells lacking both HMG-17 and HMG-14a proteins show no obvious change in phenotype with respect to the parental DT40 cells. Furthermore, no compensatory changes in HMG-14b or histone protein levels were observed in cells lacking both HMG-14a and HMG-17, nor were any alterations detected in such hallmarks of chromatin structure as DNaseI-hypersensitive sites or micrococcal nuclease digestion patterns. It is concluded that the HMG-14a and HMG-17 proteins are not required for normal growth of avian cell linesin vitro, nor for the maintenance of DNaseI-hypersensitive sites in chromatin. 相似文献
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J M Pash J S Bhorjee B M Patterson M Bustin 《The Journal of biological chemistry》1990,265(8):4197-4199
The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis. 相似文献
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The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells. 相似文献
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Chromosomal protein HMG-14. Complete human cDNA sequence and evidence for a multigene family 总被引:7,自引:0,他引:7
D Landsman T Srikantha R Westermann M Bustin 《The Journal of biological chemistry》1986,261(34):16082-16086
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Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1. 相似文献
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Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison 总被引:7,自引:0,他引:7
A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA. 相似文献
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Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA. 总被引:2,自引:2,他引:0
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Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17. 相似文献
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