三种提取HMG染色体非组蛋白方法的比较

非组蛋白是在染色体中组蛋白以外的蛋白质。它除了含有基因的调控蛋白外,还具有许多与核酸代谢有关的酶和结构蛋白。非组蛋白从染色体复合物上分离下来后,很容易聚集在一起,也很容易被蛋白水解酶降解。所以,它们很难在溶液中保持原状。目前,非组蛋白中能够较好地分离、纯化,并对其生物学性质及结构功能加以详细研究的是一组称为高泳动因子的染色体非组蛋白,简称HMG染色体非组蛋白。     

小鼠Hep.A肝癌染色质磷酸化非组蛋白的研究——Ⅰ.正常小鼠肝和Hep.A腹水肝癌染色质磷酸化非组蛋白的比较

研究了Hep.A腹水肝癌染色质磷酸化非组蛋白的变化。Hep.A肝癌染色质非组蛋白,磷酸化非组蛋白的含量以及非组蛋白被磷酸化的比例都较正常小鼠肝增加,分别达到正常肝的2.2,4.4和2.0倍。非组蛋白含磷量也增加,达到正常肝的1.36倍。结果表明Hep.A肝癌染色质非组蛋白磷酸化比例增加。但单位重量非组蛋白及磷酸化非组蛋白所含磷酸基团反而下降,仅分别为正常肝的65%和32%。Hep.A肝癌染色质磷酸化非组蛋白的SDS-凝胶电泳图谱显示分子量为69,000道尔顿的蛋白部份明显增加,此外还有一正常细胞缺乏的分子量为47,000道尔顿的蛋白部份出现。等电聚焦电泳表明等电点偏低的蛋白部份增加。氨基酸组成分析证明两种细胞磷酸基团的接受体基本相同。实验结果表明Hep.A肝癌染色质磷酸化非组蛋白与正常小鼠肝有质与量的差别。     

小鼠Hep.A肝癌染色质磷酸化非组蛋白的研究——Ⅰ.正常小鼠肝和Hep.A腹水肝癌染色质磷酸化非组蛋白的比较

研究了Hep.A腹水肝癌染色质磷酸化非组蛋白的变化。Hep.A肝癌染色质非组蛋白,磷酸化非组蛋白的含量以及非组蛋白被磷酸化的比例都较正常小鼠肝增加,分别达到正常肝的2.2,4.4和2.0倍。非组蛋白含磷量也增加,达到正常肝的1.36倍。结果表明HeP.A肝癌染色质非组蛋白磷酸化比例增加。但单位重量非组蛋白及磷酸化非组蛋白所含磷酸基团反而下降,仅分别为正常肝的65%和32%。Hep.A肝癌染色质磷酸化非组蛋白的SDS-凝胶电泳图谱显示分子量为69,000道尔顿的蛋白部份明显增加,此外还有一正常细胞缺乏的分子量为47,000道尔顿的蛋白部份出现。等电聚焦电泳表明等电点偏低的蛋白部份增加。氨基酸组成分析证明两种细胞磷酸基团的接受体基本相同。实验结果表明Hep.A肝癌染色质磷酸化非组蛋白与正常小鼠肝有质与量的差别。     

三种提取HMG染色体非组蛋白方法的比较

非组蛋白是在染色体中组蛋白以外的蛋白 质。它除了含有基因的调控蛋白外,还具有许 多与核酸代谢有关的酶和结构蛋白。非组蛋白 从染色体复合物上分离下来后,很容易聚集在 一起,也很容易被蛋白水解酶降解。所以,它们 很难在溶液中保持原状〔31。目前,非组蛋白中能 够较好地分离、纯化,并对其生物学性质及结构 功能加以详细研究的是一组称为高泳动因子的 染色体非组蛋白,简称HMG染色体非组蛋白。 HMG蛋白质分子量较低（MW < 30, 000),可 以用稀盐溶液或高氯酸从细胞核提取,并能溶 于2沁三氯乙酸溶液中。HMG蛋白质主要含 有4种主要成份： HMG HMG2, HMGl4和 HMG,,, HMG蛋白质与组蛋白有相似之处,它 们的碱性氨基酸残基的含量很高（25-30多）; 与组蛋白不同的是它们的酸性氨基酸残基的含 量也很高（20-30务）。碱性和酸性氨基酸的 含量加在一起占HMG蛋白质氨基酸含量的一 半左右^[5,11,13]     

~(60)Co-γ辐照对离体大白鼠胸腺染色质蛋白的影响

人们对DNA的辐射损伤效应已作了较为深入的研究,然而,在真核生物的染色质内,DNA与组蛋白、非组蛋白是紧密结合在一起以核蛋白的形式存在的,核蛋白复合物具有高度的辐射敏感性已为人们所公认。因此,在谈到细胞的辐射损伤时,不仅是DNA,还必须考虑到与DNA密切结合的染色质蛋白,其中组蛋白     

SET蛋白家族分类、功能及其在血液系统中研究进展

SET蛋白家族包含保守的SET结构域,很多家族成员可以利用S-腺苷甲硫氨酸对底物进行甲基化修饰.SET蛋白家族主要对组蛋白进行甲基化修饰,包括组蛋白H3K4、K9、K27、K36以及组蛋白H4K20,调控真核生物中基因的激活和沉默.除组蛋白外,部分SET蛋白家族成员对各种非组蛋白也具有催化酶活性.SET蛋白突变所导致的...     

北京鸭卵清蛋白基因调控的研究——Ⅰ.停产与产蛋的北京鸭肝脏和输卵管染色质蛋白质的比较

从停产和产蛋的北京鸭的肝和输卵管制备纯染色质。用0.4NH_2SO_4抽提组蛋白和酸溶性非组蛋白,剩余的非酸溶性非组蛋白用牛胰DNaseI消化DNA法制备。对染色质大分子含量的测定表明,非组蛋白和RNA的含量在产蛋鸭染色质中明显地增加了。用乙酸脲电泳分析,核心组蛋白成份在所有实验样品中都是恒定的,但在产蛋鸭肝和输卵管染色质组蛋白H_1呈两条区带,并且出现较多条酸溶性非组蛋白区带。用SDS电泳分析,产蛋鸭肝和输卵管染色质中出现分子量约20,000的非酸溶性非组蛋白。非组蛋白的这些变化,启示它们可能是控制基因活性的调节因素。     

重组p300组蛋白乙酰转移酶结构域的表达及应用

组蛋白乙酰化修饰是基因起始转录的关键步骤. p300等组蛋白乙酰转移酶（HATs）催化组蛋白和非组蛋白的乙酰化. HATs具有多种细胞功能,而且乙酰化对底物蛋白的功能改变也具有重要功能. 组蛋白乙酰转移酶p300可乙酰化多种细胞内蛋白,某些病毒蛋白与p300有相互作用并促进病毒复制. 因此, p300是细胞内具有广泛功能的转录激活因子. 组蛋白乙酰转移酶结构域（HAT区）是p300乙酰化酶活性的最小中心功能域,在p300乙酰化底物中具有重要功能. 本文重组表达了对应p300 HAT区的GST-p300 HAT蛋白,对其乙酰化酶的活性进行检测. 结果证实,p300 HAT蛋白在体外可高效乙酰化组蛋白H3. 随后,对体外乙酰化反应的条件进行优化. 总之,本文构建了一种简单高效、非放射性体外乙酰化体系,适用于对潜在底物蛋白的乙酰化水平和机制进行分析,以及乙酰化蛋白的相关功能的研究.     

蚕丝腺体染色质的研究——Ⅲ.染色质功能与结构蛋白的关系

本文比较了五龄三天及眠期的蓖麻蚕后丝腺体染色质的结构蛋白与转录活性,结果表明非组蛋白的单相凝胶电泳有显著的变化,转录活性亦有差异。染色质经DNase Ⅱ处理后分离的可溶性染色质与不溶部分的染色质结构蛋白电泳图谱不同。对重组染色质的转录活性进行了研究,初步结果表明同源和异源的组蛋白对DNA转录有抑制作用,去H_1的组蛋白抑制作用显著减少。非组蛋白能恢复部分被抑制的转录活性。     

蚕丝腺体染色质的研究 Ⅲ.染色质功能与结构蛋白的关系

本文比较了五龄三天及眠期的蓖麻蚕后丝腺体染色质的结构蛋白与转录活性,结果表明非组蛋白的单相凝胶电泳有显著的变化,转录活性亦有差异。染色质经DNase Ⅱ处理后分离的可溶性染色质与不溶部分的染色质结构蛋白电泳图谱不同。对重组染色质的转录活性进行了研究,初步结果表明同源和异源的组蛋白对DNA 转录有抑制作用,去H_1的组蛋白抑制作用显著减少。非组蛋白能恢复部分被抑制的转录活性。     

人胚肝发育过程中染色质组份的变化

从14-,17-,21-,27-,34-,及38-周令的人胚肝细胞核分离出柒色质,分别对其中的RNA、DNA、组蛋白(HP)及非组蛋白(NHP)进行测定。在胚胎发育过程中肝柒色质HP/DNA比值变化不大。但是,NHP/DNA与NHP/HP比值发生显著改变。人胚肝NHP量的变化一直保持在整个胚胎发育过程中,NHP量的高峰位于21-及34-周。用SDS-聚丙烯酰胺凝胶板电泳分析处于不同发育阶段的人胚肝总染色质蛋白。电泳图谱显示出染色质NHP组分在质与量上有所改变。     

Changes in nonhistone chromosomal proteins in phytohemagglutininstimulated lymphocytes

Stimulation of bovine lymphocytes with phytohemagglutinin results in quantitative as well as qualitative changes in the nonhistone chromosomal proteins. Analysis of these proteins by hydroxyapatite chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis shows not only a selective increase in the amount of some nonhistone proteins but also a decrease of other nonhistone protein bands. This observation is compatible with the view that nonhistone proteins have an inhibitory as well as an activating function at the genome level.     

Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Phosphorylation of nonhistone chromatin proteins during sea urchin development

The phosphorylation of nonhistone chromatin proteins during development was studied in the sea urchin, $\text{Strongylocentrotus purpuratus}$. The rate of phosphorylation was found to be maximal during gastrula, slightly lower during prism and almost 70% lower in pluteus stage embryos. Analysis of the phosphorylated nonhistone chromatin proteins by SDS-acrylamide gel electrophoresis showed significant variations in the labeling pattern during different stages of development. A specific protein which is actively phosphorylated during gastrula and prism stages is nearly absent from the pluteus stage.     

Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

A comparative study of the nonhistone proteins of rat liver euchromatin and heterochromatin

Rat liver was fractionated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by controlled shearing and glycerol gradient centrifugation. The histone and nonhistone proteins associated with each fraction were compared. No qualitative differences in histone content were observed, but heterochromatin contained 1.5 times more histone protein than did euchromatin. The nonhistone proteins of each chromatin fraction were fractionated on the basis of salt solubility into loosely bound (those extracted by 0.35 m NaCl), tightly bound (those extracted by 2.0 m NaCl), and residual nonhistone proteins (those not extracted by 2.0 m NaCl). Euchromatin contained 3.7 times more loosely bound nonhistone proteins than did heterochromatin, while the latter contained twice as much residual nonhistone protein. Euchromatin was devoid of tightly bound nonhistone protein, a component of heterochromatin. Electrophoretic analysis of these nonhistone protein fractions revealed marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

Carbohydrate composition of rat hepatocyte nuclear membrane as compared to normal, Morris hepatoma 7800, and phenobarbital-induced microsomal membranes

The composition of the nonhistone nuclear proteins of rat liver, brain, thymus, and kidney has been analyzed by isoelectric focusing in polyacrylamide gel. Approximately 20–30 components were separated with a wide range of isoelectric points (pl) in the 3- to 10-pH region.Different extraction procedures applied to liver nuclei removed protein mixtures with similar components present in varying amounts. 8 m Urea 50 mm phosphate, pH 7.6, was the most successful and removed most of the nonhistone protein.The thiol groups of proteins extracted from the nuclei of liver, brain, thymus, and kidney with 8 M urea, 50 mM phosphate were labeled with [¹⁴C]N-ethylmaleimide. Although there was a slight variation in the overall thiol content of these tissue proteins, separation of the mixture by isoelectric focusing and SDS polyacrylamide gel electrophoresis showed complex patterns indicating greater heterogeneity than was apparent from the Coomassie blue dye binding.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.