Distribution of tightly bound proteins in the chicken ovalbumin gene region.
Distribution of tightly bound proteins in the chicken ovalbumin gene region.
复制标题
鸡卵清蛋白基因区域紧密结合蛋白的分布。
DOI：
10.1021/bi00531a019
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Kuo,MT
中科院分区：
文献类型：
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作者：
Kuo,MT
细胞核和染色体的制备材料和方法。根据所描述的程序从鸡红细胞中分离出细胞核（Wilhelm & Champagne, 1969）。用Hewish & Burgoyne（1973）的方法从蛋鸡的输卵管或肝脏中分离出细胞核。从鸡淋巴母细胞MSB-1中分离染色体和细胞核的详细步骤将在其他地方发表（Kuo, 1982）。剪切染色质的柱层析。除非另有说明，所有手术均在4℃下用硅化玻璃器皿或塑料器皿进行。细胞核悬浮在含有15 mM Tris-HCl、pH 7.4、60 mM KC1、15 mM NaCl、15 mM/3-巯基乙醇、0.15 mM精胺、0.5 mM亚精胺和0.34 M蔗糖的溶液中，吸光度为24（以2 M NaCl测量）。在3 mL的核悬浮液中，以恒定涡流的方式滴入2 mL的5 M NaCl。核裂解液通过20号针头剪切了15次。以完整的腺病毒2型DNA作为标记，用0.4%琼脂糖凝胶电泳分析，从剪切样品中分离的DNA分子量大于35千碱基（kb）。剪切后的样品搅拌30分钟，然后通过Sepharose C1-4B （Pharmacia）色谱柱（2.5 X 60 cm）进行色谱分析，色谱柱用含有2 M NaCl， 10 mM Tris-HCl, pH 7.9和1 mM乙二胺四乙酸（EDTA）的溶液平衡，并用相同的溶液洗脱。测定各组分（5ml）在230和260 nm处的吸光度。限制性内切酶：DNA的酶切。从thespharose柱层析中提取的DNA片段在Beckman SW 41转子上以40000 rpm的转速，在1 ml蔗糖缓冲液中加入2.4 M蔗糖，10 mM Tris-HCl, pH 7.9和1 mM EDTA，离心浓缩
Materials and MethodsPreparation of Nuclei and Chromosomes. Nuclei from chicken erythrocytes were isolated according to the described procedure (Wilhelm & Champagne, 1969). From laying hen oviducts or livers, nuclei were isolated by the procedure of Hewish & Burgoyne (1973). The detailed procedure for isolation of chromosomes and nuclei from chicken lymphoblastoid cell MSB-1 will be published elsewhere (Kuo, 1982). Column Chromatography of Sheared Chromatin. All procedures were performed at 4 C with siliconized glassware or plasticware unless otherwiseindicated. The nuclei were suspended in a solution containing 15 mM Tris-HCl, pH 7.4, 60 mM KC1, 15 mM NaCl, 15 mM/3-mercaptoethanol, 0.15 mM spermine, 0.5 mM spermidine, and 0.34 M sucrose at a 260-nm absorbance of 24 (measured in 2 M NaCl). To 3 mL of nuclear suspensionwas added dropwise 2 mL of 5 M NaCl with constant vortexing. The nuclear lysate was sheared by passing it through a 20-gauge needle 15 times. The molecular weight of DNA isolated from the sheared sample was greater than 35 kilobases (kb) as analyzed by 0.4% agarose gel electrophoresis using intact adenovirus 2 DNA as a marker. The sheared sample was stirred for 30 min and then chro-matographed through a Sepharose C1-4B (Pharmacia) column (2.5 X 60 cm) which had been equilibrated with a solution containing 2 M NaCl, 10 mM Tris-HCl, pH 7.9, and 1 mM ethylenediaminetetraacetic acid (EDTA) and eluted with the same solution. Absorbances at 230 and 260 nm in each fraction (5 mL) were measured. Restriction Enzyme Digestion of DNA. The DNA fractions from theSepharose columnchromatography were pooled and concentrated by centrifugation onto a 1-mL sucrose cushion containing 2.4 M sucrose, 10 mM Tris-HCl, pH 7.9, and 1 mM EDTA in a Beckman SW 41 rotor at 40000 rpm for 7