Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.
Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.
复制标题
由含有或缺乏糖皮质激素反应元件的 DNA 片段形成的人糖皮质激素受体复合物的表征。
DOI：
10.1021/bi00431a003
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Cidlowski,JA
中科院分区：
文献类型：
--
作者：
Tully,DB;Cidlowski,JA
材料和方法化学品。[6, 7-3H]曲安奈德 ([3H] TA)，比活度 43.6 Ci/mmol，购自 New England Nuclear (Boston, MA)。[a-32P] dCTP，比活度 3000 Ci/mmol，购自 ICN Radiochemicals (Irvine, CA)。培养基和补充剂来自 GIBCO 实验室（纽约州格兰德岛）。限制酶和 DNA 聚合酶 I 的 Klenow 片段来自 GIBCO-BRL (Gaithersburg, MD)。小牛胸腺 DNA-纤维素和试剂级化学品购自 Sigma Chemical Co.（密苏里州圣路易斯）。细胞培养和细胞质制备。如前所述（Cidlowski 和 Richon，1984）从 HeLa S3 细胞制备胞质溶胶，但略有修改。简而言之，HeLa S3 细胞在 37°C 的 Joklik 基本必需培养基 (JMEM) 中悬浮培养，其中含有 2 mM 谷氨酰胺、75 单位/mL 青霉素 G、50 单位/mL 硫酸链霉素和 3.在 4℃ 下，在 Sorvall GSA 转子中以 2500g 收获 0% 的胎牛血清和小牛血清的 1:1 (v/v) 混合物，在冷的、未补充的 JMEM 中洗涤，并重悬于 4 mL JMEM 中。使用 Coulter Model ZM 细胞计数器（Coulter Electronics, Inc., Hialeah, FL）对细胞悬浮液的等分试样进行计数，并根据需要调整细胞悬浮液的体积，以产生 2-4 X 107 个细胞/mL 的最终细胞密度。将大约 2-4 X 108 个细胞与 40 nM [3H]-曲安奈德 ([3H] TA) 在 0°C 下轻轻搅拌孵育 2 小时。将类固醇处理的细胞在冷冻离心机中以 2000g 沉淀，重悬于等体积的冰冷缓冲液 A [20 mM 磷酸钠，pH 7.0，50 mM NaCl，10%(v/v) 甘油，2 mM 2-巯基乙醇，1 mM EDTA]中，并使用预冷的 Tekmar Ultra Turrax 匀浆器匀浆（Tekmar Co.，辛辛那提，俄亥俄州）进行 3 次 10 秒爆发，并在冰上休息 5-10 秒。立即将匀浆在 Beckman 50 Ti 转子中于 0°C 下以 165000g 离心 1 小时。将高速上清液（胞质溶胶）与 0.05 g 小牛胸腺 DNA-纤维素（4 mg DNA/g DNA-纤维素）在 4°C 下孵育 5-10 分钟，然后与等体积的葡聚糖包被悬浮液中的沉淀一起孵育 5 分钟。木炭 [0.1%(w/v) 葡聚糖、1.5 mM MgCl2 中的 1.0%(w/v) 活性炭]，或直接与葡聚糖包被的木炭一起孵育。将细胞质-木炭悬浮液在0℃、12000g下离心10分钟，并收集上清液。炭剥离的细胞溶胶要么维持在冰上（未激活），要么通过在 27°C 下孵育 30 分钟来热激活。根据该实验室的早期工作，根据细胞计数计算粗制细胞溶胶中 GR 浓度的估计值，该工作表明 HeLa S3 细胞含有大约 20000 个受体/细胞（Cidlowski 和 Cidlowski，1981）。质粒 DNA 和放射性标记。这些研究中使用的三个 DNA 片段先前已被描述过 (Tully & Cidlowski, 1987)。简而言之，MMTV LTR DNA 片段是一个 326-bp 片段，包含相对于小鼠乳腺肿瘤病毒 5' 长末端重复中的初级转录起始位点的 222 至+104 个核苷酸（Majors & Varmus，1983）。该 DNA 片段包含三个序列区域，在使用纯化 GR 的核酸酶保护实验中受到不同程度的保护（Payvar 等人，1983 年；Scheidereit 和 Beato，1984 年）。从 pBR322 的 Taq I 消化中获得了另外两个片段。 368 bp Taq I-DDNA 片段从 pBR322 的核苷酸 4019 延伸至 24，并包含通过计算机搜索 pBR322 DNA 序列识别出的单个 GRE 位点（Tully & Cidlowski，1987；Sutcliffe，1978；Peden ...
Materials and Methods Chemicals.[6, 7-3H] Triamcinolone acetonide ([3H] TA), specific activity 43.6 Ci/mmol, was obtained from New England Nuclear (Boston, MA).[a-32P] dCTP, specific ac-tivity 3000 Ci/mmol, was from ICN Radiochemicals (Irvine, CA). Culture media and supplements were from GIBCO Laboratories (Grand Island, NY). Restriction enzymes and the Klenow fragment of DNA polymerase I were from GIBCO-BRL (Gaithersburg, MD). Calf thymus DNA-cellulose and reagent grade chemicals were obtained from Sigma Chemical Co.(St. Louis, MO). Cell Culture and Cytosol Preparation. Cytosols were prepared from HeLa S3 cells as previously described (Cidlowski & Richon, 1984), except for minor modifications. Briefly, HeLa S3 cells grown in suspension culture at 37 C in Joklik’s minimal essential medium (JMEM) containing 2 mM glutamine, 75 units/mL penicillin G, 50 units/mL streptomycin sulfate, and3. 0% of a 1: 1 (v/v) mixture of fetal calf serum and calf serum were harvested at 2500g in a Sorvall GSA rotor at 4 C, washed in cold, unsupplemented JMEM, and resuspended in 4 mL of JMEM. Aliquots of the cell suspensions were counted by using a Coulter Model ZM cell counter (Coulter Electronics, Inc., Hialeah, FL), and the volumes of the cell suspensions were adjusted, if necessary, to yield a final cell density of 2-4 X 107 cells/mL. Approximately 2-4 X 108 cells were incubated with 40 nM [3H]-triamcinolone acetonide ([3H] TA) for 2 h at 0 C with gentle agitation. The steroid-treated cells were pelleted at 2000g in a refrigerated centrifuge, resuspended in an equal volume of ice-cold buffer A [20 mM sodium phosphate, pH 7.0, 50 mM NaCl, 10%(v/v) glycerol, 2 mM 2-mercaptoethanol, 1 mM EDTA], and homogenized by using a prechilled Tekmar Ultra Turrax homogenizer (Tekmar Co., Cincinnati, OH) for three 10-s bursts with 5-10-s rests on ice. The homogenate was immediately centrifuged at 165000g in a Beckman 50 Ti rotor for 1 h at 0 C. The high-speed supernatant (cytosol) was either incubated with 0.05 g of calf thymus DNA-cellulose (4 mg of DNA/g of DNA-cellulose) for 5-10 min at 4 C, followed by incubation for 5 min with the pellet from an equal volume of a suspension of dextran-coated charcoal [1.0%(w/v) activated charcoal in 0.1%(w/v) dextran, 1.5 mM MgCl2], or incubated directly with the dextran-coated charcoal. The cytosol-charcoal suspension was centrifuged at 12000g for 10 min at 0 C, and the supernatant was collected. Charcoalstripped cytosol was either maintained on ice (nonactivated) or heat activated by incubation for 30 min at 27 C. Estimates for the concentration of GR in the crude cytosols were cal-culated from cell counts, based on earlier work from this laboratory indicating that HeLa S3 cells containapproximately 20000 receptors/cell (Cidlowski & Cidlowski, 1981). Plasmid DNA and Radiolabeling. The three DNA frag-ments used in these studies have been previously described (Tully & Cidlowski, 1987). Briefly, the MMTV LTR DNA fragment is a 326-bp fragment that includes nucleotides-222 to+ 104 relative to the primary transcription start site in the 5'longterminal repeat of the mouse mammary tumor virus (Majors & Varmus, 1983). This DNA fragmentcontains three regions of sequence protected to varying degrees in nu-clease protection experiments with purified GR (Payvar et al., 1983; Scheidereit & Beato, 1984). Two additional fragments were obtained from a Taq I digest of pBR322. The 368-bp Taq I-DDNA fragment extends from nucleotides 4019to 24 of pBR322 and contains a single GRE site identified by a computer search of the pBR322 DNA sequence (Tully & Cidlowski, 1987; Sutcliffe, 1978; Peden …