IDENTIFICATION OF A DISTINCTION BETWEEN CYTOPLASMIC HISTONE SYNTHESIS AND SUBSEQUENT HISTONE DEPOSITION WITHIN NUCLEUS
IDENTIFICATION OF A DISTINCTION BETWEEN CYTOPLASMIC HISTONE SYNTHESIS AND SUBSEQUENT HISTONE DEPOSITION WITHIN NUCLEUS
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DOI：
10.1021/bi00701a017
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发表时间：
1974-01-01
期刊：
影响因子：
2.9
通讯作者：
CHALKLEY, R
中科院分区：
文献类型：
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作者：
OLIVER, D;GRANNER, D;CHALKLEY, R
材料和方法细胞培养和同位素标记。肝癌组织培养 (HTC) 细胞在改良的 Swim’s S77 培养基中悬浮培养，细胞密度为 4 至 6 X 105 个细胞/ml，其中含有 50 mM 三甲基甘氨酸 [7V-tris（羟甲基）甲基甘氨酸]缓冲液以及胎牛和小牛血清，终浓度各为 5%（Granner 等人，1968）。 HTC 细胞组蛋白在 20 分钟孵育期间用 0.5 mCi 的 [3H] 赖氨酸/l 进行标记。细胞。室温下以 160g 离心 5 分钟去除标记的细胞，洗涤。来自爱荷华大学内科 (D.G.) 和生物化学系和爱荷华州退伍军人医院，爱荷华市，爱荷华州 52242。收稿日期：1973 年 7 月 30 日。D.G. 得到美国公共卫生服务拨款号 CA 12191 和退伍军人管理局临床支持。调查。 R.C. 得到了美国公共卫生服务拨款号 CA 10871 和职业发展奖号 GM 46410 的支持。DO 是国家科学基金会内分泌学博士后研究员。帐篷。这个游离组蛋白池的位置是核外的，有证据表明它不在内核膜和外核膜之间的核周空间中。用未标记的培养基进行一次，然后重悬于新鲜的 S77 培养基中，温热至 37 进行追踪期。 Novikoff 细胞在大鼠腹膜腔内体内生长，或在含有改良 Swim's S77 培养基的体外培养瓶中生长。对于体内实验，将0.5 mCi的[3H]赖氨酸注射到大鼠充满细胞的腹膜腔中。脉冲结束时，取出放射性标记的细胞，用标准培养基洗涤一次，并重新注射到第二只未接种的大鼠的腹膜腔中进行追逐期。在不同时间从后者动物中取出等份细胞，如上所述离心，并快速冷冻。在体外实验中，从动物体内取出Novikoff细胞，离心一次，重悬于100ml温热的培养基中。将细胞与0.5 pd的[3H]赖氨酸在37℃孵育20分钟。脉冲后，将细胞离心，洗涤一次，并重悬于非放射性培养基中进行追踪期。在适当的时间，从培养介质中取出细胞，离心，洗涤一次，然后在 17 ℃下冷冻保存。通过将 2 ml 等份的 HTC 或 Novikoff 细胞（浓度为 5 X 105 个细胞/ml）与 3.3 MCi 的[3H]胸苷在 37 ℃下孵育 10 分钟来进行标记。[3H]胸苷的体内掺入通过注射将放射性同位素（2 MCi/ml 细胞）注入大鼠腹腔，孵育 15 分钟，此时取出等分试样并冷冻细胞。 [3H]胸苷掺入DNA的比放射性的估计通过使用Schmidt-Thannhauser (1945)程序的修改来确定。组蛋白分离和电泳。通过Panyim等人(1971)的方法从HTC和Novikoff肿瘤细胞中分离出整个组蛋白。 4 X 108 HTC 细胞的产量约为 6 mg，从相同数量的 Novikoff 细胞获得了相当的产量。如前所述，在含有 2.5 m 尿素和 0.9 n 乙酸的 9 cm 凝胶（15% 丙烯酰胺）中，在 130 V 下电泳分离每个组蛋白组分 3.5 小时（Balhorn 等人， 1971).[3H] 通过从凝胶上切下每个条带、用 30% 过氧化氢消化并在 Bray 溶液中计数来确定每个组蛋白组分中赖氨酸的掺入情况 (Bray, 1960)。 [3H] 组蛋白与细胞核的结合。大约 1 克...
Materials and MethodsCell Culture and Isotopic Labeling. Hepatoma tissue culture (HTC) cells were grown in suspension culture at cell densities between 4 and 6 X 105 cells/ml in a modified Swim’s S77 culture medium containing 50 mM tricine [7V-tris (hydroxy-methyl) methylglycine] buffer and fetal calf and calf sera at final concentrations of 5% each (Granner et al., 1968). HTC cell histone was labeled during 20-min incubationswith 0.5 mCi of [3H] lysine/l. of cells. The labeled cells were removed by centrifugation at 160g for 5 min at room temperature, washed t From the Departments of Internal Medicine (D. G.) and Biochemistry, University of Iowa and Veterans Hospital, Iowa City, Iowa 52242. Received July 30, 1973. D. G. was supported by U. S. Public Health Service Grant No. CA 12191 and by a Veterans Administration Clinical Investigatorship. R. C. was supported by U. S. Public Health Service Grant No. CA 10871 and by Career DevelopmentAward No. GM 46410. DO was a National Science Foundation Postdoctoral Fellow in Endocrinology. tent. The site of this free histone pool is extranuclear, and evidence suggests that it is not in the perinuclear space be-tween the inner and outer nuclear membranes. once with unlabeled medium, and then resuspended in fresh S77 medium warmedto 37 for the chase period. Novikoff cells were grown either in vivo within the peritoneal cavity of the rat or in vitro in culture flasks containing the modified Swim’s S77 culture medium. For the in vivo ex-periments, 0.5 mCi of [3H] lysine was injected into the cellladen peritoneal cavity of a rat. At the conclusion of the pulse, the radiolabeled cells were removed, washed once with the standard culture medium, and reinjected into the peri-toneal cavity of a second, uninoculated rat for the chase period. Aliquots of cells were removed from the latter animal at various times, centrifuged as described above, and quickly frozen. In the in vitro experiments, Novikoff cells were re-moved from the animal, centrifuged once, and resuspended in 100 ml of warm culture medium. Thecells were incubated with 0.5 pd of [3H] lysine for 20 min at 37. After the pulse, the cells were centrifuged, washed once, and resuspended in nonradioactive culture medium for the chase period. At appropriate times, cells were removed from the incubation medium, centrifuged, and washed once and then stored frozen at-17.DNA was labeled by incubating 2-ml aliquots of either HTC or Novikoff cells, at a concentration of 5 X 105 cells/ml, with 3.3 MCi of [3H] thymidine for 10 min at 37. The in vivo incorporation of [3H] thymidine was determined by injecting the radioisotope (2 MCi/ml of cells) into the peritoneal cavity of the rat for a 15-min incubation at which time an aliquot was removed and the cells frozen. Estimation of the specific radioactivity of the incorporation of [3H] thymidine into DNA was determined by using a modification of the Schmidt-Thannhauser (1945) procedure. Histone Isolation and Electrophoresis. Whole histone was isolated from HTC and Novikoff tumor cells by the method of Panyim et al.(1971). The yield from 4 X 108 HTC cells was approximately 6 mg and a comparable yield was obtained from the same number of Novikoff cells, Electrophoretic sep-aration of each of the histone fractions was accomplished in 9-cm gels (15% acrylamide) containing 2.5 m urea and 0.9 n acetic acid at130 V for 3.5 hr as previously described (Balhorn et al., 1971).[3H] Lysine incorporation into each of the histone fractions was determined by slicing each band from the gel, digesting with 30% hydrogen peroxide, and counting in Bray’s solution (Bray, 1960). Binding of [3H] Histone to Nuclei. Approximately 1 g of …