Initiation of replication at a mammalian chromosomal origin.
Initiation of replication at a mammalian chromosomal origin.
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在哺乳动物染色体起源处开始复制。
DOI：
10.1101/sqb.1993.058.01.053
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Lin,HB
中科院分区：
文献类型：
--
作者：
Hamlin,JL;Mosca,PJ;Dijkwel,PA;Lin,HB
材料和方法细胞培养，以及同步和放射性标记方案。CHO细胞和甲氨蝶呤耐药CHO细胞系CHOC 400按照之前的描述进行维持（Milbrandt等人，1981）。在涉及复制中间体分离的实验中，将细胞接种在15厘米的组织培养皿上。为了获得同步化群体，培养物被剥夺异亮氨酸45小时以诱导G 0停滞，随后释放到含有400 μ M含羞草碱的完全培养基中14小时;将药物洗出并替换为无药物培养基，使细胞以同步波进入S（Dijkwel和Hamlin 1992）。对于图5所示的同位素标记研究，将培养物接种到24孔丛皿中，并用1/xCi/ml 3 H-胸苷脉冲（80 Ci/mM; Dupont/新英格兰核公司），根据图例中所示的方案，用0.2 μ g/ml冷胸苷稀释。通过加入柠檬酸至0.2 M停止脉冲，并如前所述处理样品（莫斯卡等人，1992）。
MATERIALS AND METHODSCell culture, and synchronizing and radiolabeling regimens. CHO cells and the methotrexate-resistant CHO cell line, CHOC 400, were maintained as de: scribed previously (Milbrandt et al. 1981). In experiments involving the isolation of replication intermediates, cells were plated onto 15-cm tissue culture dishes. To obtain synchronized populations, cultures were deprived of isoleucine for 45 hours to induce G o arrest, followed by release into complete medium containing 400/xM mimosine for 14 hours; the drug was washed out and replaced with drug-free medium, allowing cells to enter S in a synchronous wave (Dijkwel and Hamlin 1992).For the isotopic labeling studies presented in Figure 5, cultures were plated into 24-well cluster dishes and were pulsed with 1/xCi/ml 3H-thymidine (80 Ci/mM; Dupont/New England Nuclear Corporation) diluted with 0.2/xg/ml cold thymidine according to the protocols indicated in the figure legend. The pulses were stopped by the addition of citric acid to 0.2 M, and samples were processed as described previously (Mosca et al. 1992).