G1-PHASE AND S-PHASE MAMMALIAN-CELLS SYNTHESIZE HISTONES AT EQUIVALENT RATES
G1-PHASE AND S-PHASE MAMMALIAN-CELLS SYNTHESIZE HISTONES AT EQUIVALENT RATES
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DOI：
10.1016/0092-8674(80)90127-0
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发表时间：
1980-01-01
期刊：
影响因子：
64.5
通讯作者：
COFFINO, P
中科院分区：
文献类型：
--
作者：
GROPPI, VE;COFFINO, P
为了确定细胞周期位置对蛋白质合成的影响，同步化的细胞群体进行代谢标记，并通过二维凝胶电泳检测碱性蛋白质（包括组蛋白）的合成。通过离心淘洗、选择性有丝分裂分离、荧光激活细胞分选或这些方法的组合，将指数生长的S49小鼠淋巴瘤或中国仓鼠卵巢（CHO）细胞分离成G1和S期细胞群，并用放射性标记的氨基酸进行脉冲标记。通过O '' Farrell、Goodman和O '' Farrell（1977）的NEPHGE [非平衡二维凝胶电泳]技术的改进，全细胞裂解液中的游离组蛋白和染色质结合组蛋白与约300种其他碱性多肽完全分离。G1期和S期标记细胞样品的匹配放射自显影比较显示，S49和CHO细胞的细胞周期中组蛋白合成速率相等。从脉冲标记的G1期S49细胞分离的细胞核含有13-15%的S期细胞核中存在的新合成的核小体组蛋白。从G1期细胞制备的细胞核经脉冲标记，然后追踪5小时，含有全细胞裂解物中存在的标记的核小体组蛋白的> 90%。随着细胞周期的进行，组蛋白合成速率的差异性改变显然不会发生到显著的程度，但是新合成的组蛋白与DNA的结合发生在DNA复制开始之后。
To determine the effect of cell cycle position on protein synthesis, synchronized cell populations were metabolically labeled and the synthesis of the basic proteins, including histones, was examined by 2-dimensional gel electrophoresis. Exponentially growing S49 mouse lymphoma or Chinese hamster ovary (CHO) cells were separated into G1 and S phase populations by centrifugal elutriation, selective mitotic detachment, fluorescence-activated cell sorting, or a combination of these, and pulse-labeled with radiolabeled amino acids. The histone proteins, both free and chromatin-bound, were completely resolved from some 300 other basic polypeptides in whole-cell lysates by a modification of the NEPHGE [nonequilibrium 2-dimensional gel electrophoresis] technique of O''Farrell, Goodman and O''Farrell (1977). Comparisons of matched autoradiograms from samples of G1 and S phase labeled cells revealed an equivalent rate of histone synthesis through the cell cycle of both S49 and CHO cells. Nuclei isolated from G1 phase S49 cells that were pulse-labeled contained 13-15% of the newly synthesized nucleosomal histones present in S phase nuclei. Nuclei prepared from G1 phase cells that were pulse-labeled and then chased for 5 h contained > 90% of the labeled nucleosomal histones present in whole-cell lysates. Differential alterations in the rate of histone synthesis apparently do not occur to a significant degree as cells proceed through the cycle, but the association of newly synthesized histones with DNA takes place after the onset of DNA replication.