A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation.
A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation.
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用于研究大分子合成的渗透性动物细胞制剂。
DOI：
10.1021/bi00599a021
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发表时间：
1978
期刊：
影响因子：
2.9
通讯作者：
A. Pardee
中科院分区：
文献类型：
--
作者：
M. R. Miller;J. Castellot;A. Pardee
本发明描述了一种用溶血素选择性透化中国仓鼠卵巢细胞的方法。这些细胞保持其一般形态，完整的细胞器; 100%的DNA和75%的总蛋白质。当与适当的底物和辅因子结合时，它们合成蛋白质、RNA和DNA，其中许多不能穿透完整的细胞。这些可渗透的细胞将是有用的研究的作用，非穿透分子，如底物，效应，和抑制剂的细胞代谢过程，包括大分子合成，在相对生理条件下。作为初步研究，DNA合成的一些性质进行了研究。DNA合成进行了初始速率至少一样高，在完整的细胞，并持续至少2小时，和20%的基因组可以复制。首先将DNA制成小（4S）片段，其快速连接到4-21 X 106道尔顿DNA。BrdUTP掺入后密度增加证明了正常的保守合成。清洗渗透性细胞缩短了S期的起始和DNA复制的调控的生化事件知之甚少。脱氧核糖核苷酸的供应可能是启动S期的一个重要控制机制，这一可能性在最近的几篇综述中已经讨论过（Edenberg & Huberman，1975; Prescott，1976）。Bjursell & Reichard（1973）和Reichard（1977）提出dCTP或类似物可能在启动多瘤病毒复制中起重要作用。为了更好地研究这个问题和其他关于DNA复制调控的问题，我们希望开发一种亚细胞系统，该系统将允许将脱氧核苷酸和其他化合物引入动物细胞中，这些细胞可以在细胞周期的各个时期（包括Gj）高度同步。理想的亚细胞系统应保持在体内以保守的速率合成DNA并复制大量基因组的能力。分离的细胞核（De Pamelo和贝格，1975; Friedman和Mueller，1968; Krokan等，1975; Krokan和Ericksen，1977; Lynch等人，1970; Thompson & Mueller，1975; Tseng & Goulian，1975）和细胞裂解物（Brown等人，1977; Lazarus，1973）在研究DNA复制的某些方面被证明是非常有用的。但是，一个对外源性化合物具有渗透性并且仍然保持高度的
A method for selectively permeabilizing Chinese hamster ovary cells with lysolecithin is described. These cells retained their general morphology, intact organelles; 100% of their DNA and 75% of their total protein. They synthesized protein, RNA and DNA when suppliedwith appropriate substrates and cofactors, many of which donot penetrate intact cells. These permeable cells will be useful for studying the roles of nonpenetrating molecules such as substrates, effectors, and inhibitors of cellular metabolic processes, including macro-molecular syntheses, under relatively physiological conditions. As an initial study, some properties of DNA synthesis were investigated. DNA synthesis proceeded at an initial rate at least as high as in intact cells and continued for at least 2 h, and 20% of the genome could be replicated. The DNA was first made as small (4S) pieces that were rapidly ligated to 4-21 X 106 dalton DNA. Normal, semiconservative synthesis was demonstrated by showing increased density followingBrdUTP incorporation. Washing the permeablecells shortened theThe biochemical events responsible for initiating the S period and regulating DNA replication are poorly understood. The possibility that the supply of deoxyribonucleotides may be an important control mechanism in initiating the S period has been discussedin several recent reviews (Edenberg & Huberman, 1975; Prescott, 1976). Bjursell & Reichard (1973) and Reichard (1977) proposed that dCTP or an analogue may be important in initiating polyoma virus replication. To better examine this and other questions regarding the regulation of DNA replication, we wanted to develop a subcellular system which would allow the introduction of deoxynucleotides and other compounds into animal cells which could be highly synchronized in various periods of the cell cycle, including Gj. Ideally the subcellular system should retain the capacity to synthesize DNA semiconservatively at in vivo rates and rep-licate extensiveamounts of the genome. Isolated nuclei (De Pamphilis & Berg, 1975; Friedman & Mueller, 1968; Krokan et al., 1975; Krokan & Ericksen, 1977; Lynch et al., 1970; Thompson & Mueller, 1975; Tseng & Goulian, 1975) and cell lysates (Brown et al., 1977; Lazarus, 1973) have proven very useful in studying some aspects of DNA replication. But a subcellular system which is permeable to exogenous compounds and still retains a high degree of