Genome exposure and regulation in mammalian cells.

T T Puck, P Webb, R Johnson
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引用次数: 7

Abstract

A method of measurement of exposed DNA (i.e. hypersensitive to DNase I hydrolysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nuclei of mammalian cells is described. While cell cultures exhibit some differences in behavior from day to day, the general pattern of exposed and sequestered DNA is satisfactorily reproducible and agrees with results previously obtained by other methods. The general pattern of DNA hydrolysis exhibited by all cells tested consists of a curve which at first rises sharply with increasing DNase I, and then becomes almost horizontal, indicating that roughly about half of the nuclear DNA is highly sequestered. In 4 cases where transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with its more normal homolog (3T3, and the reverse transformed versions of CHO, HL60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid, and nerve growth factor [NGF] respectively), the transformed form displayed less genome exposure than the nontransformed form at every DNase I dose tested. When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased exposure reverting to that of the transformed forms. Therefore Ca++ appears necessary for maintenance of the DNA in the more highly exposed state characteristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested. Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increase of exposure of CHO by DBcAMP but it must be administered before or simultaneously with the latter compound. Measurements on mouse biopsies reveal large differences in exposure in different normal tissues. Thus, the exposure from adult liver cells was greater than that of adult brain, but both fetal liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human fibroblasts as revealed by in situ nick translation reveals a nuclear distribution pattern around the periphery, around the nucleoli and in punctate positions in the nuclear interior in parts of both S and G1 phases of the cell cycle. The same exposure pattern is duplicated by the pattern of DNA synthesis in S cells. It would appear that these nuclear regions represent positions of special activity. The previously proposed theory of genome regulation in mammalian cells is supported by these findings. The theory proposes that: a) gene activity requires exposure of the given locus followed by action of transcription factors on the exposed genes; b) the fiber system of the cell (cytoskeleton, nuclear fibers, and extracellular fibers) are required for normal exposure; c) active sites for gene expression and replication consist of the nuclear periphery where differentiation genes particularly are exposed; the nucleoli where at least some housekeeping genes are exposed; and possibly also punctate regions in the interior; d) noncoding sequences play a critical role in genome regulation, possibly including the transport of loci to be activated to appropriate exposure transcriptional and replicating locations. Cancer cells have lost specific differentiation gene activities, at least sometimes because of mutation of appropriate exposure genes; at least some protooncogenes and tumor suppressor genes are responsible for exposure and transport of specific differentiation gene loci to their appropriate exposure sites in the nucleus and for inducing exposure.

哺乳动物细胞的基因组暴露和调控。
本文描述了一种测量暴露的DNA(即对DNA酶I水解过敏)的方法,而不是在哺乳动物细胞的分离细胞核中隔离的(抗水解的)DNA。虽然细胞培养每天都表现出一些不同的行为,但暴露和隔离DNA的一般模式是令人满意的可重复性,并与以前通过其他方法获得的结果一致。所有被测试的细胞所显示的DNA水解的一般模式由一条曲线组成,该曲线首先随着DNA酶I的增加而急剧上升,然后几乎变成水平,这表明大约有一半的核DNA被高度隔离。在4例转化细胞(Raszip6, CHO, HL60和PC12)中,每个细胞都与其更正常的同源物(3T3, CHO, HL60和PC12的反向转化版本,分别由二丁基环AMP [DBcAMP],维甲酸和神经生长因子[NGF]获得)进行比较,在每次DNase I剂量测试中,转化形式比非转化形式显示出更少的基因组暴露。当在Raszip6-3T3和CHO-DBcAMP体系中将Ca++从水解介质中排除时,正常细胞形式失去了其增加的暴露,恢复到转化形式。因此,Ca++对于维持DNA处于非转化表型的高度暴露状态似乎是必要的。LiCl增加了所有转化细胞的DNA暴露。硫酸葡聚糖和肝素都可以增加几种不同癌症的DNA暴露。秋碱可防止DBcAMP对CHO的暴露增加,但必须在DBcAMP之前或与后者同时施用。小鼠活组织检查的测量结果显示,不同正常组织的暴露量存在很大差异。因此,成人肝脏细胞的暴露量大于成人大脑细胞,但胎儿肝脏和胎儿大脑细胞的暴露量均明显高于成人细胞。原位缺口翻译显示,暴露在正常的人成纤维细胞中,在细胞周期的S期和G1期,细胞核分布在周围、核仁周围和核内部的点状位置。S细胞的DNA合成模式复制了相同的暴露模式。这些核区域似乎代表着特殊活动的位置。这些发现支持了先前提出的哺乳动物细胞基因组调控理论。该理论提出:a)基因活性需要暴露于特定位点,然后转录因子作用于暴露的基因;B)细胞的纤维系统(细胞骨架、核纤维和细胞外纤维)是正常暴露所必需的;C)基因表达和复制的活性位点包括细胞核外周,其中分化基因特别暴露;核仁中至少有一些内务基因暴露在核仁中;也可能是内部的点状区域;D)非编码序列在基因组调控中发挥关键作用,可能包括将被激活的基因座转运到适当的转录和复制位置。癌细胞已经失去了特定的分化基因活动,至少有时是因为适当的暴露基因发生突变;至少一些原癌基因和肿瘤抑制基因负责特定分化基因位点的暴露和运输到其在细胞核中的适当暴露位点,并诱导暴露。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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