LONG REGIONS OF SINGLE-STRANDED-DNA IN HUMAN-CELLS
LONG REGIONS OF SINGLE-STRANDED-DNA IN HUMAN-CELLS
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DOI：
10.1038/280420a0
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发表时间：
1979-01-01
期刊：
影响因子：
64.8
通讯作者：
LINDAHL, T
中科院分区：
文献类型：
--
作者：
BJURSELL, G;GUSSANDER, E;LINDAHL, T
当通过各种标准程序从活跃生长的动物细胞中提取DNA时，1-2%的总DNA被回收为单链形式1-5。脉冲标记实验已经证实，大部分单链DNA不是新合成的3-5。然而，这种DNA片段的一小部分由分离6-7期间分支迁移释放的新复制的DNA的短片段组成。对同步的人类细胞的研究表明，单链DNA主要在DNA合成3-4的过程中被发现。与此相一致，一种用于免疫荧光的变性DNA的特异性抗血清显示，在人淋巴样细胞系的细胞核中，在S期存在单链DNA，而在G1期则不存在。DNA杂交测量表明，单链序列属于DNA的非重复部分，并且至少部分材料包含在vivo2中转录的序列。我们已经开发了温和的分离方法来从EB病毒(EBV)9转化的人淋巴细胞中纯化大的、共价闭合的环状DNA分子。这些方法不会导致线性双链DNA转化为单链形式5、9。我们反复在电子显微镜下观察到宿主DNA的长单链延伸，最初与富含鸟嘌呤胞嘧啶的病毒上体DNA共纯化。在Burkitt淋巴瘤患者和健康供者的几个不同的淋巴样细胞系中，这种单链DNA约占细胞总DNA的1%，而同一株系中的EBV DNA序列仅占总DNA的0.02-0.1%。显然，单链序列主要或完全来自宿主。在两个不同来源的EBV阴性的人淋巴细胞系BJAB10和Molt-4中也发现了与EBV转化细胞中难以区分的单链DNA长区域。11)。这份报告着眼于这些单链DNA分子，它们比之前从动物细胞中分离出的任何分子都要大得多。
WHEN DNA is isolated from actively growing animal cells by a variety of standard procedures, 1–2% of the total DNA is recovered in single-stranded form1–5. Pulse labelling experiments have established that the large majority of the single-stranded DNA is not newly synthesised3–5. However, a minor part of this DNA fraction consists of short fragments of newly replicated DNA released by branch migration during isolation6–7. Studies with synchronised human cells have shown that single-stranded DNA is mainly found during the period of DNA synthesis3,4. In agreement with this, a specific antiserum against denatured DNA used for immunofluorescence has revealed the presence of single-stranded DNA in nuclei of a human lymphoid cell line during the S phase but not during the G1 phase8. DNA hybridisation measurements indicate that the single-stranded sequences belong to the non-repetitious fraction of the DNA and that at least part of this material contains sequences transcribedin vivo2. We have developed gentle isolation methods to purify large, covalently closed circular DNA molecules from human lymphocytes transformed by the Epstein–Barr virus (EBV)9. These methods do not cause the conversion of linear double-stranded DNA to single-stranded forms5,9. We have repeatedly observed by electron microscopy long single-stranded stretches of host DNA that initially co-purified with the guanine–cytosine-rich viral episomal DNA. This single-stranded DNA accounted for about 1% of the total cellular DNA in several different lymphoid cell lines derived from Burkitt lymphoma patients or from healthy donors, while the EBV DNA sequences in the same lines only constituted 0.02–0.1% of the total DNA. Clearly, the single-stranded sequences were mainly or exclusively of host origin. Long regions of single-stranded DNA, indistinguishable from those present in EBV-transformed cells, were also found in similar amounts in two EBV-negative human lymphoid cell lines of different origin, BJAB10and Molt-4 (ref. 11). This report looks at these single-stranded DNA molecules, which are considerably larger than any previously isolated from animal cells.