Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction.
Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction.
复制标题
病毒转导后 c-myc 5 侧翼 DNA 中的保守染色质结构。
DOI：
10.1016/0022-2836(91)90736-p
复制
发表时间：
1991
影响因子：
5.6
通讯作者：
Leffak,M
中科院分区：
文献类型：
--
作者：
Kumar,S;Leffak,M
研究了局部序列信息在建立人类c-mycupstream区域（MUR）染色质结构中的作用。腺相关病毒（AAV）介导的基因转导被用来引入一个额外的未重排拷贝的2·4 kbHindIII-XhoI片段的MUR到一个新的位置在基因组中的两个克隆的HeLa细胞系。用于扩增MUR的基于AAV的rep −cap−病毒载体SKMA仅保留了1.4 kb（24%）的AAV基因组，可容纳2.4 kb的插入片段。SKMA能够感染HeLa细胞并以单拷贝数整合到宿主基因组中。用间接末端标记法，在HeLa细胞中定位了转导的c-mycRNA和内源性c-mycloci上的DNase I和微球菌核酸酶（MNase）切割位点。在每个克隆中，在转导的c-mycocus中发现了一个类似有序的染色质结构域，从c-mycpromoter P0延伸到5′。13 MNase切割位点和5 DNase I超敏感位点，最初确定在非转导细胞中的内源性MUR的位置和相对灵敏度，被证明是保守的，当这个DNA被移动到一个新的染色体位点。一个保守的DNA酶I超敏感位点也被定位到左AAV末端重复序列和AAV启动子P5之间的区域。这些结果表明，所需的信息，以建立特定的染色质结构的MUR驻留在该地区的本地DNA序列。
The role of local sequence information in establishing the chromatin structure of the human c-mycupstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2·4 kbHindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-basedrep−cap−viral vector SKMA used to transduce the MUR retained only 1·4 kb (24%) of the AAV genome and could accommodate inserts as large as 2·4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19.Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-mycsequences and the endogenous c-mycloci in infected HeLa cells. A similarly ordered chromatin domain, extending 5′ from c-mycpromoter P0, was found to exist at the transduced c-myclocus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.