Dissection of a replication origin of Xenopus DNA.
Dissection of a replication origin of Xenopus DNA.
复制标题
非洲爪蟾 DNA 复制起点的解剖。
DOI：
10.1073/pnas.79.18.5572
复制
发表时间：
1982
影响因子：
11.1
通讯作者：
Taylor,JH
中科院分区：
文献类型：
--
作者：
Chambers,JC;Watanabe,S;Taylor,JH
将先前克隆的用于增强其载体质粒复制的非洲爪蟾基因组DNA的503个碱基对（bp）EcoRI片段移至pBR 322的EcoRI位点。将该质粒命名为pJCC 31和通过切割与分散的重复序列相关的503-bp片段和亚克隆而制备的5个其它克隆与pBR 322进行比较，以通过显微注射到非洲爪蟾卵中进行复制。通过掺入32 P标记的核苷酸以及在EcoRI位点处N6-甲基腺嘌呤的保守分离和稀释测量的复制显示pJCC 31的效率是pBR 322的约15倍。下一个最有效的亚克隆，pJCC 31 -2，包含一个完整的320-bp的分散重复序列的插入，由一个8-bp的直接重复括号。这一观察结果，沿着我们先前的报告，即人类基因组中Alu家族的重复序列增强载体质粒的复制几乎与假定的非洲爪蟾起源一样多，导致脊椎动物中短分散重复序列子集的成员作为染色体复制的起源的假设。初步研究还表明，假定的非洲爪蟾起源含有RNA聚合酶启动子，当它被显微注射到非洲爪蟾卵母细胞中时，该启动子增加了质粒的转录。
A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322. This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs. The replication measured by incorporation of a 32P-labeled nucleotide as well as semiconservative segregation and dilution of N6-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322. The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat. This observation, along with our previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication. Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.