A SIGNATURE ELEMENT DISTINGUISHES SIBLING AND INDEPENDENT MUTATIONS IN A SHUTTLE VECTOR PLASMID
A SIGNATURE ELEMENT DISTINGUISHES SIBLING AND INDEPENDENT MUTATIONS IN A SHUTTLE VECTOR PLASMID
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DOI：
10.1016/0378-1119(92)90482-5
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发表时间：
1992-08-01
期刊：
影响因子：
3.5
通讯作者：
SEIDMAN, MM
中科院分区：
文献类型：
--
作者：
PARRIS, CN;SEIDMAN, MM
我们开发了一种新的穿梭载体质粒，用于研究哺乳动物细胞中的诱变，可以证明相同突变的独立性。基因中某些位点的突变发生频率高于其他位点，并且在哺乳动物细胞单次转染的突变质粒集合中，相同的突变可能会出现多次。然而，那些由独立事件引起的事件无法与初始事件的兄弟事件区分开来。新的载体系统（pSP189）是一群质粒，每个质粒都包含一个 8 bp 的“特征序列”。该序列赋予每个质粒独特的识别标签，并允许通过独特的签名来识别各个成员。该质粒还携带大肠杆菌细菌supF基因作为诱变标记，以及支持在灵长类（包括人类）细胞和大肠杆菌中复制的序列。我们使用 pSP189 系统在人类 DNA 修复缺陷细胞（着色性干皮病，互补 A 组）的单板中复制后，在 suF 中产生紫外线诱导的突变谱。利用特征序列，我们能够确定转染产生的相同突变是独立起源还是同源起源。最强热点处有八个相同的突变，所有突变都有不同的特征序列。在之前的实验中只报告了其中一个事件。该质粒减少了产生由 DNA 损伤剂引起的一系列突变所需的工作，并允许更准确地评估突变热点强度。
We have developed a new shuttle vector plasmid for studying mutagenesis in mammalian cells that permits proof of independence of identical mutations. Mutations occur more frequently at some sites in a gene than in others, and in a collection of mutant plasmids from a single transfection of mammalian cells the same mutation may appear several times. However, those arising from independent events cannot be distinguished from siblings of an initial event. The new vector system (pSP189) is a population of plasmids, each of which contains an 8-bp `signature sequence'. This sequence confers a unique identification tag to each plasmid and allows individual members to be identified by a distinctive signature. The plasmid also carries the Escherichia coli bacterial supF gene as a marker for mutagenesis, as well as sequences which support replication in primate (including human) cells and E. coli. We have used the pSP189 system to generate a UV-induced spectrum of mutations in supF following replication in a single plate of human DNA-repair-deficient cells (xeroderma pigmentosum, complementation group A). With the signature sequence, we were able to determine whether identical mutations derived from the transfection were of independent or sibling origin. There were eight identical mutations at the strongest hotspot, all of which had different signature sequences. Only one of these events would have been reported in previous experiments. This plasmid reduces the effort required to generate a spectrum of mutations caused by a DNA-damaging agent and allows a more accurate assessment of mutational hotspot intensity.