Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.
Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.
复制标题
大型动物病毒基因组上基因的精确定位：使用 lambda gt11 和单克隆抗体绘制巨细胞病毒蛋白家族的基因图谱。
DOI：
10.1073/pnas.82.4.1266
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发表时间：
1985
影响因子：
11.1
通讯作者：
Michael,N
中科院分区：
文献类型：
--
作者：
Mocarski,ES;Pereira,L;Michael,N
我们描述了一种有效的程序，它使用针对特定病毒蛋白的单克隆抗体，用于大DNA病毒基因组上基因的精确定位。我们已经使用的技术来定位的基因编码的抗原相关的DNA结合蛋白的240-内切酶对人类巨细胞病毒（CMV）基因组上的家庭。使用原核载体λ gt 11产生CMV DNA片段的随机文库，该载体在感染的大肠杆菌中表达开放阅读框作为β-半乳糖苷酶融合蛋白。用针对感兴趣的基因产物的单克隆抗体的混合物筛选文库。通过使用来自免疫反应性λ gt 11的DNA作为探针，将感染细胞蛋白36（ICP 36）的编码序列定位于CMV（Towne）和CMV（AD 169）基因组上的2800个碱基对的EcoRI片段（图谱坐标0.228-0.240）。在CMV生长周期的早期和晚期检测到来自该区域的5000个核苷酸的转录物。当在体外进行杂交选择和翻译时，该转录物指导ICP 36家族的主要成员的合成。用初始定位中使用的相同单克隆抗体对体外翻译产物进行免疫沉淀，证实了ICP 36基因的位置。这些研究确立了λ gt 11表达系统用于CMV基因（或其它大型动物病毒基因）的快速和精确定位的实用性，所述CMV基因编码存在血清学试剂的蛋白质。
We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.