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T4噬菌体基因组的分子克隆:尾丝基因簇34 - 38的组织与表达

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38.

作者信息

Revel H R

出版信息

Mol Gen Genet. 1981;182(3):445-55. doi: 10.1007/BF00293934.

DOI:10.1007/BF00293934
PMID:6457960
Abstract

lambda T4 derivatives that carry T4 tail fiber genes 34-38 have been isolated and characterized by genetic, structural and functional analysis. 32 lambda T4 recombinants were identified by a marker rescue screen of 310 lambda T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved lambda vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the lambda vectors. The cloned tail fiber genes are expressed efficiently from lambda promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34--gp38 have been detected by SDS polyacrylamide gel electrophoresis of 35S-labeled extracts of appropriate lambda T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34--38 and their mutant variants that affect the assembly, structure and function of the tail fibers.

摘要

携带T4尾丝基因34 - 38的λT4衍生物已通过遗传、结构和功能分析进行了分离和表征。通过对310个λT4克隆进行标记拯救筛选鉴定出32个λT4重组体,这些克隆是通过用HindIII或EcoRI对部分含胞嘧啶的T4 DNA进行酶切并连接到适当酶切的λ载体中产生的。这些测试根据回收的基因组连续片段定义了15个重组类别。限制性内切酶结构分析鉴定出7个HindIII片段和7个EcoRI片段,建立了覆盖约11 kb的限制性图谱,并表明了λ载体中DNA插入片段的方向。克隆的尾丝基因在λ启动子的作用下高效表达,并在体内对携带尾丝基因琥珀突变的T4噬菌体起到互补作用。通过对适当的λT4重组体感染经紫外线处理的宿主细胞的35S标记提取物进行SDS聚丙烯酰胺凝胶电泳,检测到了与gp34 - gp38相对应的多肽。T4尾丝基因簇的遗传、结构和功能图谱已相互关联,为对影响尾丝组装、结构和功能的基因34 - 38及其突变变体进行基因定向DNA序列分析提供了合理的方法。

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