Wang C C, Yeh L S, Karam J D
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70115, USA.
J Biol Chem. 1995 Nov 3;270(44):26558-64. doi: 10.1074/jbc.270.44.26558.
We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated froms of gp43 that, although inactive for replication, can still exhibit biological specificity.
我们描述了一种系统发育方法的应用，用于分析噬菌体T4的单链（898个氨基酸）多功能DNA聚合酶的模块组织。我们鉴定了噬菌体RB69（T4的远亲）的DNA聚合酶基因（基因43），将其克隆到表达载体中并进行了测序。推导的RB69蛋白（RB69 gp43）的一级结构与T4 gp43的不同，在短序列的离散簇中存在差异，这些短序列簇与两种蛋白之间高度相似的簇相互穿插。尽管存在这些差异，但两种酶在噬菌体DNA复制中可以相互替代，不过T4 gp43确实对其自身基因组表现出偏好。T4和RB69之间序列差异较大的55个氨基酸的gp43内部片段，在RB69 gp43中可以被T4的相应片段取代，而不会丧失复制功能。T4 gp43片段的反向嵌合体和缺失突变体对复制均无活性，且对T4野生型等位基因具有特异性抑制作用（“显性致死”）。结果表明，系统发育标记可用于构建gp43的嵌合体和截短形式，这些形式虽然对复制无活性，但仍可表现出生物学特异性。
