Analyses of the Relationship between Amino Acid Substitution and Phenotype of the Tail Sheath Protein of Bacteriophage T4

已经报道了5种具有18号基因突变的突变体表型，分别为琥珀色（am）、温度敏感（ts：生长在30 C，但不能生长在42 C）、冷敏感（cs：生长在37 C，但不能生长在25 C）、热不稳定或敏感（hs）和聚乙二醇不敏感（CBW）。我们从原作者那里收集了这些突变体，并确定了基因18上的突变位点。为了确定突变位点，采用非对称PCR法扩增突变基因18，并采用双脱氧法直接测序。已经鉴定出17个am、3个cs、3个ts、3个CBW和4个hs突变体的突变位点。琥珀色突变在分子的n端和c端都被定位。另一方面，被认为与亚基间相互作用有关的hs和cs突变被定位在分子c端一半的一个相当有限的区域，除了一个hs突变被定位在gp18分子的n端附近。Ts突变被定位在分子的n端一半。对于琥珀色突变体，使用大肠杆菌天然和人工抑制菌株在每个琥珀色位点插入一个氨基酸（Ser、Gln、Tyr、Ala、Cys、Glu、His、Phe、Pro或Arg），并检测其功能恢复及其温度依赖性。对于错义突变体（cs, ts， hs和CBW），重新研究了每种表型与氨基酸替代之间的关系。结果与我们的gp18的三级和三级结构模型一致，其中蛋白酶抗性结构域（残基数在80 ~ 320之间）构成尾鞘的突出部分，分子的n端部分（ca. 1 ~ 80）和c端部分（ca. 320 ~ 658）构成尾鞘的内部部分，与gp18的其他原聚体和管状结构接触。

Five phenotypes of mutants which have a mutation in gene 18 have been reported, namely amber (am), temperature sensitive (ts : grow at 30ﾟC, but not at 42ﾟC), cold sensitive (cs : grow at 37ﾟC, but not at 25ﾟC), heat labile or sensitive (hs) and polyethylene glycol insensitive (CBW). We collected these mutants from original authors and have identified the mutation sites on gene 18. In order to determine the mutation sites, the mutant gene 18 were amplified by the asymmetric PCR method and directly sequenced by the dideoxy method. Mutation sites of 17 am, 3 cs, 3 ts, 3 CBW and 4 hs mutants have thus been identified. Amber mutations were mapped both in N-terminal and in C-terminal half of the molecule. On the other hand, hs and cs mutations which are considered to be related to inter-subunit interactions were mapped in a rather restricted region in the C-terminal half of the molecule except for one hs mutant which was mapped close to the N-terminus of gp18 molecule. Ts mutations were mapped in the N-terminal half of the molecule. For amber mutants, E. coli natural and artificial suppressor strains were used to insert an amino acid (Ser, Gln, Tyr, Ala, Cys, Glu, His, Phe, Pro or Arg) into each amber site and recovery of the function and its temperature dependance were examined. For missense mutants (cs, ts, hs and CBW), the relationship between each phenotype and the amino acid replacement were reinvestigated. The results were all consistent with our model of the tertiary and quartenary structure of gp18, in which the protease-resistant domain (residue numbers between ca. 80 and 320) constitutes the protruding part of the tail sheath and the N-terminal part (ca. 1-80) and the C-terminal half of the molecule (ca. 320 - 658) constitutes the inner part of the tail sheath in contact with other protomers of gp18 and the tube structure.