Duda R L, Gingery M, Ishimoto L K, Eiserling F A
Department of Microbiology, University of California, Los Angeles 90024.
Virology. 1990 Dec;179(2):728-37. doi: 10.1016/0042-6822(90)90140-m.
A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.
一种用于研究噬菌体T4尾部组装的互补系统已被开发出来，并用于测试无活力突变对特定T4尾部蛋白gp48功能的影响。该互补系统可测定gp48的组装功能，而无需产生有活力的噬菌体，从而规避了维持这种裂解性噬菌体无活力突变体所带来的操作问题。待测试的蛋白在被挑战噬菌体感染之前，先从宿主细胞中的克隆基因预表达。通过监测一种尾部组装中间体（缺乏gp48的基板）转化为含有gp48的基板或由此类基板组装而成的管状基板（或带鞘尾部）来测定组装活性。使用gp48特异性抗血清确认了gp48在这些结构中的特异性掺入，并且在直接免疫电子显微镜实验中使用相同的血清将gp48定位到T4尾管靠近基板的一端，即尾管和尾鞘与基板结合的部位。先前已证明蛋白gp48是一种基板蛋白，也是一种与尾管相关的蛋白，并对其作为尾长卷尺蛋白的可能作用进行了测试。对gp48缺失变体的测试没有得出明确结论，因为该蛋白无活性。由于内部重复，一种比野生型蛋白长20%的gp48变体在我们的组装互补系统中被发现具有部分功能。这种异常延长的蛋白允许几个组装步骤进行，包括正常长度T4尾部的组装，这意味着它并不决定尾长。gp48的插入重复变体似乎在与尾鞘蛋白的相互作用方面存在缺陷，导致尾鞘异常收缩。
