Ensinger M J, Rovinsky M
J Virol. 1983 Nov;48(2):419-28. doi: 10.1128/JVI.48.2.419-428.1983.
The physical map locations of 62 temperature-sensitive mutations of vaccinia virus WR have been determined by marker rescue experiments, using cloned HindIII fragments of wild-type DNA. Since vaccinia virus DNA is not infectious, marker rescue was performed by infecting monolayers of cells at the nonpermissive temperature with a low multiplicity of the mutant to be rescued and transfecting with calcium phosphate-precipitated recombinant DNA. Wild-type recombinants were measured by using either a direct plaque assay technique or a two-step procedure in which the final yield of virus from the transfected cells was assayed at the permissive and nonpermissive temperatures. Mutants that had been previously assigned to the same complementation-recombination group were rescued by the same HindIII fragment, with the exception of three mutants in one group that were rescued by either one of two adjacent fragments. A comparison between the genetic linkage map of the temperature-sensitive mutations in 30 mutants with their physical locations demonstrated that not only was the order of the genetic map correct but also recombination frequencies generally reflected actual physical distances.
利用野生型DNA的克隆HindIII片段，通过标记拯救实验确定了痘苗病毒WR的62个温度敏感突变的物理图谱位置。由于痘苗病毒DNA无感染性，因此通过在非允许温度下用低感染复数的待拯救突变体感染单层细胞并用磷酸钙沉淀的重组DNA进行转染来进行标记拯救。通过使用直接蚀斑测定技术或两步法来测量野生型重组体，其中在允许温度和非允许温度下测定转染细胞的最终病毒产量。先前被归为同一互补重组组的突变体由相同的HindIII片段拯救，但有一组中的三个突变体由两个相邻片段之一拯救。对30个突变体中温度敏感突变的遗传连锁图谱与其物理位置进行比较表明，不仅遗传图谱的顺序正确，而且重组频率通常反映了实际物理距离。
