DeFilippes F M
Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, MD.
Biotechniques. 1994 Jan;16(1):122-7.
The vaccinia virus genome encodes a DNA polymerase that is similar to other DNA polymerases. A mutation in the polymerase gene at a site that is adjacent to conserved residues allows viral replication in the presence of aphidicolin. Since wild-type virus is converted to aphidicolin-resistance by site-directed mutagenesis, it was feasible that active virus with substituted conserved residues could be detected by linking alterations to the aphidicolin-resistance mutation. Altered DNA, from a PCR, was introduced into virus by a marker transfer procedure. DNA from plaques of drug-resistant virus was amplified, and the product was sequenced to check for the conserved residue alteration. An alteration that introduced a Bg1I site was designed to facilitate the selection of drug-resistant virus containing substituted residues. One positive result was the replacement of two amino acids, tyrosine and alanine, by tryptophan and threonine. The failure to substitute aspartic acid for tyrosine indicates that drastic changes of the conserved sequence are not tolerated. Although the limitations associated with negative results apply, the method provides an in vivo assay for selecting a polymerase with conserved residue changes.
痘苗病毒基因组编码一种与其他DNA聚合酶相似的DNA聚合酶。聚合酶基因中与保守残基相邻位点的突变使得病毒能够在存在阿非科林的情况下进行复制。由于野生型病毒通过定点诱变转化为对阿非科林具有抗性，因此通过将改变与阿非科林抗性突变联系起来，检测具有取代保守残基的活性病毒是可行的。通过标记转移程序将来自PCR的改变后的DNA引入病毒。对耐药病毒噬菌斑的DNA进行扩增，并对产物进行测序以检查保守残基的改变。设计引入Bg1I位点的改变以促进对含有取代残基的耐药病毒的选择。一个阳性结果是酪氨酸和丙氨酸被色氨酸和苏氨酸取代。未能用天冬氨酸取代酪氨酸表明保守序列的剧烈变化是不被容忍的。尽管与阴性结果相关的局限性仍然存在，但该方法提供了一种在体内筛选具有保守残基变化的聚合酶的测定方法。
