McKnight S L, Gavis E R
Nucleic Acids Res. 1980 Dec 20;8(24):5931-48. doi: 10.1093/nar/8.24.5931.
When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene.
当用含有单纯疱疹病毒（HSV）胸苷激酶（tk）基因的重组质粒注射非洲爪蟾卵母细胞核时，可观察到tk酶活性增加100倍。三条证据表明tk活性的这种增加是HSV tk基因表达的结果。第一，酶活性被针对HSV tk蛋白产生的抗血清的IgG组分选择性地灭活。第二，在聚丙烯酰胺凝胶上与真实的HSV tk迁移率相同的一种多肽，是由注射了HSV tk基因的卵母细胞独特合成的。第三，在注射的卵母细胞中发现的诱导tk活性能够磷酸化脱氧胞苷，脱氧胞苷是HSV tk利用但细胞tk不利用的一种底物。我们利用这些观察结果建立了一种检测HSV tk基因突变体活性的方法。在体外构建了两组tk基因的缺失突变体。一组缺失不同量的5'侧翼序列和基因内序列。另一组在基因的3'端缺失。通过在卵母细胞注射试验中检测每个突变体的活性，我们划定了与HSV tk基因5'和3'末端相对应的功能边界。
