Manavathu E K, Suryanarayana K, Hasnain S E, Leung M, Lau Y F, Leung W C
Department of Medicine, University of Alberta, Edmonton, Canada.
Gene. 1987;57(1):53-9. doi: 10.1016/0378-1119(87)90176-4.
We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.
我们构建了一种质粒载体pSV2neo-MKαG，其中单纯疱疹病毒胸苷激酶（HSV-TK）的结构tk基因位于金属硫蛋白-I启动子的下游。该载体还包含选择标记氨基糖苷3'-磷酸转移酶（Km）。此载体能够转化丝状真菌两性绵霉，并获得了对G-418有抗性的菌落。Southern印迹分析显示，在转化体的基因组DNA中存在多条与HSV tk基因探针杂交的条带。通过凝胶电泳分析，其中一个转化体表现出与HSV-TK电泳迁移率相似的TK活性。在该转化体中还观察到[3H]胸苷摄取并掺入细胞DNA的量增加了约40%。这项研究表明，HSV tk基因可以在两性绵霉中表达，两性绵霉可被视为进一步进行基因表达研究的候选宿主细胞。
