人体胸苷激酶(TK)基因在共转染过程中结构改变的研究

用磷酸钙沉淀法,我们把带有人体TK基因片段的重组噬菌体DNA共转染小鼠Ltk~-细胞,得到TK~+转化细胞克隆。同时用HeLa细胞DNA转染Ltk~-细胞,得到第一代TK~+转化细胞,再进行第二轮、第三轮转染,得到第二代、第三代TK~+转化细胞。比较其转化效率,结果基因组DNA转化率大于基因两个片段的共转化率,更大于不加携带者DNA的共转化率。限制性内切酶消化各种TK~+转化细胞的DNA,与TK基因探针作Southern印迹杂交,结果表明两个TK基因片段共转染Ltk~-细胞时,它们可以在受体细胞里重建成一个具有完整功能的遗传单位,但在连接过程中结构可以发生改变。当用HeLa纽胞DNA转染Ltk~-细胞时,虽然连续三代转染,每一代TK~+转化细胞中人TK基因的结构未发现变化。但也不能排除基因结构改变的频率很低未能有效检出的可能性。

STUDIES ON THE STRUCTURAL ALTERATIONS OF HUMAN THYMIDINE KINASE GENE OCCURING IN THE PROCESS OF COTRANSFECTION

By using the method of calcium phosphate precipitation,we have transfected thymidine kinase (TK) deficient mouse recipient cell line Ltk with human TK gene fragment containing recombinant bacteriophage DNA,followed by selection for TK expresion.These phage DNAs are in pairwise combinations and divided into two groups-with or without carrier DNA during the process of cotransfection.We also transfected Ltk cells with total DNA from HeLa cells,and then used the DNA isolated from primary TK transformant thus resulted as donor DNA to produce secondary TK transfor-mant In the same way,tetiary TK transformants were obtained.Number of TK molecules needed to produce one TK transformant was used as an index of transformation efficiency.The order of trans-formation efficiency is as follow: transfection with genomic DNA containing intact target gene,cotransfection with two fragments of agene,cotransfection without carrier DNA.DNA was extracted from all of these TK transfor-mants and Southern hybridized to TK gene probes.The results showed that there is no common band among the TK transformants DNA generated by cotransfection of TK gene fragments with and without carrier DNA.It means that the gene fragments recombined as a functional unit expressing TK activity but the gene structure has been altered.However,no changes were detected in the structure of human TK gene in the genome of primary,secondary and tetiary TK~+ transformants by Southern blotting analysis.It seems that the fate of intact gene transfected in the recipient ells may be different from that of cotrans-fected gene fragments.