用DNA分子杂交研究两种人体胸苷激酶基因间的同源性

本实验从人体细胞质胸苷激酶(TK-C)基因分离出不含Alu重复顺序的3个DNA片段,分别次级克隆在质粒pBR322上,定名为pRR0.92,pXR1.5和pHK1.25。用pXR1.5和pHK1.25为探针,作Southern印迹杂交分析,都不能与小鼠细胞Ltk、CD-1、A9和BALB/c的DNA杂交。但是pXR1.5能与中国仓鼠细胞E36 DNA 23kb Bam HI片段杂交,出现信号很弱的杂交带.这提示以TK-C基因来说,人同中国仓鼠间的同源程度似大于人同小鼠的同源程度。在降低杂交严紧度的条件下,pHK1.25能与含人体16号染色体而不含17号染色体的人鼠杂种细胞DNA杂交,只是杂交信号极弱。我们推测人体TK-C基因(位于17号染色体)与人体TK-M基因(位于16号染色体)可能有某种程度的同源性,pHK1.25对进一步克隆TK-M基因也许是有用的。

Study on Homology Between Two Kinds of Human Thymidine Kinase Gene By Southern Blot Analysis

In this paper,we described the investigation of the possible homology between human cy-tosolic thymidine kinase (TK-C) gene (mapped to chromosome 17) and mitochondrial thymi-dine kinase (TK-M) gene (mapped to chromosome 16).Three Alu repetitive sequence-free fragments were isolated from human TK-C gene and then subcloned into plasmid pBR322.These recombinant plasmids were designated as pRR0.92 (Apt Tct),pXR15,(Apr Tcs) and pHK1.25 (Apr Tcs).Results of Southern blot hybridization showed that DNAs isolated from mouse cell lines Ltk,CD-1,A9 and BALB/c can not be hybridized to 33P-labelled,nick translated pXR1.5 and pHK1.25,but Chinese hamster cell line E36 DNA showed a 23 kb fragment after Bamlil digestion and then probing with pXR1.5.It suggested that there may be more homology between human beings and Chinese hamster in TK-C gene than that between human' beings and mouse.Under less stringent condition,DNA samples of human cells and mouse-human hybrid cell lines 16-17+(on human chromosome 17 only),16+17-(on human chromosome 16 only) and 16-17-(having no human chromosomes 16 and 17) were digested with HindIII and hybrized to the pHK1.25.Prominent bands were shown in the lanes of human DNA and 16-17+DNA,but nothing can be seen in the lane of 16-17-DNA.However,we can detect a faint band with a molecule size of about 7.5kb in the lane of 16+17-DNA.Thus,we inferred that there may be some homology existing between human TK-C gene and TK-M gene,and TK-C gene HindIII/KpnI 1.25kb fragment may be helpful for further investigation,of TK-M gene.