人肺腺癌SPC-A-1细胞系mtDNA限制酶切分析

RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA FROM HUMAN ADENOCARCINOMA CELL LINE SPC-A-1

为了探讨线粒体ＤＮＡ（ｍｔＤＮＡ）在细胞癌变中的作用，实验采用一步法快速制备癌细胞系ｍｔＤＮＡ，用ＰｖｕⅡ、ＸｈｏⅠ、ＰｓｔⅠ、ＥｃｏＲⅠ、ＢｓｔＥⅡ、ＨｉｎｄⅢ、ＨｐａⅠ、ＢｃｌⅠ、ＥｃｏＲⅤ、ＳｃａⅠ和ＸｂａⅠ共１１种限制性内切酶对ＳＰＣ－Ａ－１细胞系的ｍｔＤＮＡ进行了限制性片段长度多态性（ＲＦＬＰ）分析，并根据单酶切及双酶切结果，构建了ＳＰＣ－Ａ－１细胞系ｍｔＤＮＡ限制性酶切图谱。结果发现，ＳＰＣ－Ａ－１细胞ｍｔＤＮＡ基因编码区的３２个酶切位点均无变异，仅在其ｍｔＤＮＡ非编码区第１６２７６位核苷酸处出现了ＥｃｏＲⅤ新的酶切位点。结果表明ＳＰＣ－Ａ－１细胞ｍｔＤＮＡ基因编码区结构相当稳定。

To understand the role of mitochondrial DNA(mtDNA) in carcinogenesis, Single step Method was used to isolate the mtDNA from human adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism(RFLP) with 11 kinds of restriction endonuclease, which were PvuⅡ,XhoⅠ,PstⅠ EcoRⅠ,BstEⅡ,HindⅢ,HpaⅠ,BclⅠ,EcoRⅤ,ScaⅠand XbaⅠ. Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double digestionn method. It was found that any variation at 32 restriction sites failed to be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276(EcoRV) within the noncoding region. These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly conserved. Whicle the major variation of nucleotide is probably located in the noncoding region.