自噬基因APG5基因结构的生物信息学分析

Bioinformatics Analysis of Autophagy 5 Gene Structure

利用生物信息学手段对一种与人类细胞凋亡有关的基因———自噬基因 (Autophagy 5 ,简称APG5 )的基因组全序列进行拼接和基因结构分析 ,同时该基因的一个新的可变性剪切变异体 (APG5 β)被首次证实及报道。利用PCR技术从人胚脑cDNA文库和B细胞cDNA文库中调取APG5基因 ,装入绿色荧光蛋白pEGFP C1表达载体 ,测序确定其核苷酸序列 ,进行数据库搜索。该基因组全序列分散在核酸序列数据库GenBank的几个不同序列条目中。将相关克隆的基因组序列做相似性比对 ,用DOTPLOT方法确定其相互位置关系 ,并进行序列拼接 ,得到全长基因。利用GenScan、Genefinder等基因识别软件确定其内含子、外显子、转录起始位点、加尾信号等 ,分析其基因结构 ,在此基础上进一步分析其cDNA中外显子数目和排列顺序 ,证实了从B细胞cDNA文库中克隆所得的APG5为一种可变性剪切变异体。利用生物信息学工具 ,成功地拼接出全长为 1 5 0kb人类APG5基因基因组全序列 ,确定其有 8个外显子。根据剪切位点的特性找出其在序列中的位置 ,分别计算出内含子、外显子的长度 ,首次证实并报道APG5 β是第 3外显子缺失的可变性剪切变异体。将验证确实的APG5 β转染至人肝细胞株和HeLa细胞株 ,在共聚焦激光显微镜下可观察到其在细胞凋亡时的特异表达。

In order to study the relationship between autophagy and ap optosis,APG5 gene structure was revealed by bioinformatics analy sis and mean time a new isoform resulted from alternative splicing of the hAPG5 gene was confirmed,which was hereby designated as human autophagy 5β(hAPG5 β;LOCUS AF293841,GenBank). We cloned and sequenced the cDNAs from fetal brain and B cel l cDNA libraries using the known hAPG5 cDNA open reading frame seque nces as prim ers. The cDNA obtained from the human fetal brain cDNA library was identical to the known hAPG5 cDNA. However, the cDNA from adult brain cDNA li brary was 129bp s horter in length,lacking the sequence corresponding to those from positions 434 to 563 of the hAPG5 cDNA. Through search in public database and sequ ence compari sons and assembly 4 related sequences,APG5 genomic sequence was obtained. W e found that the hAPG5 gene had 8 exons,and those sequences miss ing in hAPG5 β cDNA exactly corresponded to exon 3.By bioinformatics software,the struct ure of introns,exons,splicing sites,promoter and polyA were demonstrated. Moreov e r,we were able to express both hAGP5 and 5β cDNA clones in human hepatocyte s and HeLa cells using pEGFP-C1 vector. In conclusion,our data indicate that a systematic bioinformatics method of finding protein diversity from alternative s plicing is a good approach in post-genome biology.