Dnm t3b cDNA及其在小鼠胚胎组织中选择剪接异构体的克隆

Cloning of Full-length Dnmt3b cDNA and Its Alternative Splicing Isoforms in Mouse Embryo

目的 克隆与小鼠胚胎发育相关的新的全长cDNA。方法 从小鼠胚胎cDNA文库中筛选出一个新的EST,以8～9d龄小鼠胚胎组织mRNA为模板进行RACE(rapidamplificationofcDNAends),结合PCR筛选cDNA文库,获得其全长cDNA序列后进行序列同源检索、结构分析和功能预测。根据整合后的cDNA两端序列设计引物进行RT-PCR,克隆PCR产物,并对产物进行全序列测定,验证整合序列的客观性。结果 (1)克隆的序列为小鼠DNAC5胞嘧啶特异的重新甲基转移酶(denovoDNAcytosine5′-specificmethyltransferase,Dnmt)基因cDNA,发现该基因比已报道的Dnmt3bcDNA序列在5′方向延伸了152bp,其起始密码子上游存在一个新的选择剪接区域。(3)从小鼠胚胎组织中克隆出8种Dnmt3bcDNA选择剪接异构体,并均已获得Genbank序列接收号。结论 获得了Dnmt3bcDNA的4kb全长序列,发现Dnmt3b基因上游存在新的5′选择剪接序列,可能通过选择剪接形成上游终止密码子的切换;Dnmt3b在小鼠胚胎发育过程中存在着复杂的选择剪接表达。

To clone the novel full length cDNA related to mouse embryonic development. Methods Starting from a new expression sequence (clone 2#) out of a mouse embryonic cDNA library, its full length cDNA were obtained by rapid amplification of cDNA ends with polyA RNA isolated from mouse 8～9 day embryonic tissue as template, combined with screening of mouse embryonic cDNA library by PCR. Based on the sequences at the two ends of the cDNA, the primers for long range RT PCR were designed to confirm its factuality. Results The cDNA isolated in our hands was one of the alternative splicing isoforms of mouse de novo DNA cytosine 5′ specific methyltransferase gene (Dnmt3b) reported in July, 1998. A new alternative splicing region upstream of its initiate codon was found 5′ forwards 152 bp over the Dnmt3b cDNA sequence. Eight alternative splicing isoforms of Dnmt3b from mouse embryonic tissue were cloned (Genbank accession number are AF151969, AF151970, AF151971,AF151972,AF151973,AF151974,AF151975,AF151976 respectively).Conclusions The 4 kb full length Dnmt3b cDNA was isolated. Dnmt3b may have novel exons at its 5′ upstream and present a complex alternative splicing pattern during mouse embryonic development.