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An Attempt to Clone Amplified Genes which are Overexpressed

克隆过度表达的扩增基因的尝试

基本信息

批准号：
05454179
负责人：
FUKUMOTO Manabu
金额：
$ 4.03万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454179/
关键词：
Esophageal Carcinoma Gene amplification Human Chromosome 11q13 Cyclin D1 RT-PCR Gene expression 染色体11g13

项目摘要

Gene amplification was analyzed in human esophageal carcinoma cell lines. c-myc gene amplification was significantly more detected in well-differentiated type compared with other types and was accompanied with amplification of other genes. A cell line with cyclin D1 (CCND1) gene amplification without its expression was found and they showed hyperexpression of the CCND2 gene. These suggest that distinctively different genetic changes contribute to histologically different subtypes of esophageal carcinomas, that CCND1 gene is not necessary a target gene of chromosome 11q13 amplification and that CCND family genes contribute to esophageal carcinogenesis complimentarily each other. In order to clone amplified gene we tried a novel method which is combination of In-gel renaturation and RT-PCR based cDNA library. Bias of amplified fragments during PCR amplification interferes magnitudes of each gene expression. However, we established quantification of gene expression by RT-PCR.The promoter region of the amplified CCND1 gene was compared between cells with CCND1gene expression and those without expression. So far no difference was found in 1.3 kbp region, however, structure of amplicon is complicated and is thought to be composed of tandem repeat of amplified units of which structures are alike but not similar.A modified version of In-gel renaturation method was developed to clone amplified sequences. This method allowed us to clone some repeated DNA sequences. Its refinements to clone amplified sequences are underway.

在人食管癌细胞系中分析基因扩增。c-myc基因扩增在高分化型中显著高于其他类型，并伴有其他基因的扩增。细胞周期蛋白D1（CCND 1）基因扩增而不表达的细胞系被发现，他们表现出CCND 2基因的高表达。这些结果表明，不同的遗传变异导致不同的食管癌组织学亚型，CCND 1基因不是染色体11 q13扩增的靶基因，CCND 1家族基因在食管癌发生中的作用是互补的。为了克隆扩增的基因，我们尝试了一种新的方法，即凝胶内复性和基于cDNA文库的RT-PCR相结合的方法。PCR扩增过程中扩增片段的偏倚干扰每个基因表达的幅度。然而，我们建立了定量的基因表达的RT-PCR，扩增CCND 1基因的启动子区进行比较与CCND 1基因表达的细胞和那些没有表达。在1.3 kbp的区域没有发现差异，但扩增子的结构比较复杂，可能是由多个结构相似但不相似的扩增单元串联而成。这种方法使我们能够克隆一些重复的DNA序列。克隆扩增序列的改进工作正在进行中。