An Attempt to Clone Amplified Genes which are Overexpressed

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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.