|Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
[Background] We identified a new genetic alteration in human hepatocellular carcinoma by using a high resolution technique, restriction landmark genomic scanning (RLGS). The aim of this study was to identify new genetic alterations in colorectal cancer using the same method.
[Methods] High molecular weight genomic DNAs were extracted from 25 colorectal cancerous tissues and corresponding normal mucosae. Each DNA was cleaved with the restriction enzyme Not I, size fractionated by 1st-dimensional electrophoresis using Pvu II, and the DNA fragments were then cleaved by Pst I as the third enzyme in gel and separated by 2-dimensional electrophoresis. By comparing each pair of RLGS profiles, we detected 6 common changed spots. The most frequently changed spot was directly cloned from the gel. Nineteen of 25 (76.0%) colorectal cancer cases showed a spot with the same alteration, which never appeared in normal mucosae. A 410-bp DNA fragment corresponding to this spot was cloned. This sequence was found to be 69% homologous to part of the human fatty acid synthase gene (FAS), and mapped to 95% of the 3rd chromosome of the human draft sequence. The three cloned spots (Spots B, C and F) were observed in 44%(11/25), 24% (6/25) and 28% (7/25) of the cases, respectively, and found to be 271-, 168-, and 374-bp DNA fragments. The DNA sequences from spots B, C and F were also homologous to the genomic contig of the first and the ninth chromosomes, and human Not I clones, respectively.
[Conclusion] Four spots were cloned, one of which was found to homologous human fatty acid synthase gene. Relations between these gene mutations and clinicopathological features will be studied in the future by making antibodies of these spots.