| Objective: Occurrence of tumor results flvm activation of pm-oncogene and inactivation of tumor suppressor gene. Study of the oncogene and the tumor suppressor genes has great impoitance in exploring the mechanism of the tumorigenesis, and in finding the new ways for tumor pmphylaxis and therapy. The incidence of colorectal tumor is high among gastrointestinal trmtors, with easy to recurrence and metastasis. Up to now, the molecular mechanism of colorectal tumozigenesis is not very clear, many studies indicate that the colorectal tumor is associated with the change of a series of genetic events, such as overexpression of oncgene, mismatch repair of gene and tumor suppressor gene. In order to provide theoretical evidences in eaxiy diagnosis and gene therapy of the colorectal tumor, the aim of this study is to isolate mid clone the colorectal tumor related genes by differential display method which developed recently. Methods: Total RNAs were extracted fivm colorectal tumor and a4jacent noncancenms mucosa, separately. cDNA fragments were anqlified by PCR with arbitrary primers AP1-6, and anchor primers HTl 1CIA/G by differential display PCR The amplified PCR products were separated on 6% non denaturing polyacrylamide gel electn)phoresis, and autoradiographed with X-ray at -7CYC. The special eDNA bands for colorectal tumor were recovered from the polyactylane gel, and were reamplified using the corresponding primer sets, sub-cloned, sequenced, and canied out on RNA dot blotting. Results: More than ten cDNA bands, which might represent the colorectal tumor differentially expressed genes, were obtained by DD-PCR method. Five of them were selected to reaniplification by PCR, and were cloned into the PUC18 plasmids or TA. The positive clones were identified by screening of blue-white dots and confimied by digestion with restriction endonuclease. The cloned eDNA fragments were sequenced When compared those parlial nucleotide s&piences with the sequences deposited in the GenBank data, one eDNA fragment showed more than 600/0 seqince homology to the RaM gene, one cDNA fragment showed more than 80% sequence homology to the tyn)sine protein kinase gene, two cDNA fragments dii not show any significant homology, and the last one cDNA figment showed that it was a small repeated sequence of transport osomal RNA. The eDNA fragment, which showed homology to Raf- 1 gene, was labeled as a probe Using the,probe, RNA dot blotting was done among the total RNAs which extracted from colorectal cells (HT-29). colorectal tumor and adjacent noncancerous mucosa , separately. The expression level of RNA was high u-i colorectal cells and colorectal tumor, but it was low in the colorectal adjacent noncancetous mucosa. We presume that the eDNA fragment might be a colorectal tumor related oncogene and named it CG-1. What we will do next is to predict the full-length mRNA by Northern blot analysis, to investigate the expression of the colorectal tumor in detail by in situ hybridization, to obtain the full-length eDNA sequences by the RACE amplification, and to derive the function by the amino-acid sequence. The similar experiment will be done for others differentially expressed eDNA fragments. Conclusion: More then ten eDNA fragments, which might be related colorectal tumor differentially expressed genes, are identified by the method of DD-PCR. One of those is furlher confirmed by RNA dot blotting and might be a new colorectal tumor related concogene.
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