Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation / 生物工程学报

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

The Molecular Biologic Study about the Role of Deubiquitination Gene in Bladder Tumor Progression / 대한비뇨기과학회지

PURPOSE: Ubiqutin-proteasome-mediated proteolysis is an important pathway of nonlysosomal protein degradation, which controls the timed destruction of cellular regulatory proteins of the p21, p27, p53 gene (p53), Retinoblastoma gene (Rb), E2F-1, E2F-4 and cyclin etc. The deubiqutination (DUB) gene prevents ubiquitin-proteasome- mediated proteolysis by removal of ubiquitin from ubiqutinated proteins. We investigated the role of the DUB gene (DFFRY or DFFRX) in the progression of a bladder tumor associated with Rb and p53, which follows ubiquitin-proteasome-mediated proteolysis. MATERIALS AND METHODS: RT4 (bladder pailloma cell line), 5637 (superficial bladder transitional cell carcinoma cell line), and HT1376 (invasive bladder transitional cell carcinoma cell line) were cultured. mRNA was extracted from each cell line by the Poly-A tract mRNA isolation system. Relative quantitative RT-PCR (reverse transcription-polymerase chain reaction) for the DUB gene (DFFRY or DFFRX) was performed. Northern blotting for the DUB gene, p53 and Rb were performed. Proteins were harvested at 80% confluence from each cell line. Western blotting for the p53 and Rb were performed. RESULTS: The relative quantitative RT-PCR for the DUB gene showed it to be expressed most strongly in RT4, and most weakly in HT1376, which were identical to the findings of the Northern blotting. The p53 was expressed at similar levels on the Northern blots of all three cell lines, whereas on Western blotting, it was expressed most strongly in 5637, most weakly in RT4, and moderately in HT1376. The Rb was expressed only in RT4 on Northern and Western blotting. CONCLUSIONS: The DUB gene might be related to the inhibition of the progression of bladder tumors associated with Rb and p53, which follows ubiquitin-proteasome- mediated proteolysis.

EXPRESSION OF Rb AND p53 GENE IN GASTRIC CARCINOMAS IN RELATION TO PROGNOSIS / 中国癌症杂志

PURPOSE Expression of Rb and p53 gene in gastric carcinomas and its relationship to prognosis as well as clinico pathology were investigated.METHODS Expression of Rb and p53 products and p53 gene mutation in gastric carcinomas were analysed by means of immunohistochemistry and in situ hybridazation.RESULTS p53 gene mutation was found in 6/20(30%).Over expression of Rb and p53 products was found in 62/85(72.94%) and 42/85(49.41%).Both the positive grades showed significant inverse correlation with patient survival( P

Analysis of p53 and Retinoblasoma(Rb) Gene Polymorphisms in Relation to Lung Cancer in Koreans / 결핵

BACKGROUND: The p53 and retinoblastoma(Rb) tumor suppressor genes are associated with the pathogenesis of several types of human cancer. Substantial. proportion of the primary lung cancers or cell lines have been reported to have the p53 and/or the Rb gene mutations. But so far there is no report on the analysis of the Rb gene polymorphism as one of the genetic susceptibility marker. This study was undertaken to establish the gene frequencies of the polymorphic genotypes of the p53 and Rb genes in Koreans to evaluate the possible involvement of these genotypes as a risk factor of lung cancer. METHODS: In this study 145 controls without previous and present tumor history and 128 lung cancer patients were subjected to analysis The two intragenic polymorphisms of the p53 gene(exon 4/AccII, intron 6/MspI) and one intron 17/XbaI polymorphism of the Rb gene were analysed by the method of polymersae chain react lion-restriction fragment length polymorphisms(PCR-RFLPS). The genotype of the intron 3/16 bp repeat polymorphism of p53 was determined by PCR and direct gel electrophoresis. RESULTS: There were no significant differences in the genotype distributions of the p53 gene between lung cart cert patients and controls. But heterozygotes(Arg/Pro) of the exon 4/AcclII polymorphisms were slightly over-represented than controls, especially in the Kreyberg type I cancer, which was known 13 be associated with smoking. The intron 3/16 bp duplication and the intron 6/MspI polymorphisms were in complete linkage disequilibrium. About 95% of the individuals were homozygotes of the common alleles both in the 16 duplication and MspI polymorphisms, and no differences were deteced in the genotype distributions between lung cancer patients and controls. Overall genotype distributions of the Rb gene polymorphisms between lung cancer patients and controls were not significantly different However, the genotype distributions in the Kreyberg type I cancer were significantly different from those of controls(p=0.0297) or adenocarcinomas(p=0.0008). It was noticeable that 73.4% of the patients with adenocarcinomas were heterozygotes(r1/r2) whereas 39.2% of the Kreyberg type I cancer were heterozygous at this polymorphisms. In the lung cancer patients, significant differences were a]so noted between the high dose smokers and low dose smokers including non-smokers(p=0.0258). The relative risk to Kreyberg type I cancer was significantly reduced in the individuals with the genotype of r1/r2(odds ratio=0.46, 95%C.I.=0.25-0.86, p=0.0124). The combined genotype distribution of the exon 4/AccII of the p53 and the intron 17 Rb gene polymorphisms in Kreyberg type I cancers were significantly different from dose of controls or adenocarcinomas. The highest odds ratio were observed in the individuals with the genotypes of Arg/pro and r2/r2(odds ratio=1.97, 95%C.I.=0.84-4.59) and lowest one was in the patients with Arg/Arg, r1/r2 genotype(odds ratio=0.54, 95%C.I.=0.25-1.14). CONCLUSION: The p53 and the Rb gene polymorphisms modulate the risk of smoking induced lung cancer development in Koreans. However, the exact mechanism of risk modulation by these polymorphism remains to be determined. For more discrete clarification of associations between specific genotypes and lung cancer risk, the evaluations of these polymorphisms in other ethnics and more number of patients will be needed.

Inhibition of Foreign Retinoblastoma Gene Mediated by Recombinant Adenovirus Vector on Growth of Bladder Carcinoma Cells / 中国肿瘤生物治疗杂志

In order to study the bladder carcinoma cell growth suppression by introduction of foreign retinoblastoma (Rb) gene and explore a gene therapy approach for bladder cancer, a replication-deficient adenovirus vector encoding a wild-type Rb, AdCMVRb, was constructed and transfected into the cultured human bladder carcinoma cell line EJ. The efficiency of gene transfection and expression was detected by immunochemical staining, Western blotting and polymerase chain reaction. The role of Rb in suppressing EJ growth was observed by cell-counting, [3H]thymidine incorporation and flow cytometry. The results showed that wild-type Rb gene could be transfected effectively into cultured EJ with Ad-CMVRb and could arrest the cells at GO/Gl phases of the cell cycle, leading to inhibition of DNA synthesis. The results demonstrated the potential of adenovirus-mediated Rb gene therapy for bladder cancer.