| Tumorigenesis is cogently associated with the activation of telomerase. It was exhibited that the telomerase was expressed in 85-90% of human tumors, but not in the majority of normal tissues except for some stem cell ,lymphocyte and germline cells. The expression of telomerase was also identified in some precancerous lesions and its level would be increased with the malignant progression. There might be some relationship between telomerase activity and tumor size, differention, clinical stage and prognosis. Patients would have a poor prognosis if they suffered from tumors with high level of telomerase activity. It was indicated in the in vitro studies that both hTERT( which played a key role in the activation of telomerase)and human telomere RNA(hTR) composed basic core of telomerase. These subunits acted in concert to elongate telomeres by reading from the RNA template sequence carried by the RNA subunit and synthesizing a complementary DNA strand. hTERT mRNA, in contrast to other subunits of telomerase, exists only in immortal or tumor cells. If we could find additional telomere-associated proteins which interact with hTERT, it would be advantageous for us to elucidate the biological function 昦nd molecular mechanism of telomerase in immortal and/or tumor cells. This would establish a sound theoretical basis for the tumor genesis, aging and prevention. Therefore yeast two-hybrid method was employed in the study to screen cDNA libraries with hTERT as bait. The main results are as follows:1. Dropout mixed amino acids [tryptophan(Trp), leucine(Leu), histidine (His)] were employed to detect the nutritional requirement of yeast strains' Supported by the National Natural Science Foundation of China (No. 39980010)IVAH109, Y187. It was indicated that the gene phenotypes of AH109 and Y187 yeast strains were stable and the strains grew well in the full nutritional supplement(adenine supplemented YPD) but did not in the single dropout supplements(-Leu, -Trp and -His). When the single strain of AH109 and Y187 yeasts was streaked in the SDAHis agar plates with different concentrations of 3-AT (0, 2.5, 5, 7.5, 10, 12.5, 15 mM) and was cultured at 30癈 for 3-4 days. There was no growth of AH 109 and Y187 yeast strains in the culture, which indicated that there was no leak expression of His in AH109 and Y187 and that 3-AT was unnecessary in the screening of cDNA libraries. The plasmids of yeast two hybrid system ( pGBKT7,pGBKT7-53, pGBKT7-Lam, pGADT7, pGADT7-T, Pcl-1) were transformed into the E. coli DH5 a by calcium chlorate and identified by restriction endonuclease. This provide essential conditions for the screening of cDNA libraries.2. hTERT cDNA was directly cloned into the multiclone sites EcoRI, NotI of pGBKT7 vector of DNA binding domain (DNA-BD) by recombinant technique. The recombinant of human telomerase catalytic subunit bait fusion gene, which was named as pGBKT7-hTERT, was identified by restriction endonuclease EcoR I and Not I. pGBKT7-hTERT was transformed into yeast strain AH109 by the method of Li acetate and the transformed strains were streaked onto SDATrp plate and grew well after cultured at 30癈 for 2-4 days. This implied that pGBKT7-hTERT bait fusion protein had no toxic effects on AH 109. It was exhibited in the print analysis of P -galactosidase colony-lift filter assay that there was no autonomous activated reporter gene in pGBKT7-hTERT fusion protein. A specific band was identified in 140KD during electrophoresis by Western blot. This indicated that pGBKT7-hTERT fusion protein could be stably expressed in AH 109 yeast strain. The constructed pGBKT7-hTERT satisfactorily fulfilled the requirement of the screening of cDNA libraries.3. The human testicle cDNA library transformed strain Y187 viability wasdetermined to be 4.5 ~ 5X 107cfu/ml, which was coincided with the screening of cDNA libraries. The mating of pGBKT7-hTERT transformed strain AH 109 with human testicle cDNA library transformed strain Y187 was made by yeast mating method. And the t...
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