Promoter Functional Study On Upstream Region Of Rad25 Gene From Halophilic Archaea In Three Domains Of Life

In this dissertation, DNA fragment designated RM10 was isolated from the chromosomal DNA of the halophilic archaea Halobacterium halobium by using the Escherichia coli promoter-probe vector pKK232-8 and was shown to confer promoter activity in Escherichia coli. sequence searches for homology in Genbank of NCBI revealed that RM10 contained the sequence of 5'upstream region of rad25 gene in Halobacterium salinarium. Sequence analysis revealed that RM10 fragment contained characteristic sequences similar to eukaryal and archaeal promoter-TATA box as well as the typical -35 and -10 box consensus sequences of bacterial promoter.The RM10-bgaH gene fusion plasmids were constructed and transformed into halophilic archaea Haloferax volcanii using the bgaH gene as the reporter genes, Through determining the enzymatic activity and RT-PCR analysis, it was confirmed that RM10 fragments conferred promoter activity in Haloferax volcanii. Deletion analysis of RM10 was performed in Haloferax volcanii, Through detecting the promoter activity of various deletion fragments of RM10, the important functional regions within RM10 which could influence the promoter activity were identified. The results revealed that the -305 to -130bp region of 5'upstream region of rad25 gene containing the typical TATA box and BRE sequences of archaeal promoter was the key region responsible for the basal promoter activity of RM10 in Haloferax volcanii.Using the cat gene as the reporter genes, the RM10-reporter gene fusion plasmid was constructed and transformed into Escherichia coli. Through detecting the antibiotic resistance level and RT-PCR analysis, it was confirmed that RM10 conferred promoter activity in Escherichia coli. Through deleting analysis of RM07 in Escherichia coli ,the important functional regions within RM10 which could influence the promoter activity were identified. The results revealed that the -305 to -250 region of 5'upstream region of rad25 containing the TATA â…  sequences was the important functional region responsible for the basal promoter activity of RM10 in Escherichia coli. The promoter functional region of Bacteria and archaeal were related.Using the neo,egfp and luc+ gene as the reporter genes, various RM10-reportergene fusion plasmids were constructed and transformed or transfeced into Saccharomyces cerevisiae and superior mammalian cell respectively. Through detecting the antibiotic resistance level, detecting EGFP fluorescence expression and determining the enzymatic activity, it was confirmed that RMIO conferred promoter activity in Saccharomyces cerevisiae and superior mammalian cell. Deletion analysis of RMIO was performed in Saccharomyces cerevisiae and superior mammalian cell. The results revealed that the -1767 to -889 region of 5'upstream region of rad25 gene containing characteristic sequences of TATA IV and GGCGG box was the functional region responsible for the strong promoter activity ,and the -889 to -403 region of 5'upstream region of rad25 gene containing characteristic sequences of TATAIII was the functional region responsible for the basal promoter activity of RMIO in Saccharomyces cerevisiae. In superior mammalian cell, the -889 to +1 region of 5'upstream region of rad25 gene containing characteristic sequences of TATAIII, TATA II and TATA I was the functional region responsible for the basal promoter activity and these functional region were related in Saccharomyces cerevisiae and superior mammalian cell.An efficient, quantitative, inexpensive and versatile microcalorimetry was successfully applied in studying the structure and function of promoter in Saccharomyces cerevisiae and Escherichia coli.