Study On Promoter Function And Deletion Analysis Of DNA Fragment RM07 From Halobacterium Halobium

The DNA fragment RM07 was isolated from halophilic archaea Halobacterium halobium, which can function as promoter not only in halophilic archaea and yeast, but also in Escherichia coli as eubacterial promoter. Sequencing analysis indicated that this fragment possessed the typical consensus sequences (-35 and -10) of bacterial gene promoter. It was further confirmed by deletion analysis. By using the promoter-probe plasmid pKK232-8, recombinant plasmids which contained partially deleted RM07 fragments were constructed and chloramphenicol resistant levels of the tranformants were detected. Furthermore, the chloramphenicol acetyltransferase activity of these recombinants were also determined. The results indicated that the integrity of its -35 and -10 sequences is indispensable to the promoter function of RM07 fragment in Escherichia coli: DNA fragment RM07a, with its -35 sequence deleted and -10 sequence left, nearly cannot initiate transcription; With both its -35 and -10 sequences, RM07b fragment sized 0.1kb could be active as promoter at a level even higher than RM07, which implied that the upstream and downstream regions of RM07b fragment were unnecessary sequences, whose function may even be of inhibition to some extent. Our research also showed that the promoter activity of RM07 fragment in Escherichia coli was under the control of environmental factors, especially its positive correlation with the increasing concentration of sodium chloride.Sequencing analysis also revealed that DNA fragment RM07 possessed three consensus sequences of halphillic archaeal promoter DPE structures, which were similar to that of eukaryotic promoter. Therefore its promoter activity in CHO cell line(superior mammalian cell) was further determined. With its reporter gene egfp, eukaryotic expressing plasmid pEGFP-N1 was used as vector to constructrecombinant plasmids, whose CMV promoter upstream of gene egfp was replaced by RM07 fragment and deleted fragment RM07c respectively. CHO cell was transfected by recombinant plasmids and observed under a fluorescent microscope. The appearance of fluorescence in CHO cells proved that fragment RM07 and RM07c can initiate the transcription of their downstream gene egfp.Therefore, RM07 DNA fragment may be potential novel promoter source for constructing double-function vectors. It also has special significance in elucidating the issues of the fusing characteristics of archaea and lateral gene transfer between archaea and bacteria.