The Impact Of Flanking Sequence Of Saccharomyces Cerevisiae ARS Replication Activity

During the process in the proliferation of the cells, the DNA replication of cells’ intervalis most important which can be influenced by many factors. Among these factors thereplication of starting point is particularly important. Its abnormal may led to a variety ofdiseases such as cancer. Yeast is a kind of model organism, which has the characteristic ofboth procaryotes and eukaryotes. And it can be used as good experiment material in theresearch of start site replication. The start site of yeast replication called autonomouslyreplication sequence (ARS). There are17ARS in the yeast’s chromosome III, which hasdifferent frequency of using. Some thing can be found though high-throughput nucleosomepositioning that the nucleosome distribution on the ARS flanking is different. This showsthat both sides of the ARS sequence may affect the activity of the ARS.By PCR (amplification) technique, this experiment amplified ARS304, ARS305andits flanking sequence on yeast chromosome III. And constructed recombinant plasmidpARS304,pARS304-500,pARS304-2000based on the integrating vector pRS405. In orderto identify the influence of ARS304and ARS305’s different flanking sequence on theself-replication of saccharomyces cerevisiae, transformed the constructed recombinants andthe plasmid pARS305,pARS305-500,pARS305-2000which keep saving in the lab intosaccharomyces cerevisiae cells though electroporation. Competent yeast cell is a kind ofdeficient-cell. Although plasmid pRS405contains labeled leucine, it must be expressed byintergrating into the yeast genome. But because the plasmid of ARS sequence can free fromgenome and express the synthesis of leucine idenpendently, so the transformation efficiencycan be calculated by verifying its transformant number. During this process, doingoptimization of technical condition for electroporation and influencing the electroporation byusing uniform design with electric field intensity, exogenous DNA quantity and yeast growthstatus. When electric field intensity was set in1750V, the OD of yeast is1.3and theexogenous DNA quantity is600ng, the transformation efficiency is highest. Determine theloss rate of plasmid under this condition. The result of the calculating is that when the transformation efficiency ofpARS304is389-412transformant/μg, the loss of plasmid’severy generation is16.2%, when the transformation efficiency ofpARS305is523-551transformant/μg, the loss of plasmid’s every generation is11.2%, when the transformationefficiency ofpARS304-500is476-497transformant/μg, the loss of plasmid’s everygeneration is14.7%, when the transformation efficiency ofpARS305-500is723-734transformant/μg, the loss of plasmid’s every generation is9.01%, when the transformationefficiency ofpARS304-2000is623-632transformant/μg, the loss of plasmid’s everygeneration is9.35%, when the transformation efficiency ofpARS305-2000is929-939transformant/μg, the loss of plasmid’s every generation is8.01%. This result explained thatall of the constructed recombinant plasmids stay instability in yeast and all of them can doautonomously replicating. Moreover ARS305and its flanking sequence have highertransformation efficiency than ARS304. And its autonomously replicating activity will behigher along with the increasing of its flanking sequence.The follow-up of this experiment will do with thepARS-500,pARS-1000, andpARS-2000. Package the recombinant plasmid with nucleosome, and research the fluency ofnucleosome distribution on the recombinant plasmid and the nucleosome positioning to thereplication starting activity. This will provide useful clue for the mechanism of researchingthe eukaryotic gene’s replication and transcription by using yeast as model organism.