Comparison Of ARS Element Forming Nucleosome On Saccharomyces Cerevisiae Ⅲ Chromosome In Vitro

The regulation of DNA replication initiation sites is the key of the replicationregulation in eukaryotes. It is necessary for DNA replication and cell division. DNAreplication initiation is result for cooperation of protein factor and the replicator. It is notclear that how eukaryotes choose the replicator and how nucleosome positioning regulatesDNA replication initiation. The study included cell synchronization, exploration ofreplication activity of the autonomously replicating sequence301and305and comparisonof the autonomously replicating sequence forming nucleosome on Saccharomycescerevisiae Ⅲ chromosome in vitro. The details were as follows:1. The alpha factor can arrest MAT-a cells in G1phase of the cell cycle. We addedAlpha factor25~200μg mL-1for0h to5h in culture of Saccharomyces cerevisiae YKN-10, observed cellular morphology under fluorescent inverted microscope per0.5h andrecorded the budding cell rate, then analyzed the budding rate of yeast cells with SPSS17.0software. The results showed that the average budding rate of yeast cells issignificantly affected by the concentration、control time of alpha factor and the interactiveeffect of them. At the same time, the synchronization state was mainly achieved whenyeast cells were suffering from alpha factor175~200μg mL-1for3.5h to4h in culture.We successfully got the synchronized cells of Saccharomyces cerevisiae YKN-10withalpha factor synchronization method. This experiment provided reference for non Δbar1mutant strain synchronization.2. The activity of the autonomously replicating sequence301and305was exploredwith the method of BrdU-labeled nascent DNA. First, we obtained the synchronized cells;second, the nascent DNA was labeled with BrdU in5min,10min,15min and20min;third, DNA was extracted by using the method of glass bead beating; fourth, the nascentDNA was isolated by immunoprecipitation and purification; fifth, information of DNAreplication initiation was analyzed by using microscope and PCR.The results showed thatthe autonomously replicating sequence301was inactive while the autonomously replicating sequence305was highly active; the length of BrdU-labeled nascent DNA wasabout300bp and450bp in5min and10min. The acitivity identification of theautonomously replicating sequence laid the foundation for choice mechanism of thereplicationsite.3. Ten the autonomously replicating sequences of Ⅲ chromosome were divided intoefficient and inactivity group. The autonomously replicating sequence and histone wereassembled into nucleosome and detected with Biotin labeling method in vitro. Then theability of forming nucleosome was analyzed with Image J software. The results indicatedthe efficient autonomously replicating sequence was more likely to form nucleosome thanthe inactive autonomously replicating sequence. It has important biological significance tounderstand the mechanism of DNA replication initiation.