Preliminary Study Of DNA Sequence-dependent Nucleosome Positioning In Saccharomyces Cerevisiae

Nucleosomes are the basic repeating units of eukaryotic chromatin. They not only playa basic structural role, but also participate in the regulation of gene expression proeessesthrough positioning in the genome and their chemical modification of the histone complex,such as DNA replication, transcription and repair. Nucleosome positioning is the importantlevel of epigenetic regulation of gene expression. Studies have shown that nucleosomepositioning is mainly affected by two factors, one of which is DNA sequence, and theother is the trans-acting factor.One of the most important mechanisms to regulate DNA replication in eukaryoticcellsis to regulate the initiation of the replication. Autonomously Replicating Sequence (ARS) ofSaccharomyces cerevisiae is the cis-acting sequence required for DNA replication initiation.ARS tends to lack of nucleosome positioning, but its activity is not entirely dependent onnucleosome location. It may be associated with the characteristics of its flankingsequence and nucleosome distribution. Nucleosome assembly method in vitro and DNAmarker technology were used to investigate the effect of the sequence characteristics onnucleosome positioning and replication activity of ARS in S. cerevisiae. The maincontents are summarized as follows:1. The growth curve of S. cerevisiae YPH499was determined. YPH499was grown tologarithm phase during12-22h in YPD medium at30℃、200r/min, and the populationdoubling time in this period is2.008h.2. DNA was extracted with lysozyme method, snail enzyme treatment stayed over, snailenzyme repeated freezing and thawing and glass bead beating method. The result indicatedthat the genomic DNA could be obtained by all-above method, but the glass bead beatingmethod had the advantages of high quality, low cost, short time and simple operation.3. While the HSV-TK and hENT1gene were integrated into genome of S. cerevisiae,the genome DNA was labeled with BrdU for1h,3h and5h in the mid-logarithmic of S.cerevisiae and then stained by DAPI. The fluorescence signal intensity at3h was strongest,and about55.3%of the S. cerevisiae genomic DNA can be integrated with BrdU.4. Histones were extracted from untreated and starving yeast cells by Acid ExtractionMethod. The qualities of histones were analyzed by SDS-PAGE electrophoresis andBradford method. The results showed that this method had the ability to extract histones withhigh purity and concentration, but containing other proteins. The concentrations of proteins extracted from different time starving treated were lower, and the concentration wasgradually decreased while the time of starving extend.5. After a gradient salt dialysis method was employed to assembly nucleosome,nucleosome reconstituted using601sequence, ARS304and ARS305were analyzed by EBstain and biotin labeling methods. The results indicated that the ability of nucleosomepositioning in ARS305is stronger than ARS304in the same situation.