The Theoretical Prediction Of Nucleosome Positioning In Yeast And The Preliminary Study On Nucleosome Reconstitution In Vitro

The genomic DNA in eukaryotic cell is packaged into nucleosomes that are the basic repeating units of eukaryotic chromatin. Nucleosomes not only play a basic structural role, but also participate in the regulation of diverse processes of life, including replication, transcription, DNA repair, and etc. Packaging of nucleosomal DNA into nucleosomes prevents other binding proteins from accessing the DNA template. Meanwhile, linker regions between nucleosomes facilitate the binding of transcription factors with DNA template, which thereby promote the expression of nearby genes. Several recent genome-scale mappings of nucleosome positioning have shown that the distribution of nucleosomes on DNA is heterogeneous. For example, nucleosomes are located less frequent in intergenic regions than in open reading frames (ORFs), and the regulatory regions, including promoters, transcription start and termination sites, regions around DNA replication origins are often depleted of nucleosomes. Additionally, there is an inverse correlation between the nucleosome occupancy in promoters and the transcription rates of downstream genes. All these findings indicated that the positions of nucleosomes on genomic DNA have a crucial role in regulating gene expression.With the breakthrough of high-throughput techniques, genome-wide nucleosome positioning has been mapped in several organisms. High-resolution data of nucleosome positions on genomic DNA provide an unprecedented opportunity for further modeling nucleosome positioning in vivo and investigating its relationship with gene regulation at genome level. The determination of nucleosome positioning purely using experimental approaches is time-consuming and expensive. Thus, the theoretical or computational methods for predicting nucleosome positioning along genome become increasingly important.The regulation of DNA replication origins is a substantially important issue in molecular biology. Autonomously Replicating Sequence (ARS) of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosome replication. Previous works have demonstrated that the structure of the ARS affects its activity, and the activity of ARS is associated with nucleosome occupancy. Investigating the effect of nucleosome positioning on the activity of ARS using in vitro nucleosome reconstruction technique is of great significance.In this dissertation, based on the characteristic of nucleotide distribution in nucleosome and linker DNA sequences, a computational model used to predict nucleosome positioning was proposed and applied to S.cerevisiae genome. Several recombinant plasmids pRS405-ARS were constructed and histones were extracted and purified from yeast. The main contributions are summarized as follows:1. Based on k-mer frequency in nucleosome DNA and linker DNA, a computational model was developed using the method of Increment of Diversity combined with Support Vector Machine (ID-SVM). This model was used to predict nucleosome DNA and linker DNA and obtained good performance in S.cerevisiae, H.sapiens and D.melanogaster.2. Based on k-mer frequency and poly(A) information in nucleosome DNA and linker DNA, a computional model was proposed using the method of Increment of Diversity combined with Quadratic Discriminant analysis (IDQD). This model was used to analyze nucleosome occupancy in S.cerevisiae genome. The average nucleosome occupancy around the transcriptional start site (TSS), transcriptional termination site (TTS and ARS was also predicted with high accuracy.3. Four methods of extracting yeast genomic DNA, such as lysozyme digestion, snail enzyme treatment over night, repeated freezing and thawing, and grinding with glass beads, were compared. ARS304(ARS305) and its flanking sequences of S.cerevisiae and plasmid pRS405 were recombined, the product was termed pRS405-ARS. Several recombinant plasmids pRS405-ARS were constructed.4. Histones were extracted from S.cerevisiae cells by Acid Extraction Method. It is the basis of nucleosome reconstitution in vitro.