摘要
真核生物中负载着遗传信息的DNA分子在细胞中被包装成核小体。核小体不仅是构成真核生物染色质的基本结构单位,更重要的是通过它在基因组上的定位及其组蛋白复合物的化学修饰调控诸如转录、DNA复制和修复等基因表达过程。由于核心DNA被包装进核小体中,阻断了那些负责最基本生命过程的蛋白与DNA的接触机会,而核小体之间的自由区域更有利于这些蛋白因子的进入并识别结合位点,从而保证蛋白因子易于接近染色质模板。目前基于全基因组的核小体定位分析,发现基因组上核小体分布极不均匀,某些区域核小体占据率高,某些区域核小体缺乏。在基因间区与基因区(ORFs)相比,核小体更加稀疏；在基因的一些调控区,如启动子区、转录起始和终止位点、复制起始等区域,通常缺乏核小体。也有一些工作表明启动子区核小体的占据与其下游的基因表达效率呈负相关。因此,核小体在基因组上的位置对基因的表达调控是非常重要的。
随着高通量实验技术的快速发展,目前已得到多种真核生物核小体定位的高分辨率实验图谱,但采用实验技术提取全基因组的核小体定位信息时,不仅耗费大量的时间和经费,而且对后续实验数据的分析也存在着局限。因此,基于现有的实验数据,发展核小体定位的理论预测模型已经成为了生物信息学领域的热点问题。
真核生物复制起始的调控是复制调控的重要环节,ARS(Autonomously Replicating Sequence)是酵母染色质复制所依赖的顺式作用元件。多项研究表明自主复制序列的结构影响其活性,且有不同活性的自主复制序列上的核小体分布不同。利用体外核小体重组技术研究核小体定位对ARS复制起始活性的研究有重要的生物学意义。
本文基于酵母核小体序列信息发展了预测核小体定位的模型,并用于酵母全基因组核小体预测中,取得较好的预测效果。用分子生物学方法构建了含酵母ARS的重组质粒pRS405-ARS,采用酸抽提法提取酵母的组蛋白,为核小体的体外重组奠定了基础。主要研究内容如下
1.基于核小体序列中k-mer频数信息,采用多样性增量与支持向量机结合(ID-SVM)的方法对酵母核小体核心DNA和连接DNA序列进行分类预测,取得了较高的预测精度。在人类和果蝇的核小体核心DNA和连接DNA序列分类预测中也取得了较为理想的效果。
2.基于核小体序列中k-mer频数信息和poly(A)信息,发展了预测核小体定位的IDQD模型,并将模型应用于酵母全基因组的核小体占据分析。在对转录起始位点(TSS)、转录终止位点(TTS)、复制起始序列(ARS)等功能区的核小体占据预测分析时IDQD模型也体现出良好的性能。
3.比较了溶菌酶法、蜗牛酶过夜处理法、蜗牛酶反复冻融法、珠磨法等4种破壁提取酵母基因组DNA的方法；利用基本分子生物学方法将含酵母ARS304(ARS305)及其两侧序列与酵母穿梭质粒pRS405重组后,命名为pRS405-ARS。得到一系列含有ARS304(ARS305)及两侧不同长度序列的重组质粒。
4.酸抽提法提取酵母细胞的组蛋白,这是核小体体外组装的基础。
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.
引文
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