【作者】 任立晨；

【导师】 张惟杰；

【作者基本信息】 上海交通大学 ， 生物化学和分子生物学， 2007， 博士

【副题名】应用小鼠干细胞分化模型探索哺乳动物分子进化的规律

【摘要】 哺乳动物基因的表达模式同其分子结构特征之间的关联以及这些结构特征的进化规律是生物学中基本问题,但这些问题仍有待深入研究。发育进程被认为对于探索进化机制至关重要,而哺乳动物的干细胞分化系统,包括干细胞,祖细胞和其分化产物,也提供了一个描述哺乳动物发育进程的理想模型。因此,本文以小鼠干细胞分化模型中与发育阶段相关的基因表达模式为基础,试图建立哺乳动物基因的表达模式与其分子结构特征之间的关联。在不同的细胞类型中,我们建立了基因表达量同密码子使用偏好的关联。我们发现,处于不同发育阶段的细胞,其优化密码子可能会有所不同。在小鼠基因中,GC结尾密码子的使用在整体上占据优势;但我们却在相当多种类的处于早期发育阶段的干细胞和祖细胞中,发现优化密码子以AT结尾。我们还发现,在不同发育阶段中富集表达(发育阶段关键基因)或特异性表达(发育阶段特异性基因)的基因,也可能具有不同的密码子使用模式。而且,对于同一个发育阶段,特异性表达的基因与富集表达的基因相比,往往使用更多的以GC结尾的同义密码子。进一步的分析暗示,上述与发育阶段相关的密码子偏好模式,特别是以AT结尾的优化密码子在很大程度上与翻译选择模型的预言相一致。在哺乳动物中,基因的密码子偏好(GC3)与基因所在基因组区域的GC含量(GCg)密切相关。我们的结果表明,小鼠基因所在基因组区域的GCg也同其发育阶段相关。进一步,我们还分析了小鼠发育阶段相关基因在基因组上分布和排列的规律。我们发现,发育阶段相关基因在各个染色体上的分布并不均匀,这可能意味着各个染色体对分化进程的贡献各异。我们还发现,很多与发育阶段密切相关的基因在染色体上呈现成簇分布的状态,并且,簇内基因与簇外基因相比,偏好于使用更多以GC结尾的同义密码子。我们接下来通过比较基因组学的方法研究了发育阶段相关基因在起源和进化上的初步规律。首先,我们的结果表明,高表达基因的进化速率要低于中—低表达基因的进化速率,这暗示在人—小鼠以及大鼠—小鼠分歧后对高表达基因的负选择仍然有效。与之相反,对于那些在某个发育阶段中选择性表达的发育阶段关键基因或发育阶段特异性基因,其进化速率往往显著高于那些表达模式不随发育阶段变化的基因。较高的进化速率暗示这些基因在人—小鼠以及大鼠—小鼠分歧后可能受到了不同的功能性制约。其次,我们发现,热血动物的直系同源基因间具有比较相似的密码子偏好模式,这暗示了小鼠的密码子使用偏好在热血动物共同祖先出现时已基本固定。最后,我们还发现,高表达基因与中—低表达基因相比,以及表达模式不随发育阶段变化的基因同在某个发育阶段中选择性表达的发育阶段关键基因或发育阶段特异性基因相比,通常能够在果蝇基因组中找到更多的直系同源基因。我们的结果表明,小鼠基因的表达模式和分子特征均与其古老程度相关。上述分析依赖于已发表的小鼠干细胞及其分化产物的高通量表达数据;而且,这些数据中仅包括来自外胚层和中胚层的前体细胞。我们接下来要探索,在内胚层的分化路径中,小鼠基因的密码子偏好,基因组GC含量,基因分布和基因年龄是否仍同与发育阶段相关的基因表达模式有关。我们在小鼠成体胰腺的体外培养体系中鉴定了一群具有增殖和多向分化能力的前体细胞,并发现这群细胞分布于胰岛中和导管周边。我们进一步分析了该群前体细胞和胰腺成熟内分泌细胞(胰岛)的基因表达谱,发现在胰腺前体细胞和胰岛细胞组成的分化模型中,基因分子特征,基因组特征和起源进化的相关规律也在相当的程度上与其所处的发育阶段有关,且基本规律与我们在外胚层和中胚层中观察到的相同或相近。在本文中,我们应用干细胞分化模型,为研究哺乳动物分子进化中的一些关键问题提供了有价值的线索。例如,我们发现了哺乳动物密码子偏好使用的新规律,提出了支持自然选择作用于哺乳动物同义位点的新证据。此外,我们的研究结果对于理解哺乳动物基因的进化历程和哺乳动物基因组GC含量的潜在生物学意义都提供了新的途径。这项研究将分子进化的理论分析引入干细胞领域,对于从结构特征的角度,在基因组尺度上理解干细胞的标志性分子和分化机理也具有一定的意义。我们的结果显示,干细胞和成熟细胞差异表达的基因在分子结构特征上存在差异;并且,在干细胞分化进程中经历结构变化的染色质片段,也可能与这些片段的GC含量有关。我们的这些结果,其深入意义还有待于进一步的研究和开掘。更多还原

【Abstract】 The identification of the correlations between the molecular features of mammalian genes and their patterns of expression, as well as the exploration of the evolutionary processes of these molecular features are fundamental biological issues and remain subjects of controversy. Developmental processes are believed to be of critical importance to the investigation of evolutionary mechanisms. Moreover, stem cells, progenitor cells and their derivates, defined by their distinct differentiation potential, provide ideal models of the mammalian developmental hierarchy. Therefore, in this thesis, we tried to establish the correlations between the molecular features of mammalian genes and their patterns of expression, based on the developmental stage-related patterns of gene expression in models of mouse stem cell differentiation.We established the correlations between the levels of gene expression and codon usage in different cell types. Our results showed that the optimal codons exhibited variation within the developmental hierarchy. Specifically, in certain mouse stem cells and progenitor cells in early developmental stages of ontogeny, the optimal codons were the AT-ending ones, while the mouse major codons are the GC-ending ones. We also found that genes that were enriched (developmental-pivotal genes, DPGs) or specifically expressed (developmental-specific genes, DSGs) at different developmental stages had different patterns of codon usage. Moreover, at the same developmental stage, DSGs generally used more GC-ending codons compared with DPGs. Further analyses suggest that the model of translational selection might be consistent with the developmental stage-related patterns of codon usage, especially for the AT-ending optimal codons.It has been indicated that codon usage (GC3) and local genomic GC content (GCg) are closely correlated in mammalian genome. Our results also showed that the variations of GCg were correlated with the developmental-stage related patterns of gene expression. We further investigated the distribution and organization of developmental stage-related genes in mouse chromosomes. We found that the distributions of genes corresponding to each developmental stage were not homogeneous among different chromosomes, suggesting the contributions of different chromosomes could be distinct at each developmental stage. We also observed that many developmental stage- related genes were organized as clusters in chromosomes. Moreover, genes within clusters generally used more GC-ending codons than genes out of the clusters. By a comparative genomics approach, we further investigated the origin and the evolutionary processes of developmental stage-related genes. First, our data showed that the evolutionary rates of highly expressed genes were lower than that of mid-lowly expressed genes, indicating the influence of selective constraints on highly expressed genes was still detectable after the human-mouse and mouse-rat divergence. In contrast with these observations, DPGs and DSGs generally had higher evolutionary rates than non-developmental-pivotal genes (NDPGs). The higher rates might infer different functional constraints of these genes after the human-mouse and mouse–rat divergence. Second, we indicated that there were similar patterns of codon usage among orthologous genes of warm-blood vertebrates. These results suggested that the codon usage biases of mouse genes were formed in the common ancestor of warm-blood vertebrates. Third, we found highly expressed genes and NDPGs, compared with mid-lowly expressed genes and developmental-selectively expressed genes (DPGs and DSGs) respectively, could find more orthologous genes in genome of fruit fly. We also showed that both expression patterns and molecular features were correlated with the age of mouse genes.The investigations above are dependent on the published high-throughput studies of stem cells and their derivates from ectoderm and mesoderm. We in turn to explore whether the codon usage, genomic GC content, gene distribution, and gene age are also correlated with the developmental stage- related patterns of gene expression in differentiation processes of endoderm. We defined a precursor cell population from adult mouse pancreas by selective culture. We found these precursor cells were located in the islets and around the pancreatic ducts. Based on the analyses of the expression profiling of both this pancreatic precursor cell population and mouse islets, our data showed that the molecular features, genomic characteristics and evolutionary processes are also correlated with developmental-stage related patterns of gene expression in endoderm, generally consistent with the observations in ectoderm and mesoderm.Using the models of stem cell differentiation, the current study provides certain useful clues of evolutionary issues at the molecular level. For example, we showed new patterns of mammalian codon usage and new evidence of the presence of natural selection at mammalian synonymous sites. Moreover, our data also assist our understanding of the biological meaning of genomic GC content and the evolutionary processes of mammalian genes.The current study has applied theoretical analyses of molecular evolution into stem cell research to gain a better understanding of stem cell differentiation at the genomic level. For example, we found that mammalian genes that specifically expressed at different developmental stages bear different molecular features. In addition, we also suggested that the genome segments that are involved in chromatin remodeling might correlate with their GC content. Further investigation will be needed to better understand the significance and implications of the findings presented here.更多还原