Role of DNA Topology and Chromatin Structure in Phaseolin Gene Regulation

9604617豆蛋白是菜豆种子的主要蛋白质，在菜豆胚胎发育过程中高度表达和积累，但在菜豆植株的营养组织中缺失。在含有β-小菜豆(PHAS)基因的烟草稳定转化株中也观察到了类似的基因表达模式。在许多植物基因中，pHAS基因启动子的空间调控所必需的顺式元件位于转录起始点附近。虽然许多已知与转录因子相互作用的DNA序列基序存在于这个启动子区域，但这些反式激活因子中没有一个是组织特异性的。事实上，通过轰击或电穿孔将PHAS/GUS结构导入叶细胞的瞬时分析中所看到的大量活性表明，存在于营养组织中的核因子可以与PHAS启动子元件有效地相互作用。相比之下，使用相同结构的稳定转化植物即使在用一系列生长调节剂处理时，也不会在营养组织或此类植物的愈伤组织中表达。至于一些动物和酵母基因，这些观察表明整合到植物染色质中在调节PHAS基因表达方面发挥了作用。这项研究将利用转化单拷贝PHAS基因的烟草来评估染色质在PHAS基因表达中的作用。在体内调节PHAS启动子的顺式元件将通过足迹方法来鉴定，这种方法比较了顺式元件与营养(叶)和种子组织中的细胞因子的相互作用。对叶片和种子中核小体位置的定位将确定核小体是否位于PHAS启动子的特定区域，这可能会通过处理导致营养组织中出现的不活跃的因子来阻止访问。本研究的目的是了解对PHAS基因启动子进行严格发育和空间调控的细胞机制，特别是确定该启动子在胚胎发育过程中如何从营养组织的完全不活跃状态转变为高度活跃状态。菜豆蛋白是菜豆种子的主要蛋白质，在菜豆胚胎发育过程中高度表达和积累，但在菜豆植株的营养组织中缺失。本研究的目的一般是了解对小菜豆蛋白编码基因进行严格的发育和空间调控的细胞机制，特别是确定该基因在胚胎发育过程中如何从营养组织中完全不活跃的状态转变为高度活跃的状态。

9604617 Hall Phaseolin, the major seed protein of the bean Phaseolus vulgaris, is highly expressed and accumulated during embryogenesis but is absent from vegetative tissues of bean plants. A similar pattern of gene expression is observed in stable transformants of tobacco with the beta-phaseolin (phas) gene. As for many plant genes, cis-elements essential for spatial regulation of the phas gene promoter lie close to the transcription start site. Although many DNA sequence motifs known to interact with transcription factors are present in this promoter region, none of these transactivators has yet been shown to be tissue specific. Indeed, the substantial activity seen in transient assays of phas/gus constructs introduced into leaf cells by bombardment or electroporation indicates that nuclear factors present in vegetative tissues can interact productively with phas promoter elements. In contrast, stable transformed plants using identical constructs show no expression in vegetative tissues or in calli from such plants even when treated with a range of growth regulators. As for several animal and yeast genes, these observations suggest the integration into plant chromatin plays a role in regulating phas gene expression. This research will use tobacco transformed with a single copy of the phas gene to evaluate the role of chromatin in phas gene expression. Cis-elements that regulate the phas promoter in vivo will be identified by footprinting methods that compare the interaction of cis-elements with cellular factors in vegetative (leaf) and seed tissues. Mapping of nucleosome position in leaves and seeds will determine if nucleosomes are positioned over specific regions of the phas promoter, which could prevent access by transacting factors resulting in the inactivity seen in vegetative tissues. The goals of this research are generally to understand the cellular mechanisms that confer stringent developmental and spatial regulation on the phas gene promoter, and specifically to determine how that promoter is converted from a totally inactive state in vegetative tissues to a highly active state during embryogenesis. Phaseolin, the major seed protein of the bean Phaseolus vulgaris, is highly expressed and accumulated during embryogenesis but is absent from vegetative tissues of bean plants. The goals of this research are generally to understand the cellular mechanisms that confer stringent developmental and spatial regulation on the gene encoding phaseolin, and specifically to determine how that gene is converted from a totally inactive state in vegetative tissues to a highly active state during embryogenesis.