MAIZE PROTEINS INDUCED BY DNA DAMAGING AGENTS ULTRAVIOLET-B AND MUTATOR ACTIVITY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。玉米和所有其他植物一样，缺乏胚种系。在生命周期的后期，花中存在的少量营养干细胞分化为生发前细胞；这些细胞经历减数分裂产生单倍体孢子，单倍体孢子通过有丝分裂形成生命周期的单倍体阶段。鉴于这一生命周期，如果不修复，营养细胞中发生的DNA损伤可能会传递给后代；如果不修复，其他形式的细胞对蛋白质、RNA或脂类的损伤可能会危及植物的生长和繁殖。为了了解基因组的完整性是如何维持的，以及玉米如何对持续的损害做出反应，我们使用了两种DNA诱变剂：UV-B辐射，阳光的一种自然成分，以及MuDR/Mu DNA转座子。转录组图谱和蛋白质组学被用来发现在不同的体细胞和生殖组织中对这些药物的适应反应的范围。二维分离(按等电点和按大小)用于分离处理组织和对照组织的主要蛋白质，提供约3000种可分辨蛋白质类型的数据。这些蛋白质通过特定的处理被归类为不变、增加或减少，并且显示一致变化的蛋白质被回收以进行鉴定。所有的蛋白质鉴定都是与加州大学旧金山分校的质谱学设施合作进行的；到目前为止，通过大量指纹和测序鉴定了大约150种蛋白质。在这些蛋白质中，有一些是从转录组图谱中预测的，以及以前没有涉及到对UV-B或转座子诱导的基因组损伤的反应的蛋白质。转录组图谱和蛋白质组学数据的结合被用来确定涉及哪些信号转导途径和细胞过程；验证实验包括对所确定的关键过程中的突变体进行遗传分析，即通过证明突变体表现出对UV-B的敏感性。目前的工作集中在阐明蛋白质的翻译后修饰，这些蛋白质没有被处理大量改变，然后用质谱仪进行鉴定。这些研究可能会突出对治疗最迅速的反应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maize like all other plants lacks a germ line. Late in the life cycle a small number of vegetative stem cells present in flowers differentiate as pre-germinal cells; these cells undergo meiosis to produce haploid spores, which divide by mitosis to form the haploid phase of the life cycle. Given this life cycle, DNA damage that occurs in the vegetative cells could be transmitted to the progeny if it is not repaired; other forms of cellular damage to proteins, RNA, or lipids could compromise plant growth and reproduction if not repaired as well. To understand how integrity of the genome is maintained and how maize responds to continuous damage, two DNA mutagens are employed: UV-B radiation, a natural component of sunlight, and MuDR/Mu DNA transposons. Transcriptome profiling and proteomics are being used to discover the range of acclimation responses to these agents in various somatic and reproductive tissues. Two-dimensional separation (by isoelectric point and by size) is used to fractionate the major proteins of treated and control tissues, providing data on ~3000 resolved protein types. The proteins are classified as unchanged, increased or decreased by specific treatments, and the proteins showing consistent alterations are recovered for identification. All of the protein identification is conducted in collaboration with the UCSF Mass Spectrometry Facility; to date, about 150 proteins have been identified by mass fingerprinting and sequencing. Among these proteins are a number predicted from the transcriptome profiling as well as proteins not previously implicated in responses to either UV-B or transposon-induced genomic damage. The combination of transcriptome profiling and proteomics data are used to determine which signal transduction pathways and cellular processes are involved; verification experiments include genetic analysis of mutants in key processes identified, i.e. by demonstrating that mutants show increased UV-B sensitivity. Current work focuses on elucidating post-translational modifications of proteins that were not altered in abundance by the treatments, followed by identification by mass spectrometry. These studies will likely highlight the most rapid responses to the treatments.