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Development and application of microarray technologies for the highly parallel analysis of the sequence specificity of DNA binding proteins.

机译：微阵列技术的开发和应用，可高度并行地分析DNA结合蛋白的序列特异性。

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摘要

The interactions between transcription factors and their DNA binding sites are a primary step in the regulatory networks within cells. Many of these are key to pharmacogenomics and other quantitative traits. The development of DNA microarrays beyond hybridization-based assays and into the realm of characterizing the sequence specificities of DNA binding proteins such as transcription factors provides a critical link connecting the field of mRNA expression analysis with the burgeoning fields of proteomics and structural genomics. This technology presents an exciting technological advance since up until now technologies that characterized these critical DNA-protein interactions have been laborious and thus have not permitted more than a handful of DNA sequences to be examined. Furthermore, the types of experiments and analyses presented in this thesis will permit a more complete delineation of the interplay of the regulatory pathways present within cells.; In this thesis, I present the development of a high-throughput technology for probing the specificity of DNA-protein interactions, as well as the technology's various applications and analysis of the data that it provides. In Chapter 2 I show that single-stranded oligonucleotide arrays can be double-stranded by DNA polymerase and further biochemically modified by dam methylase , and that the resulting double-stranded DNA is accessible for interaction with DNA binding proteins. In Chapter 3 I present the quantitative measurement of binding affinities of transcription factors using DNA microarrays. I also show that this microarray technology can distinguish transcription factors with very similar binding site preferences. In Chapter 4 I present the results of searches I performed on the human genome using the recognition sites found in Chapter 3 for the human/mouse early growth factor EGR1. I also describe the nonindependence of the nucleotide positions of transcription factor recognition sites, and show that this nonindependence is responsible for superiority of complete reference tables based on protein binding microarray data over the use of binding site weight matrices and consensus sequences. In Chapter 5, I show that microarrays spotted with DNA fragments at least 1 kb in length permit detection of binding to a single specific site. Therefore, microarrays spotted with entire intergenic regions from smaller genomes such as S. cerevisiae or segments of intergenic regions from higher eukaryotes can be used for genome-wide identifications of transcription factor binding sites and their putatively regulated genes. I also present specific binding of the S. cerevisiae transcription factors Rpn4 and Zap1 to microarrays via phage display constructs of their DNA binding domains. In Chapter 6, I present the construction of genomic binding site knockouts and their subsequent mRNA expression analyses in order to validate the regulatory roles of these sites on the putatively regulated genes. In Chapter 7, I discuss issues regarding the types of microarrays, microarray slides, and DNA sequences to use for such experiments.
机译：转录因子及其DNA结合位点之间的相互作用是细胞内调节网络的第一步。其中许多是药物基因组学和其他定量性状的关键。 DNA微阵列技术的发展超越了基于杂交的检测方法，并进入了表征DNA结合蛋白（如转录因子）序列特异性的领域，这为将mRNA表达分析领域与蛋白质组学和结构基因组学的新兴领域联系在一起提供了至关重要的联系。该技术代表了令人激动的技术进步，因为迄今为止，表征这些关键DNA-蛋白质相互作用的技术一直很费力，因此不允许检查少数DNA序列。此外，本文提出的实验和分析类型将使细胞内存在的调节途径之间的相互作用更加完整。在这篇论文中，我介绍了一种用于探测DNA-蛋白质相互作用特异性的高通量技术的发展，以及该技术的各种应用和对它提供的数据的分析。在第二章中，我证明了单链寡核苷酸阵列可以被DNA聚合酶双链修饰，并被 dam甲基化酶进一步生化修饰，并且所产生的双链DNA可与DNA结合蛋白相互作用。 。在第三章中，我介绍了使用DNA微阵列对转录因子结合亲和力的定量测量。我还表明，这种微阵列技术可以区分具有非常相似的结合位点偏好的转录因子。在第4章中，我介绍了使用第3章中针对人/小鼠早期生长因子EGR1的识别位点对人类基因组进行搜索的结果。我还描述了转录因子识别位点核苷酸位置的非独立性，并表明这种非独立性是基于蛋白质结合微阵列数据的完整参考表优于结合位点权重矩阵和共有序列的使用的原因。在第5章中，我展示了点样DNA片段长度至少为1 kb的微阵列，可以检测到与单个特定位点的结合。因此，点有来自较小基因组（如酿酒酵母）的完整基因间区域或来自高等真核生物的基因间区域的片段的微阵列可用于转录因子结合位点及其推定调控基因的全基因组鉴定。我还介绍了 S的特异性结合。酿酒酵母转录因子Rpn4和Zap1通过其DNA结合结构域的噬菌体展示构建物而进入微阵列。在第6章中，我介绍了基因组结合位点敲除的构建及其后续的mRNA表达分析，以验证这些位点在假定调控基因上的调控作用。在第7章中，我讨论了有关用于此类实验的微阵列，微阵列载玻片和DNA序列的问题。

著录项

  • 作者

    Bulyk, Martha Leonia.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Biophysics General.; Biology Genetics.
  • 学位 Ph.D.
  • 年度 2001
  • 页码 253 p.
  • 总页数 253
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;遗传学;
  • 关键词

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