Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

• 批准号: 10014354
• 负责人: DONALD COURT
• 金额: $ 165.22万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014354
• 关键词: 3' Untranslated Regions; Address; Affect; Alzheimer' s Disease; Antibiotics; Bacteria; Bacteriophage lambda; Bacteriophages; Binding; Biological Models; Cancer Etiology; Cell division; Cell physiology; Cells; Cellular Structures; Complex; Cyclic AMP; DNA; DNA Insertion Elements; DNA Repair; DNA annealing; DNA biosynthesis; DNA replication fork; DNA-Directed RNA Polymerase; Defect; Development; Disease; Distant; Down Syndrome; Enhancers; Ensure; Escherichia coli; Eukaryota; Event; Evolution; Exclusion; Failure; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Engineering; Genetic Recombination; Genetic Transcription; Genome; Genomics; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; HIV; Herpesvirus 1; Homologous Gene; Human; Immunity; In Vitro; Invaded; Lead; Length; Life; Lytic; Malignant Neoplasms; Mediating; Methodology; Mitochondria; Modeling; Modification; Molecular; Molecular Target; Mutation; Nucleotides; Oligonucleotides; Operon; Organism; Perception; Philosophy; Polymerase; Process; Prophages; Proteins; RNA Binding; RNA Folding; RNA Interference; Reagent; Replication Initiation; Research; Ribonuclease III; Ribosomal RNA; Ribosomes; Role; Running; Sensory; Signal Transduction; Site; Stretching; Structure; System; Systems Biology; Technology; Testing; Theoretical model; Transcript; Translations; Vaccines; Viral; Virus; Work; antitermination; biodefense; cell growth; ds-DNA; functional genomics; homologous recombination; in vivo; man; mathematical model; mutant; novel strategies; pathogen; prevent; promoter; prototype; recombinase; transcription factor

Recent characterizations of the lambda genetic network have provided a framework for systems biology approaches using lambda as a prototype for theoretical modeling methodologies, which have become important for addressing signal transduction, cancer development and other complex genetic networks of eukaryotes. Co-evolution of lambda with E. coli has produced genetic systems that are exquisitely connected to the host's most basic functions. By examining the interface between lambda and host systems, my lab follows the trail of the phage to understand what is most important and vital to both cellular life and viral exploitation of cellular systems. The virus provides clues as to how those cellular functions work and how to study them. All of the work in my lab has derived from this philosophy. New discoveries change our perception of the lambda genetic network and affect models describing it. Two genes, rexA and rexB, cotranscribed with the cI repressor gene, have been largely ignored for contributions to the complex lambda genetic network controlling repressor activity and its synthesis. We found a new role for RexA in this regard as it appears to interact with CI repressor to promote induction. We also have evidence suggesting that the immunity terminator overlaps the end of the rexB gene, and that translation of RexB modulates the terminator, affecting transcription levels of the cI gene. Thus, the classic Rex exclusion system is intimately involved with lambda immunity control, adding further subtlety to the bistable genetic switch model of lambda that has provided a basis for mathematical models of gene regulation. We have developed the lambda homologous recombination functions Red as reagents for recombineering, a revolutionary in vivo genetic engineering technology that has enabled new approaches for functional genomic studies from bacteria to man. Recombineering allows modification of genomic clones from any organism, and is being used for developing model systems for cancer and other disease-related research. Similar recombineering systems are being developed in other bacteria, including pathogens, and can be used to develop vaccines, molecular targets for antibiotics, phage therapy, and biodefense. We demonstrated that short single-strand oligonucleotides recombine with homologous targets on replisomes in E. coli and other bacteria. Phage recombinases, like Beta and RecT, stimulate this oligo recombination above low endogenous levels in the cell. Results suggest that the molecular mechanism for initiation of oligo recombination by the two recombinases Red Beta and RecT differ. Beta requires replication of the target DNA to initiate and generate a recombination intermediate, whereas, RecT does not require DNA replication to generate an intermediate. This supports the premise that Beta acts by ss-strand annealing at the replication fork, whereas RecT forms D-loops by strand invasion. We plan to similarly characterize initiation of recombination by other recombinases including HSV-1 ICP8. Faithful transcription of DNA is dependent on RNA polymerase (RNAPol) maintaining accuracy in matching the incoming nucleotide to the template to prevent misincorporation errors and maintaining the register between the template and the transcript to prevent slippage errors. Failure to faithfully transcribe the template has been suggested to lead to a variety of diseases including certain cancers, Down's syndrome, and Alzheimer's disease. My lab demonstrated that the RpoC D1143P polymerase misincorporation mutant caused genetic instablility of an IS2 insertion element. Instability was enhanced further when combined with defects in the GreA and GreB transcription factors. I speculate that RNAPol complexes arrest after misincorporation and interfere with DNA replication. This could lead to DNA repair with the potential for rearrangements, a common cause of cancers in higher organisms. Another type of mistake is transcriptional slippage. E. coli RNAPol makes frameshift errors when transcribing runs of As or Ts in the template DNA. My lab has demonstrated that the lambda N-Nus factor transcription antitermination complex modifies RNAPol and reduces these natural slippage events. N prevents transcriptional slippage, and since many intrinsic terminators have long U stretches, slippage and termination are likely to be interconnected processes. This is the first example of slippage being regulated, and it is possible that other N-like transcription regulators, e.g., HIV Tat, may also affect slippage. I study lambda to understand host functions with which the virus interacts, so that I can better understand their roles for the virus as well as the cell. These functions targeted by the virus are not only important for the virus but are also some of the most basic regulatory and sensory components of the cell. My work from the 1970's defined the post-transcriptional role of RNase III in retroregulation of lambda int gene expression from its 3' UTR. Several structures of RNase III have now been solved and contribute greatly to the understanding of the roles of Drosha and Dicer in RNAi 3' UTR gene regulation. We also described the role of RNase III in the processing of rRNA in coordination with the host Nus factors. Nus factors modify the RNAPol during rRNA transcription. The RNAPol-Nus transcription ensures rRNA folding, coordination of RNase III processing, and 30S ribosome assembly. Era is also intimately involved with 30S ribosome assembly. The Era-GTP complex binds to the 3' end of the maturing 16S rRNA, where it controls 16S processing, RNA folding, and the final stages of 30S subunit assembly. I have shown that mutants of Era block growth and cell division of E. coli, and I isolated a separation of function mutant that is competent for ribosome assembly and growth but is blocked for cell division. I propose that the Era-GTP/GDP cycle has check-points for growth and division, ensuring their coordination. Era homologues are conserved across all domains of life. Defects in human Era also cause dysfunction in the mitochondrial small ribosomal subunit, resulting in poor cell growth.

lambda 遗传网络的最新表征为使用 lambda 作为理论建模方法原型的系统生物学方法提供了框架，这对于解决信号转导、癌症发展和其他复杂的真核生物遗传网络变得非常重要。 lambda 与大肠杆菌的共同进化产生了与宿主最基本功能紧密相连的遗传系统。通过检查 lambda 和宿主系统之间的界面，我的实验室追踪噬菌体的踪迹，以了解什么对细胞生命和细胞系统的病毒利用最重要和最重要。该病毒提供了有关这些细胞功能如何发挥作用以及如何研究它们的线索。我实验室的所有工作都源于这一理念。新发现改变了我们对 lambda 遗传网络的看法，并影响描述它的模型。与 cI 阻遏基因共转录的两个基因 rexA 和 rexB 由于对控制阻遏蛋白活性及其合成的复杂 lambda 遗传网络的贡献而在很大程度上被忽视。我们在这方面发现了 RexA 的新作用，因为它似乎与 CI 阻遏物相互作用以促进诱导。我们还有证据表明免疫终止子与 rexB 基因的末端重叠，并且 RexB 的翻译调节终止子，从而影响 cI 基因的转录水平。因此，经典的 Rex 排除系统与 lambda 免疫控制密切相关，为 lambda 双稳态遗传开关模型增添了进一步的微妙性，该模型为基因调控的数学模型提供了基础。我们开发了 lambda 同源重组功能 Red 作为重组工程试剂，这是一种革命性的体内基因工程技术，为从细菌到人类的功能基因组研究提供了新方法。重组工程允许修改任何生物体的基因组克隆，并用于开发癌症和其他疾病相关研究的模型系统。类似的重组工程系统正在其他细菌（包括病原体）中开发，可用于开发疫苗、抗生素分子靶标、噬菌体疗法和生物防御。我们证明短单链寡核苷酸可以与大肠杆菌和其他细菌的复制体上的同源靶标重组。噬菌体重组酶（如 Beta 和 RecT）可刺激细胞内低内源水平以上的寡核苷酸重组。结果表明，两种重组酶 Red Beta 和 RecT 启动寡核苷酸重组的分子机制不同。 Beta 需要靶 DNA 复制来启动并生成重组中间体，而 RecT 不需要 DNA 复制来生成中间体。这支持了这样的前提：Beta 通过在复制叉处的单链退火发挥作用，而 RecT 通过链入侵形成 D 环。我们计划对包括 HSV-1 ICP8 在内的其他重组酶的重组起始进行类似的表征。 DNA 的忠实转录依赖于 RNA 聚合酶 (RNAPol) 维持传入核苷酸与模板匹配的准确性，以防止错误掺入错误，并维持模板和转录本之间的记录以防止滑动错误。有人认为，未能忠实地转录模板会导致多种疾病，包括某些癌症、唐氏综合症和阿尔茨海默氏病。我的实验室证明 RpoC D1143P 聚合酶错误掺入突变体导致 IS2 插入元件的遗传不稳定。当与 GreA 和 GreB 转录因子的缺陷结合时，不稳定性进一步增强。我推测 RNAPol 复合物在错误掺入后停止并干扰 DNA 复制。这可能导致 DNA 修复并可能发生重排，这是高等生物体中癌症的常见原因。另一种类型的错误是转录失误。大肠杆菌 RNAPol 在模板 DNA 中转录 As 或 T 时会出现移码错误。我的实验室已经证明 lambda N-Nus 因子转录抗终止复合物可以修饰 RNAPol 并减少这些自然滑移事件。 N 可以防止转录滑移，并且由于许多内在终止子具有长 U 延伸，因此滑移和终止很可能是相互关联的过程。这是滑移受到调节的第一个例子，其他 N 样转录调节因子（例如 HIV Tat）也可能影响滑移。我研究 lambda 是为了了解病毒与其相互作用的宿主功能，这样我就可以更好地了解它们对病毒和细胞的作用。病毒所针对的这些功能不仅对病毒很重要，而且也是细胞最基本的调节和感觉成分。我在 1970 年代的工作定义了 RNase III 在从 3' UTR 逆向调节 lambda int 基因表达中的转录后作用。 RNase III 的几个结构现已得到解决，并极大地有助于理解 Drosha 和 Dicer 在 RNAi 3' UTR 基因调控中的作用。我们还描述了 RNase III 在与宿主 Nus 因子协调的 rRNA 加工中的作用。 Nus 因子在 rRNA 转录过程中修饰 RNAPol。 RNAPol-Nus 转录确保 rRNA 折叠、RNase III 加工的协调和 30S 核糖体组装。 Era 还与 30S 核糖体组装密切相关。 Era-GTP 复合物与成熟 16S rRNA 的 3' 端结合，控制 16S 加工、RNA 折叠和 30S 亚基组装的最后阶段。我已经证明，Era 突变体会​​阻碍大肠杆菌的生长和细胞分裂，并且我分离出了​​一种功能分离突变体，它能够进行核糖体组装和生长，但无法进行细胞分裂。我建议Era-GTP/GDP循环有增长和分裂的检查点，确保它们的协调。时代同源物在生命的各个领域都是保守的。人类Era的缺陷也会导致线粒体小核糖体亚基功能障碍，导致细胞生长不良。