Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

• 批准号: 8763080
• 负责人: DONALD COURT
• 金额: $ 158.29万
• 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/8763080
• 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 Sequence Rearrangement; DNA annealing; DNA biosynthesis; 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; Reading; Reagent; Replication Initiation; Research; Ribonuclease III; Ribosomal RNA; Ribosomes; Role; Running; Sensory; Signal Transduction; Site; Staging; Stretching; Structure; System; Systems Biology; Technology; Testing; Theoretical model; Transcript; Translations; Vaccines; Viral; Virus; Work; antitermination; base; biodefense; cell growth; functional genomics; homologous recombination; human DICER1 protein; in vivo; man; mathematical model; mutant; novel strategies; pathogen; prevent; promoter; prototype; recombinase; transcription factor

gene regulation studies. 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.

基因调控研究。最近对lambda遗传网络的描述为系统生物学方法提供了一个框架，使用lambda作为理论建模方法的原型，这对于解决真核生物的信号转导，癌症发展和其他复杂的遗传网络已经变得重要。λ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，我的实验室跟踪噬菌体的踪迹，以了解对细胞生命和病毒利用细胞系统最重要和最重要的是什么。这种病毒提供了关于这些细胞功能如何工作以及如何研究它们的线索。我实验室里的所有工作都源于这一理念。新的发现改变了我们对lambda基因网络的认知，并影响了描述它的模型。与cI抑制基因共转录的两个基因rexA和rexB在很大程度上被忽视了，因为它们对控制抑制基因活性及其合成的复杂lambda遗传网络有贡献。我们在这方面发现了RexA的新作用，因为它似乎与CI抑制因子相互作用以促进诱导。我们也有证据表明免疫终止子重叠在rexB基因的末端，rexB的翻译调节终止子，影响cI基因的转录水平。因此，经典的Rex排斥系统与lambda免疫控制密切相关，进一步增加了lambda双稳态遗传开关模型的微妙性，该模型为基因调控的数学模型提供了基础。我们已经开发了lambda同源重组功能Red作为重组试剂，这是一项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的方法。重组允许对任何生物体的基因组克隆进行修改，并被用于开发癌症和其他疾病相关研究的模型系统。类似的重组系统正在其他细菌（包括病原体）中开发，并可用于开发疫苗、抗生素的分子靶标、噬菌体治疗和生物防御。我们证明了短单链寡核苷酸在大肠杆菌和其他细菌的复制体上与同源靶标重组。噬菌体重组酶，如β和RecT，刺激细胞内低水平的寡核苷酸重组。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。β需要目标DNA的复制来启动和产生重组中间体，而RecT不需要DNA复制来产生中间体。这支持了β在复制叉处通过ss-链退火起作用的前提，而RecT通过链入侵形成d -环。我们计划类似地描述其他重组酶（包括HSV-1 ICP8）的重组起始。DNA的忠实转录依赖于RNA聚合酶（RNAPol）保持将进入的核苷酸与模板匹配的准确性，以防止误结合错误，并维持模板和转录本之间的记录，以防止滑移错误。未能忠实地转录该模板已被认为会导致多种疾病，包括某些癌症、唐氏综合症和阿尔茨海默病。我的实验室证明，RpoC D1143P聚合酶错误结合突变体导致IS2插入元件的遗传不稳定。当与GreA和GreB转录因子缺陷结合时，不稳定性进一步增强。我推测RNAPol复合物在错误结合后阻滞并干扰DNA复制。这可能会导致DNA修复和潜在的重排，这是高等生物癌症的常见原因。另一种类型的错误是转录滑动。大肠杆菌RNAPol在转录模板DNA中的a或t序列时会产生移码错误。我的实验室已经证明，lambda N-Nus因子转录抗终止复合物修饰RNAPol并减少这些自然滑移事件。N阻止转录滑移，并且由于许多内在终止子具有很长的U延伸，滑移和终止可能是相互关联的过程。这是滑脱被调控的第一个例子，其他n样转录调控因子，如HIV Tat，也可能影响滑脱。我学习lambda是为了了解与病毒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。病毒针对的这些功能不仅对病毒很重要，而且也是细胞的一些最基本的调节和感觉成分。我从20世纪70年代开始的工作定义了RNase III在从其3' UTR逆转录lambda int基因表达中的转录后作用。RNase III的几个结构现在已经被解决，并有助于理解Drosha和Dicer在RNAi 3' UTR基因调控中的作用。我们还描述了RNase III在rRNA加工过程中与宿主Nus因子协调的作用。Nus因子在rRNA转录过程中修饰RNAPol。rna - nus转录确保了rRNA折叠、RNase III加工协调和30S核糖体组装。Era也与30S核糖体组装密切相关。Era-GTP复合物结合到成熟的16S rRNA的3'端，在那里它控制16S加工、RNA折叠和30S亚基组装的最后阶段。我已经证明Era突变体阻断了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，它能进行核糖体组装和生长，但不能进行细胞分裂。我建议在Era-GTP/GDP周期中设置增长和分工的检查点，确保两者的协调。时代同系物在生命的所有领域都是保守的。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。