Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

• 批准号: 8937715
• 负责人: DONALD COURT
• 金额: $ 146.71万
• 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/8937715
• 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.

基因调节研究。 LAMBDA遗传网络的最新表征为使用Lambda作为理论建模方法的原型提供了一个框架，这些方法对于解决信号转导，癌症发展和其他真核生物的其他复杂遗传网络变得很重要。 Lambda与大肠杆菌的共同进化产生了与宿主最基本功能相关的遗传系统。通过检查Lambda与宿主系统之间的界面，我的实验室遵循噬菌体的踪迹，以了解对细胞生命和细胞系统的病毒剥削最重要和至关重要的。该病毒提供了有关这些细胞功能如何工作以及如何研究它们的线索。我实验室中的所有作品都来自这种哲学。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抑制剂基因共转印的两个基因REXA和REXB在很大程度上被忽略了，这对控制抑制剂活性及其合成的复杂lambda遗传网络的贡献被忽略了。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，免疫终结子与REXB基因的结束重叠，REXB的翻译会调节终结子，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，从而为Lambda的Biscable遗传开关模型增添了进一步的微妙，该模型为基因调节的数学模型提供了基础。我们已经开发了LAMBDA同源重组功能红色作为重新组合的试剂，这是一种革命性的体内基因工程技术，为从细菌到人的功能基因组研究提供了新的方法。重组允许从任何生物体中修改基因组克隆，并用于开发用于癌症和其他疾病相关研究的模型系统。在包括病原体在内的其他细菌中也开发了类似的重组系统，可用于开发疫苗，用于抗生素的分子靶标，噬菌体疗法和生物粘液素。我们证明了短的单链寡核苷酸与大肠杆菌和其他细菌中的重新分裂的同源靶标重组。噬菌体重组酶，例如β和rect，刺激了细胞中内源性低于低内源性水平的这种寡聚重组。结果表明，两种重组酶红色β和矩形的分子机制不同。 Beta需要复制目标DNA来启动和生成重组中间体，而RECT不需要DNA复制来生成中间体。这支持了Beta在复制叉上通过SS链退火作用的前提，而Rect rect构成了链入侵。我们计划类似地表征包括HSV-1 ICP8在内的其他重组酶重组的开始。 DNA的忠实转录取决于RNA聚合酶（RNAPOL）保持准确性在将输入核苷酸与模板匹配的准确性，以防止构成错误并保持模板和转录本之间的寄存器以防止滑板误差。建议不忠实地转录模板会导致各种疾病，包括某些癌症，唐氏综合症和阿尔茨海默氏病。我的实验室表明，RPOC D1143P聚合酶失物突变体引起了IS2插入元件的遗传不稳定。当与GREA和GREB转录因子中的缺陷结合使用时，不稳定进一步增强。我推测，rnapol综合体在掺杂并干扰DNA复制后停滞。这可能会导致DNA修复，并可能重排，这是较高生物体中癌症的常见原因。另一种类型的错误是转录打滑。大肠杆菌rnapol在模板DNA中转录AS或TS时会出现移码错误。我的实验室表明，lambda n-nus因子转录抗释放复合物修饰了rnapol并减少了这些天然滑动事件。 n可以防止转录滑倒，并且由于许多固有的终止剂的拉伸很长，因此滑倒和终止可能是相互联系的过程。这是调节滑动的第一个例子，例如其他N样转录调节剂，例如HIV TAT，也可能会影响滑板。我研究Lambda以了解病毒相互作用的宿主功能，以便我可以更好地了解它们在病毒和细胞中的作用。这些由病毒靶向的功能不仅对病毒很重要，而且还是细胞中一些最基本的调节和感觉成分。我的1970年代的工作定义了RNase III在其3'UTR中逆转lambda int基因表达中的转录后作用。现在已经解决了RNase III的几种结构，并为理解Drosha和Dicer在RNAi 3'UTR基因调节中的作用做出了巨大贡献。我们还描述了RNase III在RRNA处理与宿主NUS因子协调中的作用。 NUS因子在rRNA转录过程中修改了rnapol。 rnapol-nus转录可确保rRNA折叠，RNase III加工的协调和30S核糖体组装。 ERA也与30s核糖体组装密切相关。 ERA-GTP复合物与成熟16S rRNA的3'端结合，它控制16S处理，RNA折叠和30S亚基组装的最后阶段。我已经表明，ERA阻滞生长和大肠杆菌细胞分裂的突变体，我分离了功能突变体的分离，该突变体具有核糖体组装和生长的作用，但被阻断用于细胞分裂。我建议ERA-GTP/GDP周期具有生长和分裂的核对点，以确保其协调。 ERA同源物在生活的所有领域中都是保守的。人类时代的缺陷还会导致线粒体小核糖体亚基的功能障碍，导致细胞生长差。