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Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

基本信息

批准号：
7733005
负责人：
DONALD COURT
金额：
$ 153.54万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7733005
关键词：
Address Affect Apoptosis Bacteria Bacterial Artificial Chromosomes Bacterial Chromosomes Bacteriophage lambda Bacteriophages Binding Biological Models Cell division Cells Chromosomes Complex Condition Cytolysis DNA DNA-Directed RNA Polymerase Development Development, Other Double-Stranded RNA Early Promoters Endoribonucleases Engineering Environment Escherichia coli Eukaryota Eukaryotic Cell F Factor Frequencies Gene Expression Regulation Generations Genes Genetic Genetic Engineering Genetic Recombination Genetic Transcription Genome Genomics Green Fluorescent Proteins Growth HIV Homologous Gene In Vitro Infection Laboratories Learning Left Lysogeny Lytic Lytic Virus Malignant Neoplasms Mammalian Cell Measurement Measures Mediating Methodology Modeling Numbers Nuts Oligonucleotides Operator Regions Organism Output Pathway interactions Polymerase Chain Reaction Process Prokaryotic Cells Prophages Protein Binding Proteins Proviruses RNA RNA Binding RNA Interference Rate Recombinants Recording of previous events Regulation Reporter Repression Resistance Ribosomal RNA Ribosomes Role SS DNA BP Screening procedure Signal Transduction Simplexvirus Single-Stranded DNA Site Study models System Systems Biology Theoretical model Transcription Elongation Translations Viral Virus antitermination base design endoribonuclease fascinate gene function gene therapy genetic manipulation genetic regulatory protein homologous recombination human DICER1 protein nuclease prevent promoter sensory system tool transcription termination

项目摘要

The bacterial virus lambda is widely used as a paradigm for gene regulation and is a premier system for developing theoretical modeling methodologies, which are becoming increasingly important for addressing complex genetic networks involved in signal transduction, apoptosis, cancer development, and other systems. Our laboratory uses E. coli and lambda as a model system for studying developmental circuits, the genes that regulate lambda circuitry, and host/phage interactions. Viruses of prokaryotes as well as eukaryotes use host functions to fulfill their developmental lifecycle and respond to the environmental conditions of the infected cell. We believe that the virus targets critical functions of the host for viral development and those functions are part of the basic sensory system of the host for reacting to the environment. The things we learn about host interactions with lambda is relevant for studies of eukaryotic viruses. Essentially, the viruses tell us what is most important in the host and how to study it. The temperate bacteriophage lambda is a great tool for such studies; it can develop as a lytic virus able to rapidly reproduce while destroying its host, or it can develop as a lysogen existing as a dormant provirus within the host genome. How it decides these fates has been an object of study for years, and these studies continue to reveal fascinating new discoveries about this simple system, a system that is widely used as a model for understanding and describing genetic circuitry networks for all organisms. Such model studies depend upon accurate and detailed information about its components, and lambda is a great system to build on because of its rich scientific history. Our lambda studies are multifaceted. We are attempting to describe the lysis/lysogeny decision following lambda infection by using direct readouts for the lytic and the lysogenic pathways. This readout takes the form of continuous intracellular GFP measurements following infection, which measure Q function for lytic output and CII function for lysogenic development. The number of phage infecting a cell affects the decision, as do the growth conditions. Other gene functions like CIII, CI, and Cro have effects on the activities of the Q and CII functions. We have designed a reporter system for the lambda pL and pR early promoters within the bacterial chromosome. This reporter allows us to examine the effects CI repressor and the left oL and right oR operators on repression and induction in the prophage state. Our studies have verified genetically an interaction of the two operator regions, which occurs by a cooperative binding of the repressor tetramers at each operator to form an octamer. This repressor octamerization and joining of oL with oR increases repression in the prophage state and prevents Cro action at the operators until repressor activity is eliminated by induction. This is a result that contradicts the Genetic Switch of Ptashne. N is a critical regulatory protein for the lytic pathway. The lambda N antiterminator is the paradigm used to understand the Tat transcription antiterminator protein of HIV. Classically, N is known to act as a positive regulator of transcription; we recently found that N is also a negative regulator of its own translation. As a positive regulator, N modifies the transcription elongation complexes that initiate at the pL and pR promoters by converting RNA polymerase (RNAPol) to a form that is resistant to transcription termination. N with several host proteins called Nus bind RNA sites, NUT, using the RNA as a tether to interact with the elongating RNAPol to form the antitermination complex. As a negative regulator, the N antitermination complex represses N translation. The E. coli dsRNA endoribonuclease, RNaseIII, which is the bacterial homolog of the eukaryotic dicer protein involved in RNAi, regulates N's repression of its own translation. Cellular levels of the global regulator RNaseIII are controlled by growth rate, and the level of RNaseIII coordinates the level of N. Since N antiterminator is required for other phage genes transcription, this control on N levels also affects the lytic/lysogenic development as we describe. The ability to modify the chromosome and carry out 'gene therapy' in bacteria has progressed rapidly in the last few years. Our studies with the lambda Red recombination functions have been critical for this advance. Gene therapy in mammalian cells using recombination based on the Red functions is a real possibility. Mammalian viruses, like HSV, use the same Red-like recombination functions as phage lambda. The lambda Red proteins include Exo, Beta, and Gam. We have discovered that Red function in the bacterial cell can be used for a new form of homologous recombination-dependent genetic engineering, called recombineering. Recombineering is possible because the Red functions can be used to direct in vitro-generated linear DNAs to targets in the cell based on homology. What makes the system practical for engineering is that short homologies of 50 bp are sufficient for targeting, and the recombination frequency is very high. In addition, the targeting is precise to the base and does not require any restriction sites. Linear double-strand (dsDNA) can be generated by PCR or just by annealing two synthetic oligonucleotides, and requires Exo, Beta, and Gam function for generation of recombinants. Gam inactivates host nucleases to protect the transformed DNA. Exo and Beta carry out the homologous recombination. Exo binds the dsDNA and degrades the 5' end generating 3' overhangs. Beta, a single-strand DNA (ssDNA) binding protein, binds the overhangs and anneals them to complementary ssDNA. In addition to dsDNA, short synthetic ssDNA oligonucleotides can also be directly recombined with the target in the cell. This recombination requires only the Beta protein and not Exo or Gam. It is also independent of RecA and under appropriate conditions generates recombinant bacteria at an efficiency of 25%, making screening for recombinants straightforward. We are studying the system both to optimize the aspect of genetic engineering and to understand how Red recombination of linear DNA occurs in the cell. Recombineering has become vital to all types of eukaryotic genetic studies using genomic clones on F plasmid-derived bacterial artificial chromosomes (BACs). Modified clones are generated in E. coli and reintroduced into their native genomic background for study.

细菌病毒lambda被广泛用作基因调控的范例，并且是开发理论建模方法的首要系统，这对于解决涉及信号转导，细胞凋亡，癌症发展和其他系统的复杂遗传网络变得越来越重要。我们的实验室使用大肠杆菌和λ作为模型系统来研究发育回路、调节λ回路的基因和宿主/噬菌体相互作用。原核生物和真核生物的病毒都利用宿主的功能来完成它们的发育生命周期，并对被感染细胞的环境条件作出反应。我们认为，该病毒针对宿主的关键功能进行病毒发育，这些功能是宿主对环境作出反应的基本感觉系统的一部分。我们对宿主与λ相互作用的了解与真核病毒的研究相关。从本质上讲，病毒告诉我们宿主中最重要的是什么，以及如何研究它。温带噬菌体是这类研究的一个很好的工具；它可以发展为一种裂解病毒，能够在破坏宿主的同时迅速繁殖，也可以发展为宿主基因组中作为休眠前病毒存在的溶原。它如何决定这些命运多年来一直是研究的对象，这些研究继续揭示关于这个简单系统的迷人新发现，这个系统被广泛用作理解和描述所有生物体遗传电路网络的模型。这样的模型研究依赖于关于其组成部分的准确和详细的信息，lambda是一个很好的系统，因为它有丰富的科学历史。我们的lambda研究是多方面的。我们正试图通过使用裂解和溶原途径的直接读数来描述lambda感染后的裂解/溶原决定。该读数采用感染后细胞内连续GFP测量的形式，其中测量裂解输出的Q函数和溶原发育的CII函数。感染细胞的噬菌体数量和生长条件都会影响这一决定。其他基因功能如CIII、CI和Cro对Q和CII功能的活性也有影响。我们设计了一个细菌染色体内lambda pL和pR早期启动子的报告系统。本报告允许我们检查的影响CI阻遏因子和左oL和右oR操作符在抑制和诱导在噬菌体状态。我们的研究已经证实了两个操作区域的遗传相互作用，这是通过每个操作区域的阻遏物四聚体的合作结合形成八聚体而发生的。这种阻遏物的八聚化和oL与oR的结合增加了在前噬菌体状态下的抑制，并阻止了操作符上的Cro作用，直到阻遏物活性被诱导消除。这是一个与Ptashne基因开关相矛盾的结果。N是裂解途径的关键调控蛋白。λ N抗终止蛋白是用来理解HIV的Tat转录抗终止蛋白的范例。传统上，N被认为是转录的正调节因子；我们最近发现N也是其自身翻译的负调节因子。作为一种正调节因子，N通过将RNA聚合酶（RNAPol）转化为一种抵抗转录终止的形式来修饰在pL和pR启动子处启动的转录延伸复合物。N与几种称为Nus的宿主蛋白结合RNA位点NUT，利用RNA作为系链与拉长的RNAPol相互作用形成抗终止复合物。作为负调节因子，N反终止复合物抑制N翻译。大肠杆菌dsRNA核糖核酸内切酶RNaseIII是参与RNAi的真核肿瘤蛋白的细菌同源物，调节N对其自身翻译的抑制。全局调控因子RNaseIII的细胞水平受生长速率控制，而RNaseIII的水平与N的水平相协调。由于其他噬菌体基因转录需要抗N终止物，因此这种对N水平的控制也影响了我们所描述的溶解/溶原发育。在过去几年中，在细菌中修改染色体和实施“基因治疗”的能力取得了迅速进展。我们对lambda Red重组函数的研究对这一进展至关重要。利用基于红蛋白功能的重组对哺乳动物细胞进行基因治疗是切实可行的。哺乳动物病毒，如HSV，使用与噬菌体相同的Red-like重组功能。红蛋白包括Exo、Beta和Gam。我们已经发现，细菌细胞中的红色功能可以用于一种新的形式的同源重组依赖基因工程，称为重组。重组是可能的，因为Red功能可用于根据同源性指导体外生成的线性dna到细胞中的目标。使该系统具有工程实用性的是50 bp的短同源性足以用于靶向，并且重组频率很高。此外，目标精确到基地，不需要任何限制地点。线性双链（dsDNA）可以通过PCR或仅通过退火两个合成的寡核苷酸生成，并且需要Exo， Beta和Gam功能来生成重组体。Gam使宿主核酸酶失活以保护转化的DNA。Exo和Beta进行同源重组。Exo结合dsDNA并降解5‘端，产生3’悬垂。β是一种单链DNA （ssDNA）结合蛋白，它将悬垂物结合并将其退火为互补的ssDNA。除了dsDNA外，短合成的ssDNA寡核苷酸也可以在细胞内与靶标直接重组。这种重组只需要β蛋白，而不需要Exo或Gam。它也独立于RecA，在适当的条件下以25%的效率产生重组细菌，使重组细菌的筛选变得简单。我们正在研究该系统，以优化基因工程方面，并了解线性DNA的红色重组如何在细胞中发生。利用F质粒衍生的细菌人工染色体（BACs）上的基因组克隆进行重组对所有类型的真核遗传研究都至关重要。改良的克隆在大肠杆菌中产生，并重新引入其原生基因组背景中进行研究。