Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

• 批准号: 8552671
• 负责人: DONALD COURT
• 金额: $ 168.21万
• 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/8552671
• 关键词: Address; Affect; Apoptosis; Bacteria; Bacterial Artificial Chromosomes; Bacterial Chromosomes; Bacteriophage lambda; Bacteriophages; Binding; Biological Models; Cell division; Cells; Chromosomes; Complex; Cytolysis; DNA; DNA-Directed RNA Polymerase; Development; Double-Stranded RNA; Early Promoters; Endoribonucleases; Engineering; Environment; Escherichia coli; Eukaryota; F Factor; Frequencies; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Engineering; Genetic Recombination; Genetic Transcription; Genome; Genomics; Growth; HIV; Homologous Gene; In Vitro; Infection; Laboratories; Learning; Left; Lysogeny; Lytic; Lytic Virus; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Mediating; Methodology; Modeling; Oligonucleotides; Operator Regions; Organism; Output; Pathway interactions; Process; Prokaryotic Cells; Prophages; Protein Binding; Proteins; Proviruses; RNA; RNA Binding; RNA Interference; Recombinants; Recording of previous events; Regulation; Reporter; Repression; Resistance; Ribonuclease III; 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

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 viral growth and N gene expression. 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，该方法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 lambda 作为模型系统来研究发育回路、调节 lambda 回路的基因以及宿主/噬菌体相互作用。原核生物和真核生物的病毒利用宿主功能来完成其发育生命周期并对受感染细胞的环境条件做出反应。我们认为，病毒以宿主病毒发育的关键功能为目标，这些功能是宿主对环境做出反应的基本感觉系统的一部分。我们了解到的宿主与 lambda 相互作用的知识与真核病毒的研究相关。从本质上讲，病毒告诉我们宿主体内最重要的是什么以及如何研究它。温带噬菌体 lambda 是此类研究的一个很好的工具。它可以发展为一种裂解病毒，能够在破坏其宿主的同时快速繁殖，或者它可以发展为一种溶原病毒，作为宿主基因组中的休眠原病毒存在。多年来，它如何决定这些命运一直是研究的对象，这些研究不断揭示有关这个简单系统的令人着迷的新发现，该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。此类模型研究依赖于其组成部分的准确而详细的信息，而 lambda 因其丰富的科学历史而成为一个很好的构建系统。我们的 lambda 研究是多方面的。我们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。该读数采用感染后连续细胞内 GFP 测量的形式，测量裂解输出的 Q 功能和溶原发育的 CII 功能。感染细胞的噬菌体数量和生长条件都会影响决策。其他基因功能如 CIII、CI 和 Cro 对 Q 和 CII 功能的活动有影响。我们为细菌染色体内的 lambda pL 和 pR 早期启动子设计了一个报告系统。该报告器使我们能够检查 CI 阻遏蛋白以及左 oL 和右 oR 操作符对前噬菌体状态的抑制和诱导的影响。我们的研究从基因角度验证了两个操纵基因区域的相互作用，这种相互作用是通过每个操纵基因上的阻遏物四聚体协同结合形成八聚体而发生的。这种阻遏蛋白八聚化以及 oL 与 oR 的连接增加了原噬菌体状态的阻抑作用，并阻止了操作者的 Cro 作用，直到通过诱导消除阻遏蛋白活性。这是与 Ptashne 的遗传开关相矛盾的结果。N 是裂解途径的关键调节蛋白。 lambda N 抗终止子是用于理解 HIV 的 Tat 转录抗终止子蛋白的范例。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。作为正调节因子，N 通过将 RNA 聚合酶 (RNAPol) 转化为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 与几个称为 Nus 的宿主蛋白结合 RNA 位点 NUT，使用 RNA 作为系链与伸长的 RNAPol 相互作用，形成抗终止复合物。作为负调节因子，N 抗终止复合物抑制 N 翻译。大肠杆菌 dsRNA 内切核糖核酸酶 RNaseIII 是参与 RNAi 的真核生物 dicer 蛋白的细菌同源物，可调节病毒生长和 N 基因表达。全局调节剂 RNaseIII 的细胞水平受生长速率控制，RNaseIII 的水平协调 N 的水平。由于其他噬菌体基因转录需要 N 抗终止子，因此对 N 水平的控制也会影响我们所描述的裂解/溶原性发育。在细菌中修饰染色体和进行“基因治疗”的能力在过去几年中进展迅速。我们对 lambda Red 重组函数的研究对于这一进展至关重要。使用基于红色功能的重组对哺乳动物细胞进行基因治疗是一种真正的可能性。哺乳动物病毒，如 HSV，使用与 lambda 噬菌体相同的 Red 样重组功能。 lambda Red 蛋白包括 Exo、Beta 和 Gam。我们发现细菌细胞中的 Red 功能可用于一种新形式的同源重组依赖性基因工程，称为重组工程。重组工程是可能的，因为 Red 函数可用于根据同源性将体外生成的线性 DNA 引导至细胞中的靶标。该系统在工程上的实用性在于50bp的短同源性足以用于靶向，并且重组频率非常高。此外，靶向精确到碱基，不需要任何限制性位点。线性双链 (dsDNA) 可以通过 PCR 或仅通过退火两个合成寡核苷酸来生成，并且需要 Exo、Beta 和 Gam 功能来生成重组体。 Gam 灭活宿主核酸酶以保护转化的 DNA。 Exo和Beta进行同源重组。 Exo 结合 dsDNA 并降解 5' 末端，产生 3' 突出端。 Beta 是一种单链 DNA (ssDNA) 结合蛋白，可结合突出端并将其退火至互补的 ssDNA。除了 dsDNA 之外，短的合成 ssDNA 寡核苷酸也可以直接与细胞中的靶标重组。这种重组仅需要 Beta 蛋白，不需要 Exo 或 Gam。它还独立于 RecA，在适当的条件下以 25% 的效率生成重组细菌，从而使重组体的筛选变得简单。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 Red 重组如何在细胞中发生。重组工程对于使用 F 质粒衍生的细菌人工染色体 (BAC) 上的基因组克隆进行的所有类型的真核遗传研究都至关重要。在大肠杆菌中生成修饰的克隆，并将其重新引入其天然基因组背景中以供研究。