Lambda Genetic Networks and Lambda Red-Mediated Recombination

Lambda 遗传网络和 Lambda Red 介导的重组

• 批准号: 8348981
• 负责人: DONALD COURT
• 金额: $ 158.63万
• 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/8348981
• 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被广泛用作基因调节的范例，是开发理论建模方法的主要系统，对于解决参与信号转导，凋亡，癌症发育和其他系统的复杂遗传网络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作为研究发育回路的模型系统，调节Lambda电路的基因以及宿主/噬菌体相互作用。原核生物的病毒以及真核生物使用宿主功能来实现其发育生命周期并应对感染细胞的环境条件。我们认为，病毒针对宿主的关键功能用于病毒发育，这些功能是宿主基本感觉系统的一部分，以对环境做出反应。关于与Lambda的宿主相互作用的知识与真核病毒的研究有关。从本质上讲，病毒告诉我们哪些在宿主中最重要的是什么以及如何研究。温带噬菌体lambda是进行此类研究的绝佳工具。它可以作为能够在破坏宿主时快速繁殖的裂解病毒发展，也可以作为溶菌原发展为宿主基因组中的休眠病毒。多年来，它如何决定这些命运一直是研究对象，这些研究继续揭示了有关此简单系统的引人入胜的新发现，该系统被广泛用作理解和描述所有生物体的遗传电路网络的模型。这样的模型研究取决于有关其组件的准确和详细信息，而Lambda是一个很好的系统，因为它具有丰富的科学历史。我们的Lambda研究是多方面的。我们正在尝试通过使用裂解和溶菌发生途径的直接读数来描述LAMBDA感染后的裂解/裂解性决策。该读数采取感染后连续的细胞内GFP测量的形式，该读数测量了质量输出和CII功能的Q功能，以造成溶菌发生。感染细胞的噬菌体数量以及生长条件也会影响决策。 CIII，CI和CRO等其他基因功能对Q和CII函数的活性具有影响。我们为细菌染色体内的Lambda PL和PR早期启动子设计了一个记者系统。该记者允许我们检查CI阻遏物以及左OL和右或操作员对预言状态的压制和归纳的影响。我们的研究从遗传学上验证了两个操作员区域的相互作用，这是由于每个操作员在每个操作员的协同结合而发生的，以形成八聚体。这种阻遏物八聚化和OL的连接与预言状态下的压抑或增加了抑制作用，并防止了操作员的CRO作用，直到通过诱导消除阻遏活性为止。这是与Ptashne的遗传转换相矛盾的结果。 N是裂解途径的关键调节蛋白。 lambda n抗固定器是用于理解HIV的TAT转录抗固定器蛋白的范例。从经典上讲，n被称为转录的积极调节剂。我们最近发现N也是其自身翻译的负面调节器。作为阳性调节剂，N通过将RNA聚合酶（RNAPOL）转换为对转录终止具有抗性的形式，修饰了在PL和PR启动子处启动的转录伸长络合物。 N使用RNA作为束系与延长的RNA相互作用的几种宿主蛋白​​结合了RNA位点的螺母，与伸长的rnapol相互作用，形成抗释放络合物。作为负调节剂，n抗释放复合物抑制了n翻译。大肠杆菌DSRNA内核酸酶RNAseIII，它是参与RNAi的真核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的细胞水平受增长率控制，RNAseIII的水平坐标了N。由于其他噬菌体基因转录需要N抗固定器，因此N级别的这种控制也会影响n级的控制水平，如我们所描述的那样。在过去的几年中，修饰染色体并在细菌中进行“基因疗法”的能力迅速发展。我们对Lambda红色重组功能的研究对于这一进步至关重要。使用基于红色功能重组的哺乳动物细胞中的基因治疗是实际的可能性。哺乳动物病毒（例如HSV）使用与噬菌体lambda相同的红色重组功能。 Lambda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用于一种新形式的同源重组依赖性基因工程，称为重组。重新组合是可能的，因为红色函数可用于基于同源性将体外生成的线性DNA引导至细胞中的靶标。使该系统实用的是，用于靶向的短同源物足够​​，重组频率很高。另外，目标对基础是精确的，不需要任何限制地点。线性双链（DSDNA）可以通过PCR或仅通过退火两种合成寡核苷酸来生成，并且需要EXO，Beta和GAM功能以生成重组剂。 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。 Beta是一种单链DNA（ssDNA）结合蛋白，将其悬垂并将其退火为互补的ssDNA。除dsDNA外，短合成ssDNA寡核苷酸也可以直接与细胞中的靶标重新组合。这种重组仅需要β蛋白，而不需要EXO或GAM。它也与RECA无关，在适当的条件下，它以25％的效率产生重组细菌，从而使重组者直接筛选。我们正在研究系统以优化基因工程的方面，并了解细胞中线性DNA的红色重组是如何发生的。使用基因组克隆在F质粒衍生的细菌性人工染色体（BAC）上，重新组合对所有类型的真核遗传研究至关重要。修饰的克隆在大肠杆菌中产生，并重新引入其天然基因组背景进行研究。