Recombination, Mutagenesis and Evolution of Phage T4 DNA

快速扩展的序列数据库的比较表明，已经有广泛的横向交换和转移的基因在进化的规模。 包括逆转录病毒在内的转座因子以及病毒和质粒的位点特异性重组系统已经涉及横向基因转移。 最近的研究结果表明，横向基因转移也可以使用同源重组机制，在有限同源性的序列之间启动，并且多个突变可以或多或少同时出现在相邻区域。 这表明DNA序列的横向转移可以同时导致新基因的获得、多组分控制元件（如DNA复制起点）的进化以及相当大的序列差异。 为了解释这些观察结果，提出了一个模型，其中异源双链体之间形成部分匹配的序列。 随后的异源双链修复同时产生多个突变，重组依赖性DNA复制解决重组中间产物并将一些未改变的外源DNA拼接到常驻基因组中。 强大的选择压力维持了那些编码功能性必需基因的重组体。 该模型意味着，类似的重组和修复步骤，产生了横向基因转移的“幸存者”之间的序列分歧，现在他们之间产生重组障碍。 该项目的主要目的是测试该模型，目的是更好地理解重组过程及其对基因突变和进化的影响。 噬菌体T4和相关的噬菌体（T-evens）对于这些研究具有几个优点：1）高重组潜力，2）包装的DNA末端和造成的断裂对重组的刺激，3）对明显冗余的理解，重组和DNA复制的交织和非线性途径，以及4）这些途径的酶在不同的复合物中以各种组合混合和匹配，执行不同的功能，这些功能差异地影响不同的重组途径和DNA复制的起始。 在上述假设的框架内，结合遗传学、基因组学和生物化学方法，正在解决以下问题：1）通过横向基因转移获得的基因内部或附近产生的明显序列差异达到何种程度？ 2)序列差异在多大程度上导致了某些基因被相关基因排除（例如，例如，在一个实施例中，T4排除T2，RB 69排除T4）？ 3)哪些噬菌体或宿主编码的复制、重组和修复蛋白以及限制酶参与了这种排斥？以及4）在何种程度上可以解释基因碱基序列的差异大于来自不同生物体的直系同源和旁系同源蛋白质的氨基酸序列的差异，作为通过同源重组的侧向转移的致突变潜力的结果？ 主要的策略是使用质粒和质粒含有同源的，但分歧的基因从不同的T-甚至这些基因的嵌合体。 将检测噬菌体和宿主重组以及限制性内切酶对重组的影响，包括子代的活力以及潜在异源双链体环的形成、持续或消除。 后者将通过非变性DNA的Southern印迹和包装后代DNA的测序进行监测。 了解这些重组和排斥机制是相关的进化分支，系统发育树和节奏的解释。 它也与理解可以产生抗体多样性的“错误”有关。

Comparisons of rapidly expanding sequence databases suggest that there has been extensive lateral exchange and transfer of genes on an evolutionary scale. Transposable elements, including retroviruses, and site specific recombination systems of viruses and plasmids have been implicated in lateral gene transfer. Recent results have indicated that lateral gene transfer can also use homologous recombination mechanisms, initiated between sequences of limited homology, and that multiple mutations can appear more or less simultaneously in an adjacent region. This suggests that lateral transfer of DNA sequences can simultaneously lead to acquisition of new genes, the evolution of multi-partite control elements, such as origins of DNA replication, and to considerable sequence divergence. To explain these observations, a model is proposed in which heteroduplexes are formed between partially matching sequences. Subsequent heteroduplex repair simultaneously generates multiple mutations, and recombination-dependent DNA replication resolves the recombination intermediates and splices some unchanged foreign DNA into the resident genome. Strong selection pressure maintains those recombinants that code for functional essential genes. The model implies that similar recombination and repair steps that generated the sequence divergence between the 'survivors' of lateral gene transfer now generate recombinational barriers between them. The major aim of this project is a test of this model, with the goal of better understanding recombination processes and their consequences for mutagenesis and evolution of genes. Phage T4 and related phages (T-evens) have several advantages for these studies: 1) a high recombination potential, 2) the demonstrated stimulation of recombination by packaged DNA ends and inflicted breaks, 3) an understanding of apparently redundant, interwoven and non-linear pathways of recombination and DNA replication and 4) an appreciation that the enzymes of these pathways are mixed and matched in various combinations in different complexes, performing different functions that differentially affect different pathways of recombination and of initiation of DNA replication. Within the framework of the hypotheses outlined above, the following questions are being addressed, combining genetic, genomic and biochemical approaches: 1) to what extent are apparent sequence differences generated within or in the vicinity of genes that had been acquired by lateral gene transfer? 2) to what extent are sequence differences responsible for exclusion of certain phages by related phages (e. g., T4 excludes T2, and RB69 excludes T4 )? 3) which phage- or host-encoded replication, recombination, and repair proteins and restriction enzymes participate in such exclusion? and 4) to what extent can one explain the larger differences in base sequences of genes than in amino acid sequences of orthologous and paralogous proteins from different organisms as consequences of the mutagenic potential of lateral transfer by homologous recombination? The main strategy is to use phages and plasmids containing homologous, but diverged genes from different T-even phages and chimeras of these genes. Effects of phage and host recombination and restriction enzymes on recombination will be tested on viability of the progeny and on formation, persistence or elimination of potential heteroduplex loops. The latter will be monitored by Southern blotting of non-denatured DNA and by sequencing of packaged progeny DNA. Understanding these recombination and exclusion mechanisms is relevant to interpretations of clades, phylogenetic trees and tempos of evolution. It is also relevant for understanding 'errors' that can generate antibody diversity.