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噬菌体λ DNA体外整合重组的限制分析：对闭环DNA底物的要求。

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

作者信息

Mizuuchi K, Nash H A

出版信息

Proc Natl Acad Sci U S A. 1976 Oct;73(10):3524-8. doi: 10.1073/pnas.73.10.3524.

DOI:10.1073/pnas.73.10.3524
PMID:1068464
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC431149/
Abstract

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.

摘要

已开发出一种用于DNA体外基因重组的新型检测方法。用限制性内切核酸酶切割底物DNA和产物DNA，然后通过琼脂糖凝胶电泳分离所得片段。选择底物DNA以便所研究的重组删除一段DNA。剩余的DNA会产生一个独特的限制性片段，被去除的DNA片段也是如此。该方法提供了一种方便的、基于物理而非遗传的方法来评估亲本DNA向重组DNA的转化。该方法已应用于一个进行噬菌体λ整合重组的体外系统。我们发现，在所测试的不同分子形式的DNA中，闭环DNA是唯一有效的底物。线性DNA和三种含有缺口的环状DNA充其量只是非常差的底物。讨论了这一惊人结果的意义。此外，我们表明体外重组系统完成了重组的断裂和重新连接步骤。未发现涉及交叉或断裂末端结构的稳定DNA中间体。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/ae18b6b0022b/pnas00040-0208-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/ec6d03c6b023/pnas00040-0206-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/b4b12cee9645/pnas00040-0207-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/574d95469e1f/pnas00040-0207-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/82a0ce287f40/pnas00040-0208-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/ae18b6b0022b/pnas00040-0208-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/ec6d03c6b023/pnas00040-0206-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/b4b12cee9645/pnas00040-0207-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/574d95469e1f/pnas00040-0207-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/82a0ce287f40/pnas00040-0208-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3643/431149/ae18b6b0022b/pnas00040-0208-b.jpg

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本文引用的文献

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Mechanism of action of rat liver DNA methylase. I. Interaction with double-stranded methyl-acceptor DNA.大鼠肝脏DNA甲基化酶的作用机制。I. 与双链甲基受体DNA的相互作用。
J Mol Biol. 1971 May 14;57(3):475-89. doi: 10.1016/0022-2836(71)90104-5.
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Termination point of replication of colicin E1 plasmid DNA in cell extracts.大肠杆菌素E1质粒DNA在细胞提取物中的复制终止点。
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Model for wandering restriction enzymes.游走限制酶模型。
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Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay.利用一种简单的物理检测方法对噬菌体T7在体内的基因重组进行研究。
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New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.与脱氧核糖核酸回旋酶活性相关的新的萘啶酸抗性突变
J Bacteriol. 1981 Nov;148(2):450-8. doi: 10.1128/jb.148.2.450-458.1981.
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Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property.超螺旋DNA中反向重复序列形成发夹环是一种局部且可传递的特性。
Nucleic Acids Res. 1981 Mar 25;9(6):1271-89. doi: 10.1093/nar/9.6.1271.
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Genetic recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination.细菌质粒DNA的基因重组：体外分子内重组的电子显微镜分析
Proc Natl Acad Sci U S A. 1980 Aug;77(8):4847-51. doi: 10.1073/pnas.77.8.4847.
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Integrative recombination of bacteriophage lambda: extent of the DNA sequence involved in attachment site function.噬菌体λ的整合重组：参与附着位点功能的DNA序列范围。
Proc Natl Acad Sci U S A. 1980 Jun;77(6):3220-4. doi: 10.1073/pnas.77.6.3220.
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DNA gyrase action involves the introduction of transient double-strand breaks into DNA.DNA 回旋酶的作用涉及在 DNA 中引入瞬时双链断裂。
Proc Natl Acad Sci U S A. 1980 Apr;77(4):1847-51. doi: 10.1073/pnas.77.4.1847.
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Manipulation of restriction targets in phage lambda to form receptor chromosomes for DNA fragments.对噬菌体λ中的限制靶点进行操作，以形成用于DNA片段的受体染色体。
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6
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7
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J Mol Biol. 1974 Aug 25;87(4):797-816. doi: 10.1016/0022-2836(74)90085-0.
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Virology. 1974 Jan;57(1):207-16. doi: 10.1016/0042-6822(74)90121-4.
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