Yankovsky N K, Bukanov N O, Yakubovich N V, Ermakova L M, Rebentish B A, Janulaitis A A, Debabov V G
Institute for Genetics and Selection of Industrial Microorganisms, Moscow, U.S.S.R.
Gene. 1989 Sep 30;81(2):203-10. doi: 10.1016/0378-1119(89)90180-7.
Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.
构建了噬菌粒λ pMYF131，它是噬菌体λ载体与质粒pUC19的杂交体。该噬菌粒及其衍生物被证明是在大肠杆菌细胞中构建和分析基因文库的有效载体。λ pMYF131 DNA分子包含噬菌体裂解发育所必需的所有基因和区域。由于其特定长度，该质粒无论是单体形式还是寡聚体形式都不能被包装。通过与外源DNA片段连接使DNA分子延长可使其能够被包装，这很容易通过噬菌斑形成检测到。因此，通过仅在体外包装时并基于其体外包装来选择重组DNA分子，可简化用于构建基因组文库的程序。与噬菌体载体λ L47.1AB相比，载体λ pMYF131每个载体分子的重组克隆产量高出数倍，达到每微克DNA 3×10⁶个克隆。载体和重组噬菌粒可以大量以质粒形式获得。λ pMYF131包含九个独特的限制性酶切位点，可用于克隆平端DNA片段以及用14种原型限制性酶消化获得的具有各种粘性末端类型的片段。对于λ pMYF131，克隆DNA片段的最大大小约为20 kb。噬菌粒载体用于构建牛、猪和鹌鹑基因组文库以及17种细菌的基因组文库。应用合适的方法可在这些文库中鉴定出13个单个基因。
