Groenen M A, van de Putte P
Laboratory of Molecular Genetics, University of Leiden, The Netherlands.
J Cell Sci Suppl. 1987;7:41-50. doi: 10.1242/jcs.1987.supplement_7.4.
To analyse the functional and structural requirements of Mu transposition a special mini-Mu transposon was constructed. This mini-Mu, situated on a derivative of pBR322, has an easily selectable marker gene conferring chloramphenicol resistance (Cam) cloned between the ends of the mini-Mu. The genes required for transposition are cloned outside the ends and are under control of the PL promoter of bacteriophage lambda. To obtain a high frequency of transposition the A and B products are required. Under B- conditions both cointegrate formation and simple insertions are strongly reduced. The role of B in the transposition process is unknown and is discussed. Deletion analysis of the ends of the mini-Mu has revealed the existence of multiple sites required for transposition. The same sites were found to be strong A-binding sites. In addition two weak A-binding sites were found. Point mutations introduced into these last sites show that they are at least as important for transposition as the strong binding sites. The organization of the binding sites is asymmetric in Mu. At the left end two binding sites are situated more than 100 bp away from the end. These affect specifically the frequency of transposition. The distance between these two sites and the site close to the left end is extremely critical. At the right end also three A-binding sites are found, but only two seem to be important for transposition. Transposition with low frequency can be obtained with a mini-Mu with only one end. Secondary att sites are selected which all show some homology with an A-binding site. An absolute requirement for a 5'T at the artificial end is observed. Based on these results a simple scheme for the evolution of a transposon is discussed.
为了分析Mu转座的功能和结构要求，构建了一种特殊的微型Mu转座子。这种位于pBR322衍生物上的微型Mu，在微型Mu的末端之间克隆了一个易于选择的赋予氯霉素抗性（Cam）的标记基因。转座所需的基因克隆在末端之外，并受噬菌体λ的PL启动子控制。为了获得高频率的转座，需要A和B产物。在B-条件下，共整合体形成和简单插入都大大减少。B在转座过程中的作用尚不清楚并进行了讨论。对微型Mu末端的缺失分析揭示了转座所需的多个位点的存在。发现相同的位点是强A结合位点。此外，还发现了两个弱A结合位点。引入这些最后位点的点突变表明，它们对转座至少与强结合位点一样重要。Mu中结合位点的组织是不对称的。在左端，两个结合位点位于距离末端100多个碱基对的位置。这些位点特别影响转座频率。这两个位点与靠近左端的位点之间的距离极其关键。在右端也发现了三个A结合位点，但似乎只有两个对转座很重要。用只有一端的微型Mu可以获得低频转座。选择了二级附着位点，它们都与A结合位点有一些同源性。观察到人工末端对5'T有绝对要求。基于这些结果，讨论了转座子进化的简单方案。
