van Drunen C M, Mientjes E, van Zuylen O, van de Putte P, Goosen N
Laboratory of Molecular Genetics, Gorlaeus Laboratories, Leiden University, The Netherlands.
Nucleic Acids Res. 1994 Mar 11;22(5):773-9. doi: 10.1093/nar/22.5.773.
In this paper we determine which of the A binding sites in the attachment sites of phage Mu are required for the stimulatory activity of the transpositional enhancer (IAS). For this purpose the transposition frequencies of mini-Mu's with different truncated attachment sites to an Ftet target were measured both in the presence and the absence of the IAS. The results show that in our in vivo assay the L3 and R3 sites are dispensable for functioning of the IAS. An additional deletion of L2 or R2 however abolishes the stimulating activity of the enhancer suggesting an interaction between A molecules bound to these sites and the IAS. The residual transposition activity of a IAS-containing mini Mu in which R2 (and R3) are deleted is much lower than the activity of the comparable construct without the IAS. This means that in the absence of R2 the IAS is inhibiting transposition. Such an inhibition is not observed when L2 (and L3) are deleted. This suggests that the IAS interacts with the attachment sites in an ordered fashion, first with attL and then with attR. Furthermore we show that mini-Mu transposition is enhanced when Fpro-lac is used as a target instead of Ftet. We show that this elevated transposition is dependent on the Mu A binding sites L2,L3 and R2. These sequences could possibly mediate an interaction between the mini-Mu plasmid and sequences present on Fpro-lac.
在本文中，我们确定了噬菌体Mu附着位点中的哪些A结合位点是转座增强子（IAS）刺激活性所必需的。为此，在有和没有IAS的情况下，测量了具有不同截短附着位点的微型Mu到Ftet靶标的转座频率。结果表明，在我们的体内试验中，L3和R3位点对于IAS的功能是可有可无的。然而，额外缺失L2或R2会消除增强子的刺激活性，这表明与这些位点结合的A分子与IAS之间存在相互作用。在缺失R2（和R3）的含IAS微型Mu中，其残余转座活性远低于不含IAS的可比构建体的活性。这意味着在没有R2的情况下，IAS会抑制转座。当缺失L2（和L3）时，未观察到这种抑制作用。这表明IAS以有序的方式与附着位点相互作用，首先与attL相互作用，然后与attR相互作用。此外，我们表明，当使用Fpro-lac作为靶标而不是Ftet时，微型Mu转座会增强。我们表明，这种转座增强依赖于Mu A结合位点L2、L3和R2。这些序列可能介导微型Mu质粒与Fpro-lac上存在的序列之间的相互作用。
