Sauer B
DuPont-Merck Pharmaceutical Company, Wilmington, DE 19880-0328.
J Mol Biol. 1992 Feb 20;223(4):911-28. doi: 10.1016/0022-2836(92)90252-f.
The Cre recombinase efficiently causes site-specific DNA recombination at loxP sites placed into the eukaryotic genome. Since the loxP site of phage P1 is 34 base-pairs in size, the natural occurrence of this exact sequence is unlikely in any eukaryotic genome. However, related sequences may exist in eukaryotic genomes that could recombine at low efficiency with an authentic loxP site. This work identifies such cryptic lox sites in the yeast genome using a positive selection procedure that allows the detection of events occurring at a frequency of less than 1 x 10(-7). The selection is based on the disruption/reconstruction of the yeast gene YGL022. Disruption of YGL022 confers multiple drug sensitivity. Recombination events at a loxP site 5' to the structural gene restore expression of YGL022 and result in a multiple drug resistant phenotype. These drug resistant mutants all display chromosomal rearrangements resulting from low-frequency Cre-mediated recombination with an endogenous cryptic lox site. Ten such sites have been found and they have been mapped physically to a number of different yeast chromosomes. Although the efficiency of Cre-mediated recombination between loxP and such endogenous sites is quite low, it may be possible to redesign recombination substrates to improve recombination efficiency. Because of the greater complexity of the human and mouse genomes compared with yeast, an analogous situation is likely to exist in these organisms. The availability of such sites would be quite useful in the development of alternative strategies for gene therapy and in the generation of transgenic animals.
Cre重组酶能有效地在导入真核基因组的loxP位点引发位点特异性DNA重组。由于噬菌体P1的loxP位点大小为34个碱基对，在任何真核基因组中不太可能天然出现这种精确序列。然而，真核基因组中可能存在相关序列，它们可能与真实的loxP位点以低效率发生重组。本研究利用一种阳性筛选程序在酵母基因组中鉴定出此类隐蔽的lox位点，该程序能检测到发生频率低于1×10⁻⁷的事件。筛选基于酵母基因YGL022的破坏/重建。YGL022的破坏赋予多重药物敏感性。位于结构基因5'端的loxP位点发生重组事件可恢复YGL022的表达，并产生多重耐药表型。这些耐药突变体均表现出由低频Cre介导的与内源性隐蔽lox位点重组导致的染色体重排。已发现10个这样的位点，并已将它们物理定位到许多不同的酵母染色体上。尽管loxP与此类内源性位点之间的Cre介导重组效率相当低，但有可能重新设计重组底物以提高重组效率。由于与酵母相比，人类和小鼠基因组更为复杂，在这些生物体中可能存在类似情况。此类位点的存在对于开发基因治疗的替代策略以及产生转基因动物将非常有用。
