Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.
Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.
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DOI：
10.1073/pnas.88.15.6657
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发表时间：
1991-08
影响因子：
11.1
通讯作者：
F. Rassool;T. Mckeithan;M. E. Neilly;E. van Melle;R. Espinosa;M. L. Le Beau
中科院分区：
文献类型：
--
作者：
F. Rassool;T. Mckeithan;M. E. Neilly;E. van Melle;R. Espinosa;M. L. Le Beau
脆弱位点是染色体上容易断裂的特定区域。在诱导脆弱位点表达的条件下培养的细胞中，观察到含有脆弱位点的染色体带存在高水平的染色体间和染色体内重组。为了确定特异性脆弱位点的表达是否会促进外源DNA在这些重组热点的优先整合，我们将载体pSV2Neo转染到含有衍生3号染色体作为其唯一人类成分的中国仓鼠-人体细胞杂交体中。3号染色体在3p14.2 （FRA3B）带有一个由蚜虫蛋白诱导的共同脆性位点。诱导表达FRA3B的细胞和未诱导的对照细胞都转染了pSV2Neo选择质粒。生物素标记的pSV2Neo探针与中期染色体的原位杂交显示，每个稳定转染的克隆中有一到三个整合位点。在FRA3B诱导条件下转染的13个克隆中，有4个在3p14处整合了pSV2Neo；这些克隆还表现出特异性整合到仓鼠1号染色体和CHO细胞的重排染色体特征（mar2）。而7个对照克隆的pSV2Neo整合表现出明显的随机模式。pSV2Neo与FRA3B和中国仓鼠染色体1和mar2的显著杂交在100个细胞中被发现。这些结果表明，人类和中国仓鼠易碎部位的标记DNA优先整合发生在暴露于阿霉素的情况下。脆弱位点的DNA序列的性质是未知的，尽管有许多方法，这些序列尚未被分离出来；我们的程序可能代表了一种克隆脆弱位点的方法。
Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.