Mittelsten Scheid O, Afsar K, Paszkowski J
Friedrich Miescher Institute, Basel, Switzerland.
Mol Gen Genet. 1994 Aug 2;244(3):325-30. doi: 10.1007/BF00285461.
Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.
多拷贝转基因插入片段在高等植物中的染色体整合常常伴随着表达丧失。我们分析了这种失活是否会像在粗糙脉孢菌中观察到的那样引发重复序列诱导的点突变(RIP)。我们之前已对拟南芥转基因株系进行了表征,这些株系含有潮霉素磷酸转移酶(hpt)基因,该基因在表达该基因的植株中为单一序列,或者在转化后直接对潮霉素具有抗性但在后代中表现出基因失活的克隆中为多聚体、紧密相连的重复序列。在序列水平上,我们确定了转基因插入片段的活性和无活性拷贝在经过三个有性世代后,其启动子和编码区的突变频率。在失活基因中未发现类似RIP的突变。将我们的数据与来自粗糙脉孢菌的数据进行比较表明,植物重复DNA内的序列分歧要么比粗糙脉孢菌中的慢得多,要么是由不同的机制产生的。