Part-II-2.3-重组.解读.ppt

不同点； 原核生物 (replication form) copy 的复制与转移 部分需要经过逆转录 真核生物 (cut paste form) 多数情况为切除后再整合 不需要 * 狭义的同源重组指涉及到DNA分子内断裂-复合的基因交流。 * 根据对DNA序列和所需蛋白质因子的要求，将重组分类 * * 根据对DNA序列和所需蛋白质因子的要求，将重组分类 * 根据对DNA序列和所需蛋白质因子的要求，将重组分类 * ES细胞通过培养小鼠胚泡获得。重组载体包含两个标记基因：Neo基因是具有对新霉素抗性的基因，供正选。HSV-tk是胸腺激酶基因，能将底物丙氧鸟苷转化成有毒的化合物，供负选。转基因片段和染色体的双重重组将导致靶基因被突变基因和抗药基因所取代。 * * 每个重组位点由一对对称排列的重组酶识别序列构成，识别序列中间所包围的是一个短的不对称序列，称为交换区，DNA的断裂和重新连接就在这发生。 * * * * * * * between two DNA moleculars within one DNA molecular Inverted repeats Direct repeats The structure of recombinase recognition site Inverted repeats Direct repeats e.g. ? 噬菌体DNA整合到E. Coli DNA ? att (attachment site) E.Coli attB BOB’ ? phage attP POP’ ? Int (integrase) IHF (integration host factor) ~30bp ~255bp ? Xis (excisionase) Inverted repeats Site-specific integration of l Inverted repeats Direct repeats Mechanism of Int action 系列转酯反应 2.3.3 转座重组 Transposition Recombination DNA上的一段核苷酸序列从一个位置转移到另一个位置的现象。 转座子（transposons)：发生转位的DNA片断 Barbara McClintock spent many years studying the behavior of unusual genetic elements in maize. She concluded that these elements were, in fact, mobile. Her work, all the more amazing because much of it was carried out before the structure of DNA was solved, was largely ignored until the mid 1970s when similar elements were discovered in bacteria. 1947 一. 原核生物的转座子 （一）IS (insertion sequence) Insertion Sequences or IS elements are the first transposons discovered in bacteria. the simplest mobile element consist of a fairly short (700 - 1800 bp) DNA segment flanked by a 10 - 40 bp inverted repeat sequence. The segment codes for the protein (transposase, tnpA) that catalyses the transposition event. l?? IS插入target site后 target sequence repeats directly flanked the IS （二）complex transposon l 两侧具有IR (35~40bp) l 2.5 kb 20 kb l 内部含有转座酶基因（ tnpA ）、 解离酶基因（ tnpR）、抗 生素抗性基因。 （三）composite transposon ● 2个IS插入到功能基因（抗生素或其它毒性抗性基因）两端，形成复合转座因子。 ● 每个IS可以独立转位，也可以与插入的功能基因作为整体一起转位。 ● The I