第二章克隆的载体.ppt.ppt

Rolling circle replication ◆M13 Gene Functions Bacteriophage ?: cos sequences circularize a linear genome gal bio gal bio gal bio gal bio gal bio gal bio gal bio cos位点的第二个作用与上面的完全不同。当前噬菌体脱离宿主基因组后，通过滚环复制模式（rolling circle）产生大量新的λ噬菌体DNA，这是一个新生的DNA链不断从模板DNA上滚落下来的机制。结果形成一个包含一系列线性基因组的连环体(catenane )，它们在cos位点处连接在一起。此时的cos位点是一种限制性内切酶的识别序列，该内切酶是λDNA分子中基因A的表达产物，能够在cos位点处交错切开双链DNA分子并形成单链的黏性末端，并与其他一些蛋白质一起，将每个λ基因组包裹入一个噬菌体头部的过程中发挥作用。 Rolling circle replication generates multiples (concatamers，连环体) of the phage genome, which are then processed by the phage nuclease into single genomes. This is an essential intermediate step in viral replication and allows rapid and efficient viral replication. One phage genome 5’ 在进行滚环复制时，首先在双链环状DNA分子一条链的特定位点上产生一个切口，切口的3’－羟基末端围绕着另一条环状模板被DNA聚合酶延伸，这样新合成的链就取代原来的亲本链形成滚环。 一轮过后，被置换的链到达一个单位的长度，连续延伸可以产生多个拷贝组成的连环体。被置换出的单链可以作为模板合成互补链形成双链体。 Consider a circular molecule of double-stranded DNA with a nick in one of the two phosphodiester backbones. As long as there is a free 3 OH end, this can serve as a template for DNA polymerase. When the 3 OH end is extended, the 5 end can be displaced in a manner analogous to the strand displacement reaction. Synthesis on this strand is also analogous to leading strand synthesis. The displaced strand can, in turn, serve as an template for replication as long as a suitable primer is available. Synthesis on this strand is analogous to lagging strand synthesis. If synthesis continues in this manner, the consequence of this mechanism of replication can be the production of concatemer copies of the circular molecule. As a result, multiple copies of a genome are produced. A rolling circle mode of replication is seen both during replication of bacteriophage lambda where rapid production of many copies of the genome is desired, and in the replication of bacteriophage M13 where only a single copy is produced each time. Lambda DNA is linear in virus cos sequence is 12 nucleotides and single stranded The two cos sequences are