六单元酵母的基因工程教程.ppt

* * * * 七、整合操作 * * * * One-step gene replacement in yeast. Within a cloned stretch of yeast chromosomal DNA containing two restriction enzyme sites (EndoR1 and EndoR2), a marker gene selectable in yeast is inserted by standard cloning procedures. Additional alterations, such as deletion of yeast sequences and/or point mutation of yeast sequences can also be carried out. All such alterations should be 60 bp from the restriction sites, so that at least 60 bp of homologous sequence flanks the mutated region on both sides. Then the mutated segment of yeast chromosomal DNA is excised from the vector by restriction digestion at the two sites, and it is introduced into yeast cells by standard transformation procedures. The ends of the restriction fragment are recombinogenic, and the desired double recombinants can be selected by growth on medium requiring the selectable marker. When the selectable marker and/or mutation are expected to destroy the function of a gene, it is best to carry out this procedure in diploids. Otherwise, if the destroyed gene is essential, no cells containing the desired mutation will survive. * *  * *  Two-step gene replacement. This procedure allows the experimenter to introduce any type of mutation anywhere in the yeast genome, without leaving behind vector sequences or a selectable marker. The desired mutation with flanking sequences is cloned into a conventional plasmid that also contains a marker that can be selected both for and against. URA3 and ura4+ are usually employed for this purpose in S. cerevisiae and S. pombe, respectively. Then the plasmid is digested with a restriction enzyme that cuts once within the yeast sequences flanking the mutation and is introduced into yeast by transformation. The DNA break stimulates recombination within the flanking sequences, leading to integration of all the vector sequences and the mutated yeast sequences into the chromosome of the recipient yeast cell. Note that there is a duplication of the t