Chapter6CloningVectorsforEandeukaryotes例析.ppt

* Other types of yeast cloning vector Yeast replicative plasmids (YRps): A yeast vector that carries a chromosomal origin of replication. Example: YRp7 * Artificial chromosomes can be used to clone large pieces of DNA in yeast Yeast artificial chromosome (YAC): a cloning vector comprising the structural components of a yeast chromosome and able to clone very large pieces of DNA. YAC telesome、centromere and ori sequence 200-500kb DNA replication * Key components of a chromosome Centromere: required for the chromosome to be distributed correctly to daughter cells during cell division Telomere : the structures at the ends of a chromosome needed for the ends of replication correctly and prevent the chromosome from being nibbled away by exonucleases. Origin of replication: the positions along the chromosome at which DNA replication initiates, similar to the origin of replication of a plasmid. * The structure and use of a YAC vector Example: pYAC3 Selectable marker: URA3 ,TRP1, SUP4 Origin , CEN4, TEL Cloning experiment with pYAC: * A YAC vector and the way it is used to clone large pieces of DNA * 6.3 Cloning vectors for higher plants Genetically modified (GM) crops Three types of vector system: Vectors based on plasmids of Agrobacterium Direct gene transfer using plasmid DNA Vectors based on plant viruses * Agrobacterium tumefaciens- nature’s smallest genetic engineer A.tumefaciens: a soil microorganism that cause crown gall disease in many dicotyledonous（双子叶植物） plants. Ti (tumor inducing) plasmid: 200 kb T-DNA:10-30kb, which make Ti plasmid integrated into the plant chromosomal DNA. T-DNA contains genes responsible for the cancerous properties of the transformed cells. * The Ti plasmid and its integration into the plant chromosomal DNA after A. tumefaciens infection * Using the Ti plasmid to introduce new genes into a plant cell Main problem: difficulty to find an unique restriction site in a 200kb plasmid. Solutions: