Topics to be covers Basic features present on plasmids Eukaryotic and prokaryotic plasmids lacZ complementation (blue white screening) Replicative and integrative plasmids Yeast artificial chromosome

DNA Plasmids plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA double-stranded and, in many cases, circular Typically present in bacteria and lower eukaryotes In molecular biology replicative and integrative vectors are used

Plasmids as Cloning Vectors Cloning vector plasmid pUC19 Essential features include: an ampicillin resistance marker polylinker with multiple restriction enzyme cut sites. Insertion of cloned DNA within the polylinker inactivates the truncated lacZ’ gene that encodes part of beta- galactosidase so that transformants with insert will be by white and cells without insert - blue

Principle of the lacZα complementation

Prokaryotic Expression Systems Most often E. coli for screening of libraries Prokaryotic promoter— ribosome binding site— MCS—transcription termination site Prokaryotic selectable marker Origin of replication ori

Eukaryotic expression vectors E. coli origin of replication A selectable eukaryotic marker gene A eukaryotic promoter sequence The appropriate eukaryotic transcriptional and translational stop signal A sequence that signals polyadenylation of the transcript messenger RNA (mRNA) Origin of replication Marker gene and ori for propagation of vector in E. coli Eukaryotic marker system Eukaryotic origin of replication Coding sequence

Yeast artificial chromosome (YAC) The YAC is designed to clone large fragments of DNA (100-1000kb) The YAC is maintained as a separate chromosome in the host yeast cell and is highly stable Use of YAC: the physical mapping of human genomic DNA the analysis of large transcription units the formation of genomic libraries containing DNA from individual human chromosomes

Construction of yeast artifical chromosomes (YAC) Telomere sequence required to insure stability of YAC ends DNA fragment can range from 100 to 1000kb

Integrative plasmids