Lecture 8 handout.pdf

Micro/Genome 411 Observed phenotype of IS insertions: ?Non-leaky "knockouts" Highly polar ?Non-suppressible Revertable (at abnormally low frequency) Lecture 8 TRANSPOSABLE ELEMENTS I. Introduction A. Structural classes of transposable lements B. Phenotypes associated with transposition II. Genetic analysis of Tn3 transposition A. Antibiotic resistance mutants B. Transposition mutants 1. "Mating out" transposition assay 2. Complementation groups 3. cis-dominant mutants C. Resolution mutants 1. Complementation groups 2. cis-dominant mutants III. The Shapiro model of replicative transposition IV. Tn10 transposition A. Molecular-genetic analysis of replicative vs conservative transposition B. Adaptation of the Shapiro model V. Transposition behavior of composite transposons Structural classes of transposable elements: tnp tnpTet r tnpRtnpAAmp r res Insertion sequences (IS1) Composite transposons (Tn10) Noncomposite transposons (Tn3) Lecture 8: Transposable Elements Micro/Genome 411 Donor DNA Recipient DNA Transposon Insertion ("target") site Recipient DNA Donor DNADonor DNA Recipient DNA Conservative ("cut-and-paste") transposition Replicative transposition Genetic analysis of Tn3 transposition Known features: ?It's a large piece of DNA It encodes ampicillin resistance (Amp r ) ?It transposes (randomly) Conjugation Donor (Str s Amp r ) Recipient (Str r Amp s ) Lecture 8: Transposable Elements Micro/Genome 411 Donor DNA Recipient DNA Transposon Insertion ("target") site 5' 3' 5' 3' 3'5' 3'5' 5' 3' 3' 5' Tnp-mediated "nicking" Tnp-mediated joining Conjugation Donor (Str s Amp r ) Recipient (Str r Amp s ) Transconjugant (Str r Amp r ) Genetic analysis of Tn3 transposition ?A single gene (tnpA) encodes Tn3 transposase. ?Extreme terminal sequences are essential for transpo- sition (transposase recognition / binding?). ?A second gene (tnpR) represses transposition frequency (tnpA expression? TnpA activity?). ?Transposition is replicative (!) with formation of a cointegrate intermediate. ?An internal sequence (res) is essential for cointegrate resolution. ?TnpR is a site-specific recombinase ("resolvase"). 3' 3' Tn strand separation DNA synthesis Resolution (site-specific recombination) Lecture 8: Transposable Elements Micro/Genome 411 Donor: Recipient: lacZ ConservativeReplicative Replication Transposition of Tn10 lacZ heteroduplex: 5' 3' 3' 5' 5' 3' 3' 5' Nicking of donor DNA (DNA separation) 3' 3' DNA synthesis tnpTet r Tet r tnp Kendall Gray Microsoft Word - Lecture 8 handout.doc