Transposition Flashcards
what are transposons
mobile DNA segments that hop from one part of DNA to another
what did Barbara McClintok figure our with transposons
- they dont require recA or homology to be introduced into new bacterium
what is the conservative “cut and paste model” of transposition
when a transposase enzyme cleaves transposon at inverted repeating sequences (IRS)
- enzymes scan for target site on target DNA and insert it
what are inverted repeated sequences
sequences that encode from a transposase (tnp)–> mediates transposition
what are insertion sequence elements (IS)
small transposons (750-2000 bp)
- has inverted repeats, transposase, and regulators
- they have no selectable markers, generally they are just machinery that moves DNA
what is non composite transposons
- has inverted repeats but no IS elements
- typically carry extra genes that have antibiotic resistance
( terminal inverted sequences selected for transposon within gene that carries antibiotic resistance)
–>———–<–
what is composite transposons
- has 2 IS elements in same or opposite orientation that brackets other genes
- encodes antibiotic resistance
means transposition can be mediated inside or outside of inverted repeats
<–>——————-<–>
what are suicide vectors
a DNA molecule that cannot replicate, contains transposon with selectable marker (antibiotic resistance)
what is transposition assay for suicide vectors
1) introduce suicide vector into transposon
2) plate media containing antibiotics
3) if growth is observed, then transposon with antibiotic resistance has jumped onto a DNA that can replicate
(transposon mutagenesis
what is the mating out assay
transposition detected by acquisition of antibiotic resistance in conjugate plasmid
1) donor carries AMPR and recipient carries KNR
2) conjugates for AMPR + KNR
3) AMPR+KNR donor mate with clean AMPS and KNS recipient
4) if there is any AMPR+KNR in conjugants= transposition occurred
what is hybridization analysis
1) WT dna and mutant DNA is denatured to ssDNA
2) when cooled down, BP reform, if there is one strand with mutant and one strand with WT–> observed transposon
what is required for replicative transposition
- non-composite transposons such as Tn3
- requires transposase to catalyze transposition
- resolvase to promote recombination
- res sites where resolvase works
what is the process of replicative transposition
1) transpose nicks at opposite ends of target site and transposon site to reveal 3’ ends
2) each exposed 3’ end of transposon is joined by 5’ end of target site to for cointegrate
3) DNA polymerase replicates transposon to complete double strand, exchange between 2 copies of transposon occurs mediated by resolvase at res site to separate cointegrate
explain non replicative transposition
cut and paste model
enzyme cleaves at ends of transposon and inserts into recipient
no duplication occurs
