Transposition Flashcards
transposons (def.)
mobile segments of DNA that can hop or transpose from one place in DNA to another
transposition was documented in _____ Barbara McClintock in corn
1950s
transposition is not like ________ (___ independent); does not require lots of_____ DNA
-homologous recombination
-RecA
-homologous
Transposons exist in ___ organisms; they indirectly move between different genera of _____; transposition occurs from _______ to ____ cell divisions and is _____ regulated
-all
-bacteria
-1/10^3 to 1/10^8
-highly
Transposition is a way of introducing genes from one bacterium to another where ______
little DNA sequence homology exists
Two types of transpositions
1) Conservative or “Cut and Paste” Transposition (one copy before and after transposition)
2) Replicative Transposition (One or two copies of transposon in DNA after; site specific recombination)
There are many different types of transposons; all transposons contain ______ sequences at their _____ encode a ______ internally that mediates transposition. all transposons are bracketed by ______ surrounding tranposon (a consequence of transposition)
-inverted repeat
-ends
-transposase (tnp)
-short direct repeats of target DNA
3 types of transposons
1) Insertion Sequence Elements
2) Composite Transposons
3) Non-composite Transposons
Insertion Sequence Elements are _____ transposons; 750-2000 bp. have _____ at ends, _____ enzyme and ______. almost all bacteria carry IS elemnts; ___ carries several. Eg. ___
-smallest
-inverted repeats
-transposase
-regulators
-E.coli
-IS3
IS3 was og detected as mutation in ___ genes with funny properties which were ____ (4)
-gal genes
-highly polar, cannot be induced to revert
-galactose utilization gal plasmids transferred to gal-mutants acquire mutations with similar characteristics
-these mutated plasmids are larger than wild type counterpart
Non-composite Transposons have ______ on ends (not IS elements), ______, regulator of ____, and _____ genes
- short inverted repeats
-tranposase
-transposase
-other (AbR)
Composite Transposons are 2 IS elements in ______ orientation which bracket other genes & encode _____
-same or opposite
-antibiotic resistance determinants
flanking IS elements from a composite Tpn can transpose ______ or with the ______
-individually
-entire composite transposon
composite transposon insertions in a
plasmid -> ________ can be used to transpose the plasmid DNA
inside inverted repeats
assays of transposition
1) suicide vectors
2) mating out assay
3) hybridization analysis
suicide vectors detect transposition from DNA molecule that can’t ______ by selecting for phenotype encoded on transposon after ______
-replicate
-introduction into bacterial host
suicide vector/plasmid (def.)
DNA molecule that can not replicate; carries a transposon with selectable marker (antibioticR)
transposition assay induce ____ into bacterium, plate out on media containing ____; bacteria will only grow if transposon has hopped into chromosome -> _______
-suicide vector
-antibiotics
-transposon mutagenesis
mating out assay: transposition is detected by assaying for _________ on conjugative plasmid. ex. donor carries AmpR transposon on ____ & recipient carries kanamycinR (KnR) transposon on ____ . mate donor + recipient -> grow exconjugants on _____. at low rate, transposition of KnR transposon onto ____ will occur. detect transposition by using KnR, AmpR exconjugants as donors in 2nd mating with ______ recipients
-acquisition of a selectable marker
-plasmid
-chromosome
-Kn + Amp
-plasmid
-AmpS , KnS Tn-free
Hybridization analysis: new transposon DNA in plasmids is detected microscopically as _______
-single stranded loops that fail to hybridize with parent plasmid
Hybridization analysis method: plasmids are obtained by techniques like ______. ______ hybridized with the original parent plasmid. any areas of non -homology (such as where ______) will loop out from the duplex DNA and be visible by _____
-mating-out assay
-denatured and ssDNA
-a new transposon has hopped into the plasmid
-Electron Microscopy
generally transposition has ___ target specificity; insertion of transposon always results in the duplication of _____
-low
-short nucleotide sequence or target site
replicative transposition used by ______ transposons such as ____. It requires ______ (3)
-non-composite
-Tn3
-transposase (catalyzes transposition)
-resolvase (promotes recombination)
-res site (sites where resolvase works)
replicative transposition: The original transposon sequence is _____ during transposition so that a copy of the transposon remains at the _____ and a new copy is found at a ____. This type is characterized by formation of a ______ during transposition which is when donor & recipient DNAs are _____
-duplicated
-original site
- new site
-conintegrate
- fused to make 1 molecule
replicative transposition steps (1-4)
i) - a pair of staggered, single strand breaks is made in target sequence by transposase
ii) - a pair of single strand breaks is made in complementary strands at opposite ends of the transposon (each 3’ free end of transposon is joined to a 5’ protruding end in cut target site)
iii) - replication of the transposon occurs from the free 3’ ends( remaining 3’ and 5’ ends are joined) – this forms a cointegrate
iv) - double strand exchange between 2 copies of transposon is mediated by a resolvase at a resolution site (RES SITE) to separate the cointegrate into donor and recipient
Nonreplicative Transposition: transposition occurs by a _____ mechanism - no _____ of the
transposon
-CUT AND PASTE
-duplication
Bender and Kleckner 1986 proved that non-replicative transposition occurs with _____
-Tn10
Bender and Kleckner 1986 experiment: they constructed a heteroduplex transpon that carried ____ gene; one DNA strand = lacZ- mutation, other= wildtype lacZ+ allele; examined lac phenotype following transposition (lactose utilization is followed using the _____, which turns blue upon enzymatic cleavage).
-lac
-non-metabolizable substrate X-gal
Bender and Kleckner 1986 experiment expected results: replicative transposition
- cells get a copy of either lacZor lacZ+ but not both
- bacterial colonies would be Lac+ or Lac- (blue OR white colnies)
Bender and Kleckner 1986 experiment expected results: non- replicative transposition
-cells get a copy of both lacZand lacZ+
- upon cell division, one daughter would inherit lac+ and one lac- = mixed
Lac+ /Lac- colonies (blue/white colonies)
Bender and Kleckner 1986 experiment actual results
-sectored colonies (blue/white colonies= non-replicative transposition
2 Genetic Consequences of Transposition
1) Mutation (via Insertional Inactivation)
- insertion mutations generated by transposon insertion
2) Deletions & Inversions
- often arise next to transposon insertions
- transposition occurs to a 2nd site within same circular DNA
- Recombination (resolvase or RecA) can result in either an inversion or a deletion depending on the orientation of the transposons relative to each other
