Transposition

Question 1

transposons (def.)

Answer 1

mobile segments of DNA that can hop or transpose from one place in DNA to another

Question 2

transposition was documented in _____ Barbara McClintock in corn

Answer 2

1950s

Question 3

transposition is not like ________ (___ independent); does not require lots of_____ DNA

Answer 3

-homologous recombination
-RecA
-homologous

Question 4

Transposons exist in ___ organisms; they indirectly move between different genera of _____; transposition occurs from _______ to ____ cell divisions and is _____ regulated

Answer 4

-all
-bacteria
-1/10^3 to 1/10^8
-highly

Question 5

Transposition is a way of introducing genes from one bacterium to another where ______

Answer 5

little DNA sequence homology exists

Question 6

Two types of transpositions

Answer 6

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)

Question 7

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)

Answer 7

-inverted repeat
-ends
-transposase (tnp)
-short direct repeats of target DNA

Question 8

3 types of transposons

Answer 8

1) Insertion Sequence Elements
2) Composite Transposons
3) Non-composite Transposons

Question 9

Insertion Sequence Elements are _____ transposons; 750-2000 bp. have _____ at ends, _____ enzyme and ______. almost all bacteria carry IS elemnts; ___ carries several. Eg. ___

Answer 9

-smallest
-inverted repeats
-transposase
-regulators
-E.coli
-IS3

Question 10

IS3 was og detected as mutation in ___ genes with funny properties which were ____ (4)

Answer 10

-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

Question 11

Non-composite Transposons have ______ on ends (not IS elements), ______, regulator of ____, and _____ genes

Answer 11

• short inverted repeats
  -tranposase
  -transposase
  -other (AbR)

Question 12

Composite Transposons are 2 IS elements in ______ orientation which bracket other genes & encode _____

Answer 12

-same or opposite
-antibiotic resistance determinants

Question 13

flanking IS elements from a composite Tpn can transpose ______ or with the ______

Answer 13

-individually
-entire composite transposon

Question 14

composite transposon insertions in a
plasmid -> ________ can be used to transpose the plasmid DNA

Answer 14

inside inverted repeats

Question 15

assays of transposition

Answer 15

1) suicide vectors
2) mating out assay
3) hybridization analysis

Question 16

suicide vectors detect transposition from DNA molecule that can’t ______ by selecting for phenotype encoded on transposon after ______

Answer 16

-replicate
-introduction into bacterial host

Question 17

suicide vector/plasmid (def.)

Answer 17

DNA molecule that can not replicate; carries a transposon with selectable marker (antibioticR)

Question 18

transposition assay induce ____ into bacterium, plate out on media containing ____; bacteria will only grow if transposon has hopped into chromosome -> _______

Answer 18

-suicide vector
-antibiotics
-transposon mutagenesis

Question 19

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

Answer 19

-acquisition of a selectable marker
-plasmid
-chromosome
-Kn + Amp
-plasmid
-AmpS , KnS Tn-free

Question 20

Hybridization analysis: new transposon DNA in plasmids is detected microscopically as _______

Answer 20

-single stranded loops that fail to hybridize with parent plasmid

Question 21

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 _____

Answer 21

-mating-out assay
-denatured and ssDNA
-a new transposon has hopped into the plasmid
-Electron Microscopy

Question 22

generally transposition has ___ target specificity; insertion of transposon always results in the duplication of _____

Answer 22

-low
-short nucleotide sequence or target site

Question 23

replicative transposition used by ______ transposons such as ____. It requires ______ (3)

Answer 23

-non-composite
-Tn3
-transposase (catalyzes transposition)
-resolvase (promotes recombination)
-res site (sites where resolvase works)

Question 24

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 _____

Answer 24

-duplicated
-original site
- new site
-conintegrate
- fused to make 1 molecule

Question 25

replicative transposition steps (1-4)

Answer 25

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

Question 26

Nonreplicative Transposition: transposition occurs by a _____ mechanism - no _____ of the transposon

Answer 26

-CUT AND PASTE -duplication

Question 27

Bender and Kleckner 1986 proved that non-replicative transposition occurs with _____

Answer 27

-Tn10

Question 28

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).

Answer 28

-lac -non-metabolizable substrate X-gal

Question 29

Bender and Kleckner 1986 experiment expected results: replicative transposition

Answer 29

- cells get a copy of either lacZor lacZ+ but not both - bacterial colonies would be Lac+ or Lac- (blue OR white colnies)

Question 30

Bender and Kleckner 1986 experiment expected results: non- replicative transposition

Answer 30

-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)

Question 31

Bender and Kleckner 1986 experiment actual results

Answer 31

-sectored colonies (blue/white colonies= non-replicative transposition

Question 32

2 Genetic Consequences of Transposition

Answer 32

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