Transposition Flashcards

1
Q

transposons (def.)

A

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

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2
Q

transposition was documented in _____ Barbara McClintock in corn

A

1950s

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3
Q

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

A

-homologous recombination
-RecA
-homologous

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4
Q

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

A

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

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5
Q

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

A

little DNA sequence homology exists

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6
Q

Two types of transpositions

A

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)

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7
Q

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)

A

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

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8
Q

3 types of transposons

A

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

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9
Q

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

A

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

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10
Q

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

A

-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

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11
Q

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

A
  • short inverted repeats
    -tranposase
    -transposase
    -other (AbR)
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12
Q

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

A

-same or opposite
-antibiotic resistance determinants

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13
Q

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

A

-individually
-entire composite transposon

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14
Q

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

A

inside inverted repeats

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15
Q

assays of transposition

A

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

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16
Q

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

A

-replicate
-introduction into bacterial host

17
Q

suicide vector/plasmid (def.)

A

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

18
Q

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

A

-suicide vector
-antibiotics
-transposon mutagenesis

19
Q

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

A

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

20
Q

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

A

-single stranded loops that fail to hybridize with parent plasmid

21
Q

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 _____

A

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

22
Q

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

A

-low
-short nucleotide sequence or target site

23
Q

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

A

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

24
Q

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 _____

A

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

25
Q

replicative transposition steps (1-4)

A

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

26
Q

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

A

-CUT AND PASTE
-duplication

27
Q

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

A

-Tn10

28
Q

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).

A

-lac
-non-metabolizable substrate X-gal

29
Q

Bender and Kleckner 1986 experiment expected results: replicative transposition

A
  • cells get a copy of either lacZor lacZ+ but not both
  • bacterial colonies would be Lac+ or Lac- (blue OR white colnies)
30
Q

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

A

-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)

31
Q

Bender and Kleckner 1986 experiment actual results

A

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

32
Q

2 Genetic Consequences of Transposition

A

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