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Genetics III > Lecture 4 - Site specific recombination and transposition > Flashcards

Lecture 4 - Site specific recombination and transposition Flashcards

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1
Q

What are the two types of transposon?

A

DNA transposon

Retrotransposon

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2
Q

What is the main difference between the DNA transposon and the Retrotransposon?

A

DNA transposon
-move using a ‘cut and paste’ mechanism
Retrotransposon
-move using a ‘copy and paste’ mechanism

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3
Q

What are the features of the DNA transposon?

A
  • move using a cut and paste mechanism
  • in prokaryotes
  • IS encodes the transpose
  • transposase binds to inverted repeats#
  • mechanism of transposition creates the target site repeates
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4
Q

How do DNA transposons move?

A

transposon cut out of position in the genome by transposase enzyme -> transposase cuts new target DNA and ligates the transposon to cut target

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5
Q

How do retrotransposons move?

A

RNA polymerase transcribes the retrotransposon to generate new RNA copy -> reverse transcriptase copies RNA to make dscDNA -> cDNA integrated into new site by integrase OR endonuclease

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6
Q

What is a composite transposon?

A

where two insertion sequences flank another piece of DNA
insertion sequences are usually inverted and one carries mutation to prevent it from being active, usually in the transposable gene

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7
Q

What elements can move in a composite transposon?

A

-whole composite transposon can move using the outer IR, one from the outside ends of the 2 IS elements
OR
-the just IS elements can move (normally only one as the other is inactive, and can only move using the transposase gene from active element)

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8
Q

What are IS elements?

A

Just encode the transposase gene flanked by inverted repeats

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9
Q

What type of genes might a composite transposon encode?

A

antibiotic resistance genes

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10
Q

Two examples of composite transposons?

A

Tn5

Tn10

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11
Q

What is the structure of Tn5?`

A

IS50 - kanamycin resistance gene - IS50

IS50 = two IS inverted elements

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12
Q

What is the structure of Tn10?

A

IS10 - tetracycline resistance gene - IS10

IS10 = two IS inverted elements

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13
Q

What is a simple transposon?

A

an IS element containing multiple protein coding genes between the short inverted sequences that flank the gene coding region

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14
Q

What is an example of a simple transposon?

A

Tn3

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15
Q

What is the structure of Tn3?

A

IR - transposase - resolvase - ampicillin resistance gene - IR

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16
Q

What are R plasmids?

A

plasminds that contain antibiotic resistance genes

17
Q

What is plasmid 100?

A

an R plasmid that contains 6 transposable antibiotic resistance genes and the plasmid transfers by conjugation

18
Q

Through what method of recombination do transposons and IS elements cause DNA rearrangements

A

Homologous recombination
either Deletion and Integration
OR Inversion

19
Q

How do transposon/IS elements cause DNA rearrangements by deletion and integration?

A
  • transposon/IS elements generate multiple copies in the genome (repeat sequences)
  • pairing of the direct repeats
  • recombination releases material between repeats as a circular molecule
20
Q

How do transposon/IS elements cause DNA rearrangements by inversion?

A
  • transposon/IS elements generate multiple copies in the genome (repeat sequences)
  • inverted sequences pair
  • region is inverted
21
Q

What is non-replicative transposition?

A

transposition by breakage and reunion in Tn5 or Tn10

22
Q

What is the process of non-replicative transposition by Tn5 and Tn10?

A
  1. Transposase binds to IR at the ends of of the transposon and nicks the transferred strands (DNA strands that it will bind to the target DNA, have 3’OH group)
  2. Transponase then nicks the non-transferred strands to cut out the transposon, remaining bound at the ends = paired end complex
  3. Paired end complex attacks the target site by generation of a staggered breaks through 3’OH on transferred strands acting as neutrophiles
  4. Transposase joins transferred strands to 5’ end of target site
  5. Host repair enzymes replicate through gaps to generate the target site direct repeats
23
Q

What is the paired end complex?

A

a complex containing the cut out transposon and transposase bound at the ends

24
Q

What happens to the donor DNA in the process of non-replicative transposition by Tn5 and Tn10?

A
  • host repair enzymes can repair the DNA but often introduce deletions
  • > if can directly ligate the DNA target site duplication still usually interrupts the DNA
  • > however if transposon has moved from a plasmid or rapidly growing cells a second copy of the donor DNA can repair the donor DNA
25
Q

What is an example of a transposon that varies the order of breakage and reunion in nonreplicative transposition? And how?

A

Phage Mu in E.coli

  1. Transposase binds to both ends of transposon
  2. Transferred ends are nicked by transposase at the IR
  3. Donor is nicked to give staggered break and the free 3’OH end of the donor DNA is joined to 5’ end of the target -> crossover structure
  4. Crossover structure resolved by transposase nicking the non-transferred strand to release the donor
  5. repair enzymes fill the gap in the recipient
26
Q

What is the structure of the paired end complex in Tn5 transposase?

A

2 subunits, each subunit has 2 binding sites
-one subunit is the active site where end of the transposon located
- other is DNA binding region which contacts the IR sequence
-The right end has its active site in one subunit but right end IR bound by other subunit
Geometry of active sites determine length of the staggered cut in the target sit and length of target site duplication

27
Q

What are two examples of transposons that undergo replicative transposition?

A

Tn3

phage Mu when undergoing lytic growht

28
Q

What is the pathway of replicative transposition?

A
  1. Nicks generate staggered ends in transposon and target, 3’ ends of the transposon joins to 5’ end of donor plasmid
  2. the 3’ ends of the target site serve as primers for DNA replication to form cointegrate, leading to two copies of the transposon at junctions between replicons
  3. cointegrate resolution performed by resolvases which recognise a specific site in the transposon (res)
  4. two res sites in the cointegrate recombine to leave two separate DNA circles
29
Q

Maize Ac/Ds McClintock: What were her conclusion?

A

C gene wild type : purple phenotype
c-m(ds) (no ac): stable allele colourless kernels
c-m (ds) (+Ac): spotted kernals as Ds can occasionally excise, repair of donor site leaves functional gene -> purple spots (by NHEJ or HR with sister chromatid)
c-m (Ac): spotted kernals if full Ac element located in the colour gene

30
Q

Maize Ac/Ds McClintock: What does the extent of spottyness depend on?

A

when the element excises

early: large spots
late: small spots

31
Q

Maize Ac/Ds McClintock: What are MITES found in Ds element?

A

Minature inverted repeat transposable elements (essentially the IR)

32
Q

How does the P element in drosophila cause hybrid dysgenesis?

A

Interbreeeding between certain (P and M) lines of drosophila results in many sterile progeny
Cross has to be between P male and M female
How?
-P lines have 30-50 copes of a transposon P element, activated in germlines of PmaleXMfemale crosses and cause muations in 2 ways:
1.insertion of P element into new sites
2. exision of P at the donor site

33
Q

Why doesn’t PfemaleXMmale drosophila cross result in sterile progent?

A

P female germlines carry repressor of P element transposition, which P male progeny from cross will carry for several generations

34
Q

What is the structure of the P element transposase and how is it expressed?

A

-4 exons and 3 introns
In somatic cells only introns 1 and 2 are excised so transposase not expressed as SC contain protein prevent splicing of last intron
In germline 3rd intron removed and active transposase expressed

35
Q

What represses P element transposition in P females?

A

repression of P transposition by action of regulatory RNA, piRNA which degrades the P element mRNA

36
Q

Maize Ac/Ds McClintock: What was the inital strain she was studying and why did she decided AC was a mobile element?

A

a strain where frequent chromosome 9 breakage events at specific site

  • correlated non pigmented shrunken and non waxy kernels to loss of part of chrom. 9
  • required two genetic factors: Ds (dissociation factor at breakage site) and Ac (activator - unlinked factor to activate breakage)
  • could not map Ac decided it was a mobile element
37
Q

Maize Ac/Ds McClintock: What does chrom. 9 encode for?

A

encodes for purple pigment gene

38
Q

Maize Ac/Ds McClintock: What were the second lines of maize McClinktock developed?

A

-some kernels all purple
-some have spots of pigment
deduced from loss of Ds fragment - only occur in strains with Ac element

Genetics III (12 decks)

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