Lecture 4 - Site specific recombination and transposition

Question 1

What are the two types of transposon?

Answer 1

DNA transposon

Retrotransposon

Question 2

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

Answer 2

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

Question 3

What are the features of the DNA transposon?

Answer 3

• 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

Question 4

How do DNA transposons move?

Answer 4

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

Question 5

How do retrotransposons move?

Answer 5

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

Question 6

What is a composite transposon?

Answer 6

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

Question 7

What elements can move in a composite transposon?

Answer 7

-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)

Question 8

What are IS elements?

Answer 8

Just encode the transposase gene flanked by inverted repeats

Question 9

What type of genes might a composite transposon encode?

Answer 9

antibiotic resistance genes

Question 10

Two examples of composite transposons?

Answer 10

Tn5

Tn10

Question 11

What is the structure of Tn5?`

Answer 11

IS50 - kanamycin resistance gene - IS50

IS50 = two IS inverted elements

Question 12

What is the structure of Tn10?

Answer 12

IS10 - tetracycline resistance gene - IS10

IS10 = two IS inverted elements

Question 13

What is a simple transposon?

Answer 13

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

Question 14

What is an example of a simple transposon?

Answer 14

Tn3

Question 15

What is the structure of Tn3?

Answer 15

IR - transposase - resolvase - ampicillin resistance gene - IR

Question 16

What are R plasmids?

Answer 16

plasminds that contain antibiotic resistance genes

Question 17

What is plasmid 100?

Answer 17

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

Question 18

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

Answer 18

Homologous recombination
either Deletion and Integration
OR Inversion

Question 19

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

Answer 19

• 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

Question 20

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

Answer 20

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

Question 21

What is non-replicative transposition?

Answer 21

transposition by breakage and reunion in Tn5 or Tn10

Question 22

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

Answer 22

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

Question 23

What is the paired end complex?

Answer 23

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

Question 24

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

Answer 24

• 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

Question 25

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

Answer 25

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

Question 26

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

Answer 26

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

Question 27

What are two examples of transposons that undergo replicative transposition?

Answer 27

Tn3 | phage Mu when undergoing lytic growht

Question 28

What is the pathway of replicative transposition?

Answer 28

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

Question 29

Maize Ac/Ds McClintock: What were her conclusion?

Answer 29

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

Question 30

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

Answer 30

when the element excises early: large spots late: small spots

Question 31

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

Answer 31

Minature inverted repeat transposable elements (essentially the IR)

Question 32

How does the P element in drosophila cause hybrid dysgenesis?

Answer 32

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

Question 33

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

Answer 33

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

Question 34

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

Answer 34

-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

Question 35

What represses P element transposition in P females?

Answer 35

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

Question 36

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

Answer 36

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

Question 37

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

Answer 37

encodes for purple pigment gene

Question 38

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

Answer 38

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