Transposons

Question 1

What are the key features of a transposon?

Answer 1

Key element of a transposon is the insertion sequence, often encodes transposase. this is flanked by inverted repeats.

Question 2

How often does transposition occur, and under what circumstances?

Answer 2

transposition does not occur very frequently, and does not require homology with the target DNA.

Question 3

What is the mechanism for non replicative transposition?

Answer 3

1. Transposase aligns inverted repeats and flanking DNA
2. One phosphodiester bond nicked on each strand
3. 3’OH attacks the intact ends and creates a hairpin structure.
4. Hairpins are renicked and the re-exposed 3’OH ends attack recipient DNA

Question 4

What are the dangers associated with conjugative transposons?

Answer 4

Conjugative transposons can confer antibiotic resistance, through conjugation of the transposable element containing antibiotic resistance genes. Conjugation does not require homology giving a broad range of host organisms

Question 5

How can you select for transposon insertion mutants?

Answer 5

Grow cells recombined with a temperature sensitive plasmid containing transposon carrying antibiotic resistance. Plasmid will not grow at restrictive temperature. 1 in 10^4 cells, transposon will have jumped into bacterial chromosome. These cells will grow at restrictive temperature AND carry antibiotic resistance.

Question 6

What is the purpose of Transposon Mediated Differential Hybridization

Answer 6

This technique can be used identify the position of essential genes in specific conditions in a chromosome

Question 7

Briefly describe steps of TMDH

Answer 7

1. Create a mutant library with modified promoters for TN, T7 and S6.
2. TN delivered to host chromosome with a temperature sensitive plasmid. Antibiotic resistance to select for TN mutants. (population will contain lots of different TN mutants at different loci)
3. Grow population under normal growth conditions (A)
4. Prepare genomic DNA from the population and fragment with endonuclease. (only some of these fragments will contain TN)
5. Separate DNA into two tubes, in one tube add T7 RNApol and a small amount of green fluoro labelled C residues. (whenever fluoro C incorporated, transcription stopped)
6. repeat from S6 primer with red C
7. Hybridise RNA products to microarray.
8. Grow in condition B and compare microarrays. If present in A and not in B, they are essential for growth (Tn mutagenesis at essential genes blocks growth)

Question 8

What is a DNA transposon

Answer 8

non-replicative

Question 9

What is a retro transposon

Answer 9

replicative

Question 10

How are Ds transposons a reliant system in Maize

Answer 10

Ds transposons do not encode their own transposase (they are non autonomous) they require tranposase to have been expressed from Ac transposon. Have same inverted repeats as Ac

Question 11

What is pol required for in LTR transposons

Answer 11

3 protein activities, RNase, reverse transcriptase and transposition

Question 12

What are the key steps in the mechanism of LTR transposons?

Answer 12

Generate of RNA molecule and protein products of LTR transcript by host machinery
Reverse transcriptase mechanism, involves the LTR encoded reverse transcriptase.
Creates a cDNA from mRNA template, RNase (from pol) degrades RNA
Transport of nascent DNA - genome. (creation of target site directed repeats)