SSR and transposition

Question 1

DNA-type elements

Answer 1

• Move as a DNA sequence.
• Widespread both in eukaryotes and prokaryotes.
• Have short inverted repeats at each end
• Carry the gene(s) required for transposition.
• They code for at least a transposase

Question 2

Retrotransposons

Answer 2

Move as an RNA sequence.
Make copies of DNA with reverse transcriptase activity. Only in eukaryotes. There are two types:
• LTR (Long-terminal repeat) retrotransposons
• Non-LTR retrotransposons

Question 3

Transposase

Answer 3

recognizes the inverted terminal repeats, catalyzes the transposition reaction and recognizes the target site

Question 4

Insertion sequences (IS)

Answer 4

• The simplest bacteria transposons.
• The smallest only codes for a transposase.
• IS inserts at target site of the host DNA, which is duplicated in the same orientation generating short direct repeats at both ends of the IS

Question 5

Composite elements

Answer 5

Bigger transposons flanked by two IS.
They code for antibiotic resistance genes and other markers. (e.g. Tn3, Tn10

Question 6

DNA Transposition

Answer 6

The movement of a defined DNA segment (transposon) from one genomic site to another

Question 7

LTR retrotransposons

Answer 7

• Have directly repeated long terminal repeats (LTRs) at each end.
• Code for reverse transcriptase and integrase

Question 8

Non-LTR retrotransposons

Answer 8

• Poly A at 3’ end of RNA transcript; 5’ end is often truncated.
• Code for at least reverse transcriptase end endonulcease

Question 9

Difference between retrotransposons and retroviruses

Answer 9

• Retrotransposons are confined to an intracellular cycle
• Retroviruses can generate infectious particles and have an extracellular cycle

Question 10

DNA-type elements replicative pathway
(Tn3 transposon of E.coli)

Answer 10

1. Transposon codes for transposase that creates staggered nicks in both donor and acceptor DNA
2. Both DNA molecules form a crossover structure joining ends together
3. The DNA is replicated creating a cointegrate: A single molecule that contains two copies of the transposon.
4. Cointegrate is resolved by a resolvase
5. It obtains two individual replicons that each carry one copy of the transposon.

Question 11

DNA-type elements Nonreplicative pathway

Answer 11

1. Transposon codes for transposase
2. Two transposases flank the transposon, bind the inverted terminal repeats and produce a DSB on each side of the transposon. Donor DNA must be repaired (HR or NHEJ).
3. Each transposase binds to one end of the transposon and form a loop structure that protects the transposon and moves along th genome.
4. Transposases recognize the target site and make two staggered end braks in each side.
   Transposon joins between the single strand ends.
5. Gaps are filled and sealed by cell machinery (DNA polymerase, ligase) -> generation of short direct repeats.

Question 12

Life cycle of retrovirus

Answer 12

1. External form: retrovirus has a capsid, envelope and two copies of a ssRNA genome.
2. infectious step: When it enters the cell it becomes a free ssRNA genome.
3. Cytoplasm: It transcribes for a reverse transcriptase enyme to make a dsDNA copy of its ssRNA transcript.
4. Nucleus: It integrades in the host genome with the help of own integrase. In this state it’s called a provirus.
5. Induction step: It can be activated, and it will use the RNA polymerase and the translation machinery of the cell host to transcribe the dsDNA to ssRNA and produce the proteins required to assemble new viruses

Question 13

Transposition mehanism of LINE
(Non-LTR retrotransposons)

Answer 13

1. Transcription dsDNA -> ssRNA
2. It goes to the cytoplasm
3. Translates the 2 ORFS: RNA binding proteins and a reverse transcriptase-endonuclease protein (bind to PolyA)
4. It is imported into nucleus
5. Endonuclease activity cleasves one strand of the DNA target site.
6. RT uses free 3’ end to elongate the DNA. It copies and inserts the RNA sequence of LINE in the genome.

Question 14

Life cycle of phage Lambda

Answer 14

• During the lytic cycle it replicates, synthesizes virus particles and lysis the cell to produce offspring.
• During the lysogenic cycle it stays latent as a prophage and replicates with the devision of the cell.
• During the induction event the DNA is released by an excision reaction.

Virulent phages can only follow the lytic cycle.
Temperate phages can follow both cycles

Question 15

CRE recombinase

Answer 15

• Discovered in phage 1 of E.coli.
• recognizes the recombination site loxP
• No ATP or accersoory proteins required -> standard for genome manipulation.