Transposons

Question 1

What are the two major classes of transposons?

Answer 1

DNA type elements (class II elements) and retroelements (type I elements)

Question 2

Briefly describe DNA-type elements. 7 points.

Answer 2

1. move directly from one location to another
2. found in both prokayrotes and eukaryotes
3. have inverted terminal repeats (ITR)
4. encode a transposase
5. target site is duplicated (target repeat, direct repeat, target site duplication)
6. target site might be random or specific
7. Most studied are the IS elements (bacterial insertion sequences)

Question 3

Give an example of an inverted repeat

Answer 3

5’ GGATAC —— GTATCC 3’
3’ CCTATG —— CATAGG 5’
(sequence is inversely repeated in the opposite strand)

Question 4

Why do you get direct repeats?

Answer 4

You get direct repeats in the DNA flanking the inverted repeats. The direct repeats are made from target site DNA flankning the inverted repeats.

1. Staggered nicks are made at target site
2. Transposon is joined to the single stranded ends
3. Gaps are filled in giving direct repeats

5’ AATCGT:GGATAC —- GTATCC:AATCGT 3’
3’ TTAGCA:CCTATG —- CATAGG:TTAGCA 5’

(DNA transposons)

Question 5

1. What is a transpoase? 2. How does it work?

Answer 5

1. the enzyme catalyzing the transposition
2. recognizes the inverted terminal repeats and may have endonuclease activity. May cut both target and transposon. Often a dimer.

Question 6

What kind of elements do you find in both eukaryotes and prokaryotes?

Answer 6

DNA type elements

Question 7

What is the typcial structure of a DNA transposon?

Answer 7

5’— DR> ITR> transposon —-3’

Question 8

Which two mechanisms exist for DNA type element transposition?

Answer 8

Replicative and non-replicative.

Replicative: transposon is inserted at a new site and the copy at the original site is kept. Requires a resolvase. TEGN!

Non-replicative: the transposon moves from donor site to target site (copy at donor site is lost). A double stranded break is generated at acceptor site. Tegn.

Question 9

What kinds of retrotransposons are there?

Answer 9

1. Retrotransposons (LTR retrotransposons)
2. Retroposons (non-LTR retrotransposons)
3. Non-autonomous

Question 10

Which transposons are very similar to retroviruses?

Answer 10

LTR transposons

Question 11

Which genes/sequences are found in LTR transposons?

Answer 11

Gag, Pol, (non-functional Env?)

Question 12

Retroviral genes:

Answer 12

Retrovirus genomes commonly contain these three open reading frames that encode for proteins that can be found in the mature virus:

group-specific antigen (gag) codes for core and structural proteins of the virus;
polymerase (pol) codes for reverse transcriptase, protease and integrase; and,
envelope (env) codes for the retroviral coat proteins.

Question 13

Processed pseudogenes

Answer 13

• Non-autonomous retroelements generated from spliced mRNAs

- Similar to endogenous genes, but introns are lacking

Question 14

SINEs

Answer 14

• Non-autonomous retroelement, depends on autonomous element for transposition
• Usually 5’ end is derived from PolIII-transcribed small cellular RNA (e.g. tRNA)

Question 15

Size of SINEs?

Answer 15

Rather small: 100-300 bp

Question 16

What are the first steps in insertion of DNA from retrotransposons (retroelements)?

Answer 16

Via an RNA intermediate, use a reverse transcriptase to generate the DNA to be inserted from RNA

Question 17

Describe the roles and properties of a transpoase.

Answer 17

Transpoase is the enzyme catalyzing the transposition of a DNA transposon:

• Recognizes the ends of the transposon - the inverted terminal repeats
• May have endonuclease activity generating nicks in both transposon and target
• Often a dimer

Question 18

How is the insertion site duplication formed when a transposon inserts in the genome?

Answer 18

1. Staggered nicks at target site
2. Transposon isjoined to the ss ends
3. Gaps are filled in - this results in DR

Question 19

What is the difference between an autonomous and non-autonomous element?

Answer 19

• Autonomous transposon has the full ability to transpose - may be regulated or blocked by DNA methylation
• Non-autonomous has lost the ability to transpose on its own - depends on a similar autonomous (same family) transposon somewhere else in the genome. Typically the transpoase gene is defect.

Question 20

What are inverted terminal repeats? Draw example.

Answer 20

Found at the end of DNA transposons. Inverted on oppsite strand:

ATGC——-GCAT
TACG——–CGTA

Question 21

Typical DNA transposon

Answer 21

DR>IR>transposon

Question 22

Describe the differences for replicative and non-replicative mechanisms for transposon insertion.

Answer 22

Replicative - copy of transposon inserted at new site, original copy is kept at donor site

• cointegrate which is a fusion of the donor and the target replicons
• Recombinationtion between the transposon copies regenerates the original replicon, but the recipient has also gained a copy
• Recombination event is catalyzed by a resolvase

Question 23

Non-replicative transposition:

Answer 23

Results if a crossover structure is nicked in the unrboken pair of donor strands and the target strans on either side of the transposon are ligated
Two pathways differ in if the first pair of transposon strands are joined to the target before the second pair are cut, or whether all four strands are cut before joining to the target

Question 24

Which genes/sequences are found in LTR-transposons?

Answer 24

Typical retroviral genome:
Long terminal repeats
Gag-pol-env –> Gag and pol are translated from the full length transcipt. Translation of pol requires a frameshift. Env is translated from a separate mRNA that is generated by splicing.

You get a target site duplification

Question 25

Outline how the minus and plus strands of retroviral DNA are synthesized

Answer 25

Viral RNA has direct repeats. Plus strand RNA. R-U5 and U3-R. tRNA is used as a primer and a DNA strand of the R-U5 region is synthesized in the 3'-->5' direction. This short strand is then displaced to the U3-R (base pairs with this region) and revers transcriptase resumes synthesis of minus strand DNA. tRNA primer is removed and RNA is degraded leaving fragments a primers. Plus strand DNA is then synthesised starting with the primer and the newly synthesised U3-R region of this plus strand DNA is then displaced and moved to the 3' region of the minus strand where it basepairs withthe minus strand and this is then used as a primer for complete synthesis of the plus strand.

Question 26

What is the role of a stop codon supression or reading frame shift when proteins from retroviruses and LTR transposons are formed?

Answer 26

Gag and pol are made from the same transcript, but may have different reading frames. In addition Gag is transcribed from the initiation codon to the first termination codon, but this termination codon needs to be bypassed for expression of Pol. 20 times more Gag than Pol.

Question 27

Decribe the outline/mechanism of movement of non-LTR retrotransposons

Answer 27

Two ORFs: nucleic acis binding protein and reverse transcriptase and endonuclease 1. Nick in target - provides priming end 2.RNA used as template and the DNA nicked as a primer - this primer is elongated with the cDNA from the RNA template 3. DNA replaces RNA template 4. Intron is created by recombination Target site duplications/direct repeats

Question 28

What are LINEs and SINEs?

Answer 28

- Non-LTR transposons (retroposons) - LINE - typical LINE1 is the only active non-LTR transposon in mammals. makes up 17% of the human genome. Long interspersed repeat elements SINEs - short interspersed repeats do not transcripe a reverse transcriptase, rely on other active elements. 11% of the human genome. <500 nt. Alu elements.

Question 29

Why are there often large difference in the genome size of organisms that are quite closely related?

Answer 29

Repetitive DNA makes up more than 50% of the human genome - most of this is transposons --> LINEs, SINEs, Retrovirus-like elements, DNA transposon fossils

Question 30

Give some examples of how transposons kan generate new gene functions.

Answer 30

Can generate new gene functions by formation of chimeric retrogenes, use of Alu or L1 i coding regions of genes, premature cleavage of gene transcripts at strong poly(A) signals in L1. Segmental duplications and deletions. Trans-driven insertion of processed pseudogenes.

Question 31

Briefly describe McClintock's discovery of transposons in Maize

Answer 31

Presence of transposon often gives rise to a chromosomal break, leading to gene inactivation Obseved as a region of different colour on the maize kernel, representing the cells descending from the cell where breakage took place Called the transposons controlling elements Variegation in colour

Question 32

The sleeping beauty transposon

Answer 32

almost all vertebrates are devoid of active DNA transposons, genome sequence reveal a number of inactivated ones. Based on sequence alignments of various inactivated copies from fish a consensus sequence was obtained. The sleeping beauty transposon was awakened --> able to mediate the insertion of a non-autonomous transposon containing and antibiotic resistance gene

Question 33

Briefly describe the two main classes of transposons:

Answer 33

DNA transposons (type II elements): - DNA moves directly from one location to the other - found in both prokaryotes and eukaryotes - IR and Target duplication - encode a transpoase - target site might be random or specific - bacterial insertion sequence most studied RNA transposons (type I elements): - uses an RNA intermediate - uses a reverse transcriptase - LTR, non-LTR, non-autonomous - some are very similar to retroviruses - found only in eukaryotes