Lecture 20 Flashcards Preview

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Flashcards in Lecture 20 Deck (25):
1

Transposable elements cause problems:

- They can mutagenise protein coding genes by inserting between a coding sequence
- They can increase in number, so require host cell resources
- They threaten the integrity of the genome as they can increase in copy number

2

Class 1: Retrotransposons:

- Copy and paste mechanism
- long terminal repeat sequences, pol (reverse transcriptase), gag (integrase
- RNA molecule produced by pol, and makes a DNA copy which integrates back into the host genome using gag
- increase in genome

3

Class 1b: nonviral-like retrotransposons

- Copy and paste
- Differ in that they lack long terminal repeats, but they still have an RNA indermediate
- LINEs and SINEs

4

Class 2: DNA transposons:

- Cut and paste mechanism
- Just hop around the genome, and only increase in number if they move at the right time of DNA replication

5

How can you stop transposable elements causing a problem?

- Target the RNA transcripts
- Wrap them up in chromatin

6

Distribution of TEs in the human genome:

- Retro-elements (RNA intermediate elements) cause the most problems for humans

7

IF you were a transposable element, in what tissue would you want to be most active?

- In the germ line cells so that you contribute genetic material to the next generation
- Can also be active in other cells, but germ line cells are the most logical

8

Indentifyication of fly PIWI mutants:

- Mutants displaying defects in gametogenesis and are sterile

9

PIWI (P-element induced wimpy testis):

- Spermatogeneseis defects arise from precocious activation of retrotransposons in the male germ line
- Oogenesis defects arise from a loss of stem cells as a result of DNA damage signaling

10

Mammalian PIWIs and gonad development:

- 3 PIWIs in humans
- Arrest of gametogenesis and complete sterility in males
- Activation of LINE and retrotransposons in Mili/Miwi2 mutants

11

PIWI is a member of the Argonaut family:

- PIWI's are only found in animals
- AGO's are found in animals, plants and fungi

12

PIWI's are only active in the germline

- This is clearly a pathway specific for the germline

13

piRNA's

- PIWI interacting RNAs
- Incredibly diverse (1.5 million different piRNAs in flies)
- Mostly derived from retrotransposons, transposons and repetitive elements

14

piRNA clusters in the genome:

- 80% of piRNS's map to specific genomic loci
- usually antisense to the TE mRNA, giving a strong bias for uridine at the 5' end

15

piRNA's role

- Preventing transposable element mobilisation in the germline
- Prevent DNA damage
- Prevent telomere loss (composed of retrotransposon repeats)

16

piRNA modes of action:

- Target retrotransposon/transposon RNA for endonucleolytic cleavage (slicing) in cytoplasm (post-transcriptional gene silencing)
- DNA methylation/chromatin modification of transposons in genome (transcriptional gene silencing)

17

piRNA biogenesis:

- a piRNA cluster is made up of sense and antisense RNA
- This is chopped up to be loaded into PIWI.
- The processing differs in the nucleus to the cytoplasm

18

Does piRNA need dicer in the cytoplasm?

- No, instead it uses exonucleases

19

Loading the piRNA into RISC produces active piRISC. Where do these go?

- Some go into the nucleus, where transcriptional silencing occurs with the help of Pol2
- Some go into the cytoplasm where post-transcriptional silencing occurs, using TE transcript cleavage

20

What is ping-pong amplification?

- The way that lots of piwi's can be generated
- TE sense and antisense piRNAs are associated with different subclasses of PIWIs and have a 10nt overlap

21

Ping pong cycle:

- Binding of the retrotransposon transcript with a PIWI type 1 results in slicing
- PIWI type 2 binds at the 10A sequence
- It goes into the cytoplasm for processing
- a piRISC complex forms
- It can now basepair with the pre-RNA transcript
- The sliced transcript is then recognised by the first class of PIWIs
- A secondary piRNA derived from the pre-cursor transcript binds again.

22

What does the ping-pong amplification loop achieve?

- Massive amplification of piRNAs
- Silences TE through cleave of their transcripts
- Leads to the amplification of useful piRNAs (they have to be able to target transposons)

23

Defence against transposable elements, the main problems:

- There is massive diversity within TE
- TE can evolve rapidly
- TE can jump species barriers

24

Hybrid dysgenesis:

- Crossing lab bread flies with WT flies results in F1 sterility
- This is due to TE present in the wild flies become active in the captive flies
- The captive population cannot control the TE
- If you study the F1 over time they can regain fertility, as the p-elements jump into a piRNA cluster and then piRNAs can be generated for it

25

piRNA clusters are programmable loci in a way. What does this mean?

- When a TE jumps into a piRNA cluster, a piRNA can be produced for it
- Until then, there may be no piRNA for that specific piRNA