BIOL 338 Transposable Elements

Question 1

What is DNA methylation?

Answer 1

• Methylation of cytosines that represses gene activity (silences genes)
• Typically at CG dinucleotides
• Controls retrotransposon proliferation
• Varies by cell type, tissue, and development stage.

Question 2

How can DNA methylation be detected?

Answer 2

1) Southern blot: Apply methylation-sensitive and insensitive restriction enzymes → differential cutting → gel electrophoresis to separate by size → transfer to membrane → probe
2) Bisulfite sequencing → converts unmethylated cytosines to uracils → PCR and genotype sequence or do full genotype sequencing

Question 3

How can DNA methylation be disrupted?

Answer 3

Inhibition using 5-aza-cytosine (AzadC) → incorporated into DNA and prevents methylation via deamination

Question 4

What is the effect of histone acetylation on gene expression?

Answer 4

Acetyl groups on lysines of histone H3/H4 tails (added by Histone acetyltransferases, removed by Histone deacetylases) open chromatin to euchromatin, increasing gene expression.

Question 5

What is the effect of histone methylation on gene expression?

Answer 5

Methylation on histones H3/H4 condenses chromatin into heterochromatin, reducing gene expression.

Question 6

Which modifications increase gene expression?

Answer 6

Histone acetylation → promotes euchromatin formation.

Question 7

Which modifications reduce gene expression?

Answer 7

1) DNA methylation
2) Histone deacetylation

Question 8

How can histone acetylation be studied experimentally?

Answer 8

Using Trichostatin A, which induces acetylation and allows functional effects to be observed.

Question 9

How do DNA methylation and histone acetylation interact?

Answer 9

Methyl-C binding protein MECP2 binds methylated DNA and recruits histone deacetylases, causing deacetylation and further gene repression.

Question 10

What is a Transposable Element (TE)

Answer 10

Discrete elements that can move from one DNA site

Question 11

Characteristics of DNA TEs

Answer 11

• Short inverted repeats at each end

Question 11

Classes of TEs

Answer 11

• Class 1 (RNA Elements)
• Class 2 (DNA Elements)

Question 12

How do DNA TEs transpose

Answer 12

• Cut and paste mechanism: element excises and inserts elsewhere in the genome
• Transposase binds to terminal repeats

Transposase binds to short inverted repeats at each end of the DNA TE and excises by double double-stranded break of the DNA. The transposase with the TE DNA binds to the target sequence and causes a staggered cut for the TE to insert. Host repair mechanisms repair the breakages.

Question 13

DNA TE autonomous elements

Answer 13

Code for transposase

Question 14

Non-autonomous DNA TEs

Answer 14

Do not code for transposase (use transposase from closely related autonomous elements)

Non-autonomous elements were derived from autonomous elements by internal deletion

Question 15

RNA elements

Answer 15

• mRNA as a transposition intermediate
• Often present in high copy numbers

Question 16

LTR elements

Answer 16

Contain gag and pol coding regions
- Gag codes for capsid like protein
- Pol codes for enzymes involved in transposition (reverse transcriptase, protease, RNAse, integrase)

Question 17

Non-LTR elements

Answer 17

No terminal repeats

Long Interspersed Nuclear Elements (LINEs)
- Autonomous elements: code for endonuclease and reverse transcriptase

Short Interspersed Nuclear Elements (SINEs)
- Non-autonomous: derived from LINEs via internal deletion

Question 18

DNA transposon structure and transposition mechanism

Answer 18

Draw

Question 19

LTR transposon structure and transposition mechanism

Answer 19

Draw

Question 20

Non-LTR transposon structure and transposition mechanism

Answer 20

Draw

Question 21

Target site duplication

Answer 21

Draw

Question 22

How do TEs affect genome structure?

Answer 22

• TE insertions can increase the distance between genes and increase genome sizes
• Recombination between TEs can lead to DNA deletion and decrease genome size

Question 23

Nested retrotransposons

Answer 23

• TEs that are inserted into each other
• Each TE originated later than the DNA flanking it
• This is a common occurrence because they do not disrupt genes and are thus unlikely to adversely affect the cell

Question 24

How are solo LTRs generated

Answer 24

- Due to recombination between LTRs of two different TEs on the same chromosome, brought into close proximity - Can lead to a decrease in genome size

Question 25

Draw solo LTR mechanism

Answer 25

Draw

Question 26

Effects of TE insertions

Answer 26

1) Insertion into a coding exon/splice site: Can disrupt a gene 2) Insertion into a regulatory region: Can have a positive or negative effect on gene expression (depending on the region) 3) Exonization of a TE: Insertion of TE leads to new splice sites (either contains its own splice sites, leads to mutation, or disrupts reading frame to create)

Question 27

Transcriptional silencing of TEs

Answer 27

Prevents transcription of TEs - Methylation of promoter regions → chromatin remodelling to heterochromatin

Question 28

Post-transcriptional silencing of TEs

Answer 28

Sequence-specific RNA degradation - Double-stranded RNA formation - SiRNAs

Question 29

Double-stranded RNA formation

Answer 29

Formed by antisense transcription (transcription of the opposite DNA strand to form a complementary RNA that binds to form dsRNA) and dsRNA degradation

Question 30

SiRNAs

Answer 30

Small interfereing RNAs - siRNA in association with proteins in a complex results in transcript cleavage - siRNA directs complex to target site by base pairing with the target RNA → proteins cleave