Test2: Lect 9 Sean Speese Flashcards
Transposon:
- Characteristics:
- Alternative name:
- Two types:
- Characteristics:
Flanked by inverse repeats - Alternative name:
P elements (also called “jumping genes” but not professionally) - Two types:
Autonomous: encode the genes for transposition
Non-autonomous: do not encode genes for transposition. Someone other p - element must make these products
How is class 1 transposon transposition achieved?
Transposons inverted repeats line up, making the transposon loop out. ->
Transposase catalyzes the cleavage ->
insertion random but euchromatic sites and non-coding regions
are preferred
Transposons and the brain:
Normally tissues inactivate transposons.
The brain has transposon activity during neurogenesis and in aged neurons.
- May contribute to neural diversity during neurogenesis.
- May contribute to neural degeneration in aged neurons
Class 1 transposable elements:
Class 2 transposable elements:
1: The element itself is cut out, and inserted elsewhere in the genome
2: Work through an RNA intermediate, which allows it to be copied elsewhere into the genome
- Note: both are copied!
How is class 2 transposon transposition achieved?
RNA made as normal ->
reverse transcriptase encoded for ->
Copies RNA into dsDNA ->
Inserts DNA into the genome
P elements in flies are usually what class of transposon?
Class 1 baby
Transposable mutagenesis:
- Define:
- Why not just use EMS?
- Define:
Cross a fly with non-autonomous transposable element to a fly with a transposase gene. ->
Mutations will occur as P element inserts into new areas ->
Knockout or silence the transposase gene ->
Screen for mutants - Why not just use EMS?
The advantage, is after you’ve confirmed a mutant, you can use imprecise excision to rip out your transposon and some of the genetic material around it. It makes it easier to find out where you are.
Gene insertion using a P-element for fly:
Take a P element vector, remove the transcriptase, and replace it with your gene of interest ->
Insert your modified gene transposon, and insert DNA with transcriptase (but no flanking repeats) separately into an embryo ->
gene inserted, transposase is no longer there. ->
you can check to see if the fly has the phenotype of interest
What are some uses of P-elements?
bring back wild type gene in mutant flies – reversion of the phenotype (for double knock outs)
-
for dominant mutations; introduce the mutated version in wild type
I want to know in what cell types and when hypothetical gene A is being expressed, how do I test that with P elements.
1: Make a p-element with a fluorescent marker gene with the promotor for gene A in front of the fluorescent marker gene.
2: Gene A will be transcribed whenever that promoter would be transcribed and you can see where it is fluorescing
Enhancer Trap:
- How would I make one?
- What does it do?
- Why would I make one?
- How would I make one?
Basic promoter with no enhancer region, followed by a fluorescent gene, and a gene which shows a distinct phenotype in the fly (like white, which produces red eyes) - What does it do?
It doesn’t have an enhancer, so it will only operate when located near an enhancer for it. So you can discover where enhancers are for certain regions.
Then, imprecise excision can cut it back out and you can identify which regions are near that region. - Why would I make one?
Identify where regulatory domanins are within the genome.
Protein traps:
- Describe:
- Why is it used?
- Describe:
1: A p element which likes to insert into introns.
2: A 5’ and 3’ splice site, and a fluorescent marker, like GFP. (this means that upon insertion into an intron, it will essentially make itself into a new exon)
3: This is inserted into an intron. - Why is it used?
1: Allows you to see exactly when that gene is turned on.
2: Sometimes insertion will stop the gene from making a product. Which is lethal.
3: Other times, GFP or whatever fluorescent reporter you use, is incorporated into the protein, and the protein maintains function. Then you can use this method to see where the protein is in the cell.
If a mutant has been created by P element mutagenesis, what would:
- Using precise excision do?
- Using imprecise excision do?
- Using precise excision do?
1: Should restore wild type phenotype, by cleanly removing itself from the gene. - Using imprecise excision do?
1: It would rip out the regions around the gene, allowing you to look at what was taken out.
2: It would rip out the regions around the gene, possibly creating a mutant form of that gene (if it cut out exons)
If you put a stop codon into a gene trap insertion what would happen?
Premature stop of the protein.
GaL4/UAS system:
- Define GAL4:
- Define UAS:
- Define GAL4:
Is a gene which encodes for a transcription factor which binds to the UAS enhancer sequence to activate transcription - Define UAS:
An enhancer, which can be bound to by GAL4 to activate transcription of the gene after it
