Lecture 17: Making Mutants File

Question 1

When do mutations occur ?

Answer 1

mutations can occur at any time, but are fixed at replication

Question 2

How many mutations per generations ?

Answer 2

roughly 150

Question 3

how can you mutate haploid yeast ?

Answer 3

UV (frame-shift) and EMS (point mutation)

Question 4

How would you screen east for abnormal shape/size ?

Answer 4

Use microscope and centrifuge

Question 5

How do flies achieve high alcohol tolerance levels ?

Answer 5

Drosophilia Alcohol dehydrogenase (adh) gene organisation

Question 6

What are the steps you would take to find x-linked mutations in flies ?

Answer 6

• Feed males EMS
• Mate to healthy females
• Collect F1 females
• Mate to healthy males
• Collect 10,000 F2 males
• Screen for alcohol intolerance
• Create stock of mutants
• Confirm X-linked inheritance
• Map, clone and identify gene

Question 7

What instrument could be used to filter out flies according to their alcohol tolerance ?

Answer 7

Inebriometer

Question 8

What is cheapdate ?

Answer 8

an allele of amnesiac (gene involved in learning and memory). It encodes neurpeptide and reveals biological link between alcohol tolerance and learning and memory

Question 9

Flies have cocaine tolerating genes,what are these linked to ?

Answer 9

circadian rhythm

Question 10

whats a classic mutagenesis problem ?

Answer 10

easy to mutate genes, but difficult to find, clone and identify mutant gene

Question 11

In E.coli, whats a common mutagen ?

Answer 11

Tn5 transposon

Question 12

What does Tn5 transposon do ?

Answer 12

carries kanomycin resistance, which is flanked by inverted repeats while facilitate jumping

Question 13

WHat are the stages of the Tn5 transposon jump ?

Answer 13

1. Tn5 is inside plasmid with defective oriR site
2. Tn5 transposon jumps randomly into the host chromosome
3. plasmid with defective oriR site is lost as cell replicates

Question 14

What happens after transposon mutagenesis?

Answer 14

Each bacteria has a different mutation, plate bacteria on CM to generate colonies, then replicate plate onto CM-ADE to find Ade- mutants (if screening for ade auxotrophs )

Question 15

What are the pros of transposon mutagenesis ?

Answer 15

Easy to clone the mutated gene

Question 16

What are the cons of transposon mutagenesis ?

Answer 16

1. some transposons dont jump at high rate
2. some transposons only jump into specific target sequences
3. all transposon mutations are insertions (no point mutations)

Question 17

What is an RNA interface (RNAi) ?

Answer 17

some genes make dsRNA (double stranded), the dicer protein compelx cleaves the dsRNA into siRNA (short interfering RNA)

Question 18

What is the job of the RISC protein complex ?

Answer 18

It hunts complementary strands and destroys them

Question 19

How would you exploit the RISC degredation of mRNAs ?

Answer 19

1. introduce a newly synthesised dsRNA to cell
2. it will attack and destroy homologous mRNAs

This can be targetted at specific gene (switches it off)

Question 20

What does the process of RNAi create ?

Answer 20

gene knockdowns

Question 21

What does RNAi mimic ?

Answer 21

the effect of mutations

Question 22

How would you use a targetted RNAi interference ?

Answer 22

1. find gene of interest on fly genome
2. make transgene that generates dsDNA
3. Introduce RNAi transgene to host male fly
4. Test fly for sterility