Lecutre 5

Question 1

Benzer complementaion system diagram

Answer 1

• both in rIIA thus not complemented - no plaque will form
• in different genes - plaque will form

Question 2

The outcome of benzer complementaion experiment

Answer 2

Some pairs of mutants could from progeny phage ie. each mutant was able to provide the function missing from the other: COMPLEMENTATION: mutations are in different genes.

Some pairs did not give rise to progeny phage ie. neither phage could provide the missing function: NO COMPLEMENTATION: mutations are in the same gene

Progeny phage were identical to the parental infecting page ie. had not undergone genetic changes

Question 3

What is recombination

Answer 3

A process that generates new gene combinations

Question 4

How recombination in E.coli strain B worked

Answer 4

• infected E.coli strain B with pairs of mutants (all rII mutants can grow in this strain) to allow recombination to occur

Question 5

How did they test that the recombination had occurred

Answer 5

• by infecting the progeny phage onto E.coli strain K
• rII mutants can’t grow on this strain but wild-type cam
• plaques indicated that wild-type phage had been formed by recombination between two rII mutant chromosomes inside the E.coli strain B cells

Question 6

Complementaion vs recombination

Answer 6

• complementation: lets you tell if mutations are in the same or different genes; progeny are genetically identical to parents
• recombination: results in new combinations of mutations (ie. progeny are genetically different to parents.

Question 7

Recombination involves..

Answer 7

.. the exchange of genetic material (DNA) between corresponding chromosomes
…. Breakage and rejoining of DNA

Question 8

Hollidays model for recombination

Answer 8

No recombinant = still parental strand = cleavage across horizontal?

Heteroduplex = complemetnatry pairing with the other strand

Question 9

Key concepts of recombination process

Answer 9

• alignment of DNA sequences
• breakage of DNA strands, exchange and rejoining of the DNA ( creation of the Holliday junction)
• branch migration, giving heteroduplex (hybrid) DNA where each strand is derived from a different parent molecule
• resolution leaves two DNA molecules with combinations of alleles

Question 10

How does recombination occur at the molecular level?

Answer 10

• best understood in E.coli
• involves a number of enzymes
• RecBCD protein complex nicks, unwinds and degrades the DNA to generate single- stranded DNA
• RecA protein coats the single-stranded DNA and then catalyses base-pairing of the DNA with the “target” double-stranded molecule
• proteins (RevA and RuvB) cause branch migration
• RuvC protein then resolves Holliday junctions by DNA cleavage

Question 11

Take home

Answer 11

Question 12

The brothers

Answer 12

Question 13

Wrap up