Friday, 8 February 2013

Intragenic Recombination

In other parts of this blog you saw how genes can be mapped with respect to each other using a simple rule:  the close two genes to each other, the less likely there can be a recombination event between them.  This means that the proportion of crossovers is linked to gene distance, so Sturdevant, a student of T.H. Morgan, developed the first classical mapping technique - essentially the one used today.

What's the CLOSEST two loci can be to each other?  From a theoretical standpoint, it would be to the smallest unit that can be heritable.  Scientists knew these to be "genes", and in the 1950s (before the double-helix structure of DNA was known), the biologist Seymour Benzer wanted to ask what the smallest measurable distance of crossover would be.  Genes weren't understood the way we know them now - as linear arrays of nucleotides that portray information not unlike how certain chains of letters make up words and sentences.  Perhaps they were very complicated structures, and crossover couldn't occur within them?  Or is it possible that the nucleotide letters in a gene align up between homologous regions and crossover can occur?  Benzer used rII mutant viruses to answer the question.

rII mutants cannot infect E. coli strain K12.  They *can* infect E. coli strain B, but the plaques that form have unusual phenotypes.  At high density of plaque formation, though, they're hard to distinguish from each other.  In the course of investigating the genetic nature of the rII locus, Benzer found out there are two genes there, which he named A and B.  A functional A and a functional B gene product (i.e. protein) is required to infect strain K12.  If you coinfect bacteria with an A mutant and a B mutant, lots of progeny form.  However, if recombination can occur within a single gene, two A mutants (let's call them rIIa and rIIa') can - on rare occasions- create two recombinants (we'll call them rIIa+ and rIIa'').    The trick is to find out how many wild type (rIIa+) phage are created.  So Benzer's trick is simple:  find out your total progeny by counting how many phage result from a coinfection of E. coli B strain by plating it on, well, a B strain lawn.  You can find all the wild-type phage (rIIa+) by plating them on a K12 strain lawn, and use the Studevant mapping formula:  distance = #recombinants/total progeny x 100.

However, since you don't see ALL the recombinants - only the wild type rIIa+because rIIa'' look just like the parentals - you need to multiply the number of K12 plaques by 2.  For every wild-type phage, you'll make one of those new "double mutant" alleles (A bad name for the rIIa'' genotype, but somewhat descriptive.  Hopefully you're following the logic here!).

In the two videos below I go over these concepts.  The first is the theory I've given above.  The second shows how to use the formula.  I didn't point out that coinfection must occur in E. coli strain B, but you probably could figure that out for yourself!

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