6 bacterial genetics screencasts - recombination frequency mapping: recombination between phage as an example

what is recombination?

the process where genetic markers in two separate genomes become physically linked via genetic exchange

where does homologous recombination generally occur in the chromosome?

at random along all points along the chromosome

how does two genetic markers being further apart affect their chances of being separated?

they are more likely to be separated by genetic exchange in a recombination event

how can we use recombination events to order the markers on a chromosome?

by comparing the recombination frequencies

why does screening more progeny increase the resolution of the map?

because recombination is a relatively rare event, more progeny screened increases the resolution

what mutations can the markers in alleles be based on?

substitution, addition or deletion mutations

what is the equation for the map distance?

-       the map distance = number of recombinants  x 100%

-    ————————————————————————————————————

                                 number of progeny

how did Benzer exploit the properties of deletions?

he did this to rapidly narrow down the locations of new point mutations to specific regions of DNA in a procedure called deletion mapping

what is the logic behind deletion mapping?

a point mutation cannot produce recombinants in a cross with a deletion mutant in which the location of the point mutation is within the region deleted

how was Benzer able to select mutants from the same location to produce a higher resolution map?

by narrowing down locations of mutants to specific regions

what would happen if he wasn’t able to narrow down new point mutations to specific regions?

the number of crosses required to produce the map would have been substantially higher

why were deletion mutants identifiable in Benzer’s work?

because they never reverted

describe the assay used to estimate phage numbers in a sample

• plaques

• phage sample is mixed with a large excess of bacteria

• then spread onto solid medium in a petri dish

• as bacteria grow on the plate, each phage forms a focus of infection

• successive rounds f infection result in an area where the bacteria has been killed - a plaque

• these plaques are easy to detect

do temperate (lysogenic phage also produce plaques?

yes, although they are turbid and not clear

what phage did Benzer study?

rII mutants of bacteriophage T4

what is different about the rII mutant plaques to the wild type T4 phage plaques?

they are larger plaques with sharper edges

what strain of E. coli were the rII mutants able and not able to grow on?

• able to grow on E. coli strain B

• unable to grow on E. coli strain K12 lambda

what did the rII mutants only being able to grow on E. coli strain B show?

they had an altered host range

in a cross between two rII mutants, how could the total number of progeny be determined?

by the number of plaques produced on E. coli strain B

in a cross between two rII mutants, how could the total number of recombinants be determined?

by the number of plaques produced on E. coli strain K12 lambda

what was the problem with the plaques seen on E. coli strain B in the rII mutant cross?

they were only detecting the number of wild type progeny, not the equal number of double mutant progeny

what was Benzer’s modified equation to deal with the test only detecting the number of wild type progeny?

what does 2x total number of r+ (wild type) recombinants mean in the modified equation?

the total number of recombinants

what process did Benzer do to make the map for the rII locus?

• infected strain B with a mix of two mutants in equal proportions but with phage in excess over the cells

• meaning most cells were infected simultaneously by both rII mutants

• this established the conditions for recombination between the phage genomes

• following infection, the phage progeny (a mix of parental and recombinant types) were mixed separately with strains B and K12 lambda

• so cells were now in excess over the phage and any plaques produced would be the product of a single phage

• Benzer counted the plaques and used the modified formula to produce the high-resolution map of the rII locus

what was Benzer able to do because the resolution for his rII locus map was so high?

he was able to define mutations separated by only a single nucleotide

what did Benzer prove with his high resolution rII locus map?

that mutations were distributed in many different parts of a single gene

what are the principles used to map the rII region of bacteriophage T4 also used for?

• mapping eukaryotic genes

• mapping the distance between mutations in different genes (intergenic mapping)

• mapping the distance between mutations in the same gene (intragenic mapping)