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Biology of Cells
Describe how Benzer used genetic mapping and complementation testing to
elucidate the fine structure of the rII region of bacteriophage T4
Seymour Benzer’s experiments on T4 bacteriophages and E.coli, using the
techniques of genetic mapping and complementation testing, enabled the
determination of the structure of the rII locus of the phage.
It is necessary to understand the mechanism of bacteriophage infection of
bacterial cells before the experiments Benzer used can be understood. The phage
attaches to the bacterial cell by binding to receptor proteins on the cell’s surface,
and injects its genetic information into the cell, leaving the protein coat on the
outside. The receptor proteins on the bacterial cell’s surface membrane are
complementary in shape to the regions of the phage with which they interact,
but are normally used for other essential functions. For example, a transport
protein responsible for the uptake of maltose in E.coli is the cell surface receptor
that allows the bacteriophage lambda to bind to the bacterium. After entering,
phage replicate inside the cell; its genes direct the machinery of the bacterial cell
to carry out replication of the phage chromosome and synthesis of the protein
components of the coat. Using these components new phage self assemble and
are released on cell lysis, ready to infect more bacterial cells. When susceptible
bacterial cells on a plate lyse clear regions, called plaques, form. This observation
was used in Benzer’s experiments to indicate whether there had been successful
phage attack, as a way of determining which host bacterial strains had been
infected.
Benzer’s experiments investigated the rII locus. Phage with mutations at this
locus are able to infect and lyse E.coli B, and cause rapid cell lysis, indicated by
large, clear plaques; but are unable to lyse E.coli K. Wild type T4 bacteriophages
lyse both K and B type E. coli.
Recombination between phage chromosomes is very rare, but because of the
huge number of phage that can be used in these experiments, crossovers do
occur.
Biology of Cells
When two phage with different mutations at rII locus are crossed by double
infection of an E. coli B cell, double mutant recombinants may be formed via
cutting and joining of DNA by bacterial enzymes, as well as wild type
recombinants. The wild type recombinants are able to infect E.coli K, so cause the
formation of plaques when added to the E. coli K plates. By using these
experiments to determine the recombination frequency between different
mutations Benzer analysed over 2000 mutants to create a genetic map of the rII
region.
Here, Recombination frequency = 2 x ((number of recombinant phage strains
able to infect E.coli K)/total plated )x100
Where the fraction is multiplied by two to account for double recombination.
The recombination frequency can be used to work out the relative distances
between mutations, as the further apart the mutations are, the more likely it is
that there will be crossovers producing the wild type double recombinants, able
to infect E.coli K. This can be used to work out the order of the sites of mutations
in the genes, and therefore the creation of a genetic map of the rII region.
Benzer utilised complementation testing in order to determine how many genes
the rII region contained, which could not be indicated by the genetic mapping
experiment. When E. coli K is infected simultaneously with two different rII
mutants a plaque will only form if the mutations are in different genes and
therefore there is still one functional copy of each gene being expressed between
them. This means the phage are able to direct their own replication, and the
bacterial cells lyse. The mutations are said to complement. Mutations in the same
gene do not complement as there will be no functional copy of that gene present
in the E. coli, so the bacteriophages will not be able to replicate, and the cells will
not lyse. He tested the mutations that he had previously mapped, to see whether
pairs of mutant phage with various combinations of the pairs of mutations would
lead to the wild type phenotype being expressed; and so complementation being
shown. The results of Benzer’s experiments showed that there were two rII
genes, rIIA and rIIB, since mutations in group A complemented mutations in
Biology of Cells
group B, and all mutations in group A mapped to one half of the locus, while
those in group B mapped to the other half.
In conclusion, Benzer used genetic mapping by the formation of wild type double
recombinants from many pairs of mutant phage in order to find recombination
frequencies, and complementation by the infection of E.coli simultaneously by
two different mutant phage and observing whether cell lysis occurred to
elucidate the fine structure of the rII region of bacteriophage T4. His
experiments were far more significant than determining the structure of the rII
region alone, as this was the first time that the order and separation of genes and
their internal structure had been determined.
References
Lectures: ‘Hunting the Gene’ David Summers
Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th
edition. New York: W. H. Freeman; 2000. Available from:
http://www.ncbi.nlm.nih.gov/books/NBK21989/