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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/