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Structure of R II Genes - The fine structure of a gene is essentially the linkage map of multiple alleles of a gene and it depicts the location of these alleles within the genes.

The procedure used for this purpose is basically the same as that used for preparation of linkage maps of different genes.

Benzer prepared the first fine structure map in 1961 for the R-II locus of the T4 bacteriophage of E coli.

Bacteriophage T4 is an obligate parasite that grows on E. coli. It consists of a single linear molecule of DNA about 200,000 bp long.

When a wild type T4 phage particle infects an E coli under optimal conditions, it will produce 200-400 progeny viruses in 20-30 min. by lysing the cell.

On a Petri plate consisting of lawn of bacteria, wild type T4 phage produces a clear area in the lawn. When present, such an area is called as plaque.

These plaques are fuzzy, turbid or halos. When a wild type T4 infection E. coli, it is super infected with a second T4 phage (artificial second infection by strains of the same type of T4 virus). The plaque formation is inhibited by 2 hours. This phenomenon is called as lysis inhibition.

But certain mutations in T4 bacteriophages lead to prevention of lysis inhibition, i.e., the mutants do not show lysis inhibition.

Such mutants are called as r mutants. These r mutants produce larger plaques. Benzer discovered that r-mutants are due to mutations in one of the three regions or loci, which he called as r I, r II and r III loci.

Benzer differentiated these loci due to inability of the r-mutants to grow on different subs trains of E. coli. r-mutants with mutations in r-II locus or r-II locus mutants are unable to multiply in K12(λ) strain of E. coli. But they grow on the other strain of E. coli like Band K12.

Phages carrying r-II mutants can be easily isolated by serially transferring inoculum with sterile loop from r-type plaques on E. coli B lawn to lawns of E. coli K12 (λ.), Band K12. The r-I and r-III mutants will grow on both K12 (λ) and B whereas r- II will grow only on B lawn but do not grow on K12 (A).

To confirm the mutation site, Benzer did cis-trans or complementation tests, and all the r-II mutants were found to be located in one of the two genes. For the complementation tests, Benzer developed a simplified procedure called complementation spot test.

In this method, the mutant phage which needs to be tested is mixed with E. coli K12 (λ) strain and r+ mutant grows on K12 (λ.) strain.Then he plated the mixture on the nutrient medium.

Then a large plaque is formed at the reciprocative site, i.e., if the mutation has occurred in r-III B gene, then the plaque is formed at the drop placed in r-II A gene.

If complementation does not occur, the area will be grown with the lawn of E. coli cells. R+ mutants are mutants that occur at other than the three sites (r-I, r-II and r-III).

Although, complementation tests showed that all the r-II mutations were located in two genes, recombination tests demonstrated that these mutations were located at many distinct sites within these two genes.

Recombination between r-II mutations is analysed by simultaneously infecting E. coli B cells (permissive host cells).

With the two r-II mutants in question and plating the progeny phages on a lawn of E. coli K12 (λ.) will show if any wild type recombinants have been produced.

The total number of progeny phages produced is determined by plating the lysate on an E. coli s lawn. As many as 108 progeny phages may be seen in a single Petri plate as recombination is a very rare event (2 in 100 million and can be easily be detected.)
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