Fine Structure Analysis of Bacteriophage Genes

In the 1950s and 1960s, Seymour Benzer conducted a series of experiments to examine the structure of a gene. Because no molecular techniques were available at the time for directly examining nucleotide sequences, Benzer was forced to infer gene structure from analyses of mutations and their effects. The results of his studies showed that different mutational sites within a single gene could be mapped (intragenic mapping) by using techniques similar to those just described. Different sites within a single gene are very close together; so recombination between them takes place at a very low frequency. Because large numbers of progeny are required to detect these recombination events, Benzer used the bacteriophage T4, which reproduces rapidly and produces large numbers of progeny.

Benzer's mapping techniques Wild-type T4 phages normally produce small plaques with rough edges when

8.30 T4 phage rll mutants produce distinct plaques when grown on E. coli B cells. (Dr. D. P. Snustad, College of Biological Sciences, University of Minnesota.)

grown on a lawn of E. coli bacteria. Certain mutants, called r for rapid lysis, produce larger plaques with sharply defined edges. Benzer isolated phages with a number of different r mutations, concentrating on one particular subgroup called rll mutants.

Wild-type T4 phages produce typical plaques ( Figure 8.30) on E. coli strains B and K. In contrast, the rll mutants produce r plaques on strain B and do not form plaques at all on strain K. Benzer recognized the r mutants by their distinctive plaques when grown on E. coli B. He then collected lysate from these plaques and used it to infect E. coli K. Phages that did not produce plaques on E. coli K were defined as the rll type.

Benzer collected thousands of rll mutations. He simultaneously infected bacterial cells with two different mutants and looked for recombinant progeny ( FIGURE 8.31). Consider two rll mutations, and b~, and their wild-type alleles, a+ and b+. Benzer infected E. coli B cells with two different strains of phages, one a~ b+ and the other a+ b~ (Figure 8.31, step 1). While reproducing within the B cells, a few phages of the two strains recombined (Figure 8.31, step 2). A single crossover produces two recombinant chromosomes; one with genotype a+ b+ and the other with genotype a~ b~ :

The resulting recombinant chromosomes, along with the nonrecombinant (parental) chromosomes, were incorporated

Question: How can rII phage mutants be mapped and what can they reveal about the structure of the gene?

rll mutant 2 chromosome

(feme rII mutant 1 chromosome rII mutant 1 chromosome rll mutant 2 chromosome


Only the a+ recombinant can grow on E. coli K, allowing them to be identified.

No plaques

Plaques produced by a+ b+ recombinant phage

Infect E. coli K cell

Gene-structure map of the rII region

Only the a+ recombinant can grow on E. coli K, allowing them to be identified.

No plaques

Plaques produced by a+ b+ recombinant phage

Infect E. coli K cell

No plaques

Gene-structure map of the rII region rllA

Conclusion: Mapping more than 2400 rII mutants provided information about the internal structure of a gene at the base-pair level—the first view of the molecular structure of a gene.


The frequencies of recombinants were used to map rII mutants.

Each box represents one DNA base pair.

Mutations were found at each location shown in red.

8.31 Benzer developed a procedure for mapping rII mutants. Two different rII mutants (a- b+ and a+ b-) are isolated on E. coli B cells. Neither will grow on E. coli K cells. Only the a+ b+ recombinant can grow on E. coli K, allowing these recombinants to be identified. rIIA and rIIB refer to different parts of the gene.

into progeny phages (Figure 8.31, steps 3 and 4), which were then used to infect E. coli K cells. The resulting plaques were examined to determine the genotype of the infecting phage.

The rII mutants would not grow on E. coli K, but wildtype phages could; so progeny phages with the recombinant genotype a+ b+ produced plaques on E. coli K (Figure 8.31, step 5). Each recombination event produces an equal number of double mutants (a- b-) and wild-type chromosomes (a+ b+); so the number of recombinant progeny should be twice the number of wild-type plaques that appeared on E. coli K. The recombination frequency between the two rII mutants would be:

recombination frequency =

2 X number of plaques on E. coli K total number of plaques on E. coli B

Benzer was able to detect a single recombinant among billions of progeny phages, allowing very low rates of recombination to be detected. Recombination frequencies are proportional to physical distances along the chromosome (p. 000 in Chapter 7), revealing the positions of the different mutations within the rII region of the phage chromosome. In this way, Benzer eventually mapped more than 2400 rII mutations, many corresponding to single base pairs in the viral

DNA. His work provided the first molecular view of a gene. __

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