If the rlla and rllb mutations occur at different loci that code for different proteins then, in bacterial cells infected by both mutants, the wild-type sequences on the chromosome opposite each mutation will overcome the effects of the recessive mutations; the phages will produce normal plaques on E. coli K cells ( FIGURE 8.32, steps 3, 4, and 5). (Benzer coined the term cistron to designate a functional gene defined by the complementation test.) If, on the other hand, the mutations occur at the same locus, no functional protein is produced by either chromosome, and no plaques develop in the E. coli K cells ( FIGURE 8.32 steps 6, 7, and 8). Thus, the absence of plaques indicates that the two mutations occur at the same locus.

In the complementation test, the cis heterozygote is used as a control. Benzer simultaneously infected bacteria with wild-type phage ( a+ b+ ) and with phage carrying both mutations ( a b- ). This test also produced cells that were heterozygous and cis for the phage genes:

Regardless of whether the rIIa and rIIb mutations are in the same functional unit, these cells contain a copy of the wildtype phage chromosome ( a+ b+ ) and will produce normal plaques in E. coli K.

Benzer carried out complementation testing on many pairs of rll mutants. He found that the rll region consists of two loci, designated rllA and rllB. Mutations belonging to the rllA and rllB groups complemented each other, but mutations in the rIIA group did not complement others in rllA; nor did mutations in the rllB group complement others in rIIB. __

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