Page 86 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

7.4 What Mutations Tell Us About Gene Structure 245

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(Fig. 7.24c.1). To ensure that the two kinds of phages appearance of rare rII progeny (Fig. 7.24d.1). He knew
would infect almost every bacterial cell, he added many these wild-type progeny resulted from recombination and
more phages of each type than there were bacteria. not from reverse mutations because the frequencies of the
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When tested by Benzer’s method, if the two rII muta- rII phage particles he observed, even if rare, were much
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tions were in different genes, they would complement each higher than the frequencies of rII revertants seen among
other: Each of the mutant T4 chromosomes would supply progeny produced by infecting B strain bacteria with either
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one wild-type rII gene function, making up for the lack of mutant alone (Fig. 7.24d.2).
that function in the other chromosome and resulting in These experiments were possible only because Benzer
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lysis. On the other hand, if the two rII mutations were in devised a selection for rare rII recombinants. In a selec-
the same gene, they would fail to complement: No plaques tion, conditions are such that the only survivors are the rare
would appear because neither mutant chromosome would individuals you seek to identify. Benzer’s selection condi-
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be able to supply the missing function. tion for identifying rare rII recombinant progeny was plat-
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Tests of many different pairs of rII mutations showed ing for plaques on E. coli K(λ). Benzer could assay a phage
that they fall into two complementation groups: rIIA and lysate containing tens of thousands of phage progeny on a
rIIB. However, Benzer had to satisfy one final experimen- single petri plate containing a lawn of E. coli K(λ). Because
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tal requirement: For the complementation test to be mean- none of the rII phage in the lysate could form plaques,
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ingful, he had to make sure that pairs of rII mutations that even a single rII recombinant among them could be
failed to complement were each recessive to wild type and identified as a plaque.
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also did not interact with each other to produce an rII phe- On the basis of his observations with the rII genes,
notype dominant to wild type. He checked these points by Benzer drew three conclusions about gene structure and
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a control experiment in which he recombined pairs of rIIA function: (1) A gene consists of different parts that can
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or rIIB mutations onto the same chromosome (as de- each mutate; (2) recombination can occur between differ-
scribed in the next section) and then simultaneously ent mutable sites in the same gene; and (3) a gene performs
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infected E. coli K(λ) with these double rII mutants and its normal function only if all of its components are wild
with wild-type phages (Fig. 7.24c.2). If the mutations were type. From what we now know about the molecular struc-
recessive and did not interact with each other, the cells ture of DNA, this all makes perfect sense: The different
would lyse, in which case the complementation test would mutable units are the base pairs that constitute the gene.
be interpretable.
The significant distinction between the actual comple-
mentation test and the control experiment is in the placement A Gene Is a Discrete Linear
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of the two rII mutations. In the complementation test, one Set of Nucleotide Pairs
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rII mutation is on one chromosome, while the other rII
mutation is on the other chromosome (Fig. 7.24c.1); two How are the multiple nucleotide pairs that make up a gene
mutations arranged in this way are said to be in the trans arranged—in a continuous row, or dispersed in precise
configuration. In the control experiment (Fig. 7.24c.2), the patterns around the genome? And do the various mutations
two mutations are on the same chromosome, in the so-called that affect gene function alter many different nucleotides,
cis configuration. The complete test, including the comple- or only a small subset within each gene?
mentation test and the control experiment, is known as a
cis-trans test. In the complete experiment, two mutations
that do not produce lysis in trans but do so when in cis are in Using deletions to map mutations approximately
the same complementation group. Benzer called any com- To answer these questions about the arrangement of nucle-
plementation group identified by the cis-trans test a cistron, otides in a gene, Benzer eventually obtained thousands of
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and some geneticists still use the term cistron as a synonym spontaneous and mutagen-induced rII mutations that he
for gene. needed to map with respect to each other.
With the knowledge that the rII locus consists of two To map the location of a thousand mutants through
genes (rIIA and rIIB), Benzer could look for two mutations comparisons of all possible two-point crosses, Benzer
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in the same gene and then see if they ever recombine to would have had to set up a million (10 × 10 ) matings.
produce wild-type progeny. But by taking advantage of bacteriophage strains with large
deletions, he could obtain the same information with far
fewer crosses.
Recombination between different These large deletions are mutations that remove many
mutations in a single gene contiguous nucleotide pairs along a DNA molecule. In
When Benzer infected E. coli B strain bacteria with a crosses between bacteriophages carrying a mutation and
mixture of phages carrying different mutations in the same bacteriophages carrying deletions of the corresponding re-
gene (rIIA 1 and rIIA 2 , for example), he did observe the gion, no wild-type recombinant progeny can arise, because