Page 83 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

242 Chapter 7 Anatomy and Function of a Gene: Dissection Through Mutation

Figure 7.23 How recombination within a gene could phage infection, replication, and release produce a circu-
generate a wild-type allele. Suppose a gene, indicated by the lar cleared area in the lawn, called a plaque, devoid of
region between brackets, is composed of many sites that can living bacterial cells. The process of mixing phages with
mutate independently. Recombination between mutations m 1 and m 2 bacteria to produce a lawn and plaques on a petri plate is
at different sites in the same gene produces a wild-type allele and
a reciprocal allele containing both mutations. called plating phages.
Original Recombination Resultant Most plaques contain from 1 million to 10 million
chromosomes event chromosomes descendants of the single bacteriophage that originally
Gene infected a cell in that position on the petri plate. Sequen-
tial dilution of phage-containing solutions makes it pos-
+ + m 1 +++ + + + + m 1 +++ m 2 + sible to measure the number of phages in a particular
plaque and arrive at a countable number of viral particles
Mutation 1 ++ m ++ ++ + Recombinant gene
1
with two mutations (Fig. 7.24a.4).
++ + ++ + m 2 + When Benzer first looked for genetic traits associated
+ + + + ++ m 2 + + + + + ++ + + with bacteriophage T4, he found mutants that, when
added to a lawn of E. coli B strain bacteria, produced
Mutation 2 Recombinant larger plaques with sharper, more clearly rounded edges
wild-type gene
than those produced by the wild-type bacteriophages
(Fig. 7.24b). Because these changes in plaque morphol-
between homologous chromosomes carrying different mu- ogy result from the abnormally rapid lysis of the host bac-
tations known to be in the same gene could in theory gener- teria, Benzer named the mutations r for rapid lysis. Many
ate a wild-type allele (Fig. 7.23). r mutations map to a region of the T4 chromosome known
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Because mutations affecting a single gene are likely to as the rII region; these are called rII mutations.
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lie very close together, it is necessary to examine a very An additional property of rII mutations makes them
large number of progeny to observe even one crossover ideal for the genetic fine structure mapping (the mapping
event between them. The resolution of the experimental of mutations within a gene) undertaken by Benzer. Wild-
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system must thus be extremely high, allowing rapid detec- type rII bacteriophages form plaques of normal shape and
tion of rare genetic events. For his experimental organism, size on cells of both the E. coli B strain and a strain known
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Benzer chose bacteriophage T4, a virus that infects E. coli as E. coli K(λ). The rII mutants, however, have an altered
cells (Fig. 7.24a.1). Because each T4 phage that infects a host range; they cannot form plaques with E. coli K(λ)
bacterium generates 100 to 1000 progeny in less than an cells, although as we have seen, they produce large, unusu-
hour, Benzer could easily produce enough rare recombi- ally distinct plaques with E. coli B cells (Fig. 7.24b). The
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nants for his analysis (Fig. 7.24a.2). Moreover, by exploit- reason that rII mutants are unable to infect cells of the
ing a peculiarity of certain T4 mutations, he devised K(λ) strain was not clear to Benzer, but this property
conditions that allowed only recombinant phages, and not allowed him to develop an extremely simple and effective
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parental phages, to proliferate. test for rII gene function, as well as an ingenious way to
detect rare intragenic (within the same gene) recombina-
tion events.
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The experimental system: rII mutations
of bacteriophage T4
Even though bacteriophages are too small to be seen The rII region has two genes
without the aid of an electron microscope, a simple tech- Before he could check whether two mutations in the same
nique makes it possible to detect their presence with the gene could recombine, Benzer had to be sure he was really
unaided eye (Fig. 7.24a.3). To do this, researchers mix a looking at two mutations in a single gene. To verify this, he
population of bacteriophage particles with a much larger performed customized complementation tests tailored to
number of bacteria in molten agar and then pour this mix- two significant characteristics of bacteriophage T4: They
ture onto a petri plate that already contains a bottom layer are monoploid (that is, each phage carries a single T4
of nutrient agar. Uninfected bacterial cells grow through- chromosome, so the phages have one copy of each of their
out the top layer, forming an opalescent lawn of living genes), and they can replicate only in a host bacterium.
bacteria. However, if a single phage infects a single bacte- Because T4 phages are monoploid, Benzer needed to en-
rial cell somewhere on this lawn, the cell produces and sure that two different T4 chromosomes entered the same
releases progeny viral particles that infect adjacent bacte- bacterial cell in order to test for complementation between
ria, which, in turn, produce and release yet more phage the mutations. In his complementation tests, he simultane-
progeny. With each release of virus particles, the bacterial ously infected E. coli K(λ) cells with two types of T4 chro-
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host cell dies. The agar in the top layer prevents the phage mosomes—one carried one rII mutation, the other carried
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particles from diffusing very far. Thus, several cycles of a different rII mutation—and then looked for cell lysis