Page 84 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

FEATURE FIGURE 7.24

How Benzer Analyzed the rII Genes of Bacteriophage T4
(a.1) (a.2) 1. Phage injects
its DNA into
host cell.
Viral
chromosome
Host
chromosome 2. Phage proteins
synthesized;
Sheath DNA replicated.
4. Lysis of Host chromosome
host cell
degraded.
Tail
fibers
3. Assembly of phages
within host cell
(a.3) (a.4) Pipette out
0.01 ml 0.01 ml 0.1 ml
0.1 ml
Add plating
bacteria

1 ml 1 ml 1 ml 1 ml 25 plaques
Concentrated
solution of Tubes containing medium
bacteriophages without phages
a.1: © Science Source; a.3: © McGraw-Hill Education/Lisa Burgess
(a) Working with bacteriophage T4
1. Bacteriophage T4 (at a magnification of approximately 100,000×) and in an artist’s rendering. The viral chromosome is con-
tained within a protein head. Other proteinaceous parts of the phage particle include the tail fibers, which help the phage attach to
host cells, and the sheath, a conduit for injecting the phage chromosome into the host cell.
2. The lytic cycle of bacteriophage T4. A single phage particle infects a host cell; the phage DNA replicates and directs the
synthesis of viral protein components using the machinery of the host cell; the new DNA and protein components assemble into new
bacteriophage particles. Eventual lysis of the host cell releases up to 1000 progeny bacteriophages into the environment.
3. Clear plaques of bacteriophages in a lawn of bacterial cells. A mixture of bacteriophages and a large number of bacteria in
molten agar are poured into a petri plate. Uninfected bacterial cells grow, producing an opalescent lawn. A bacterium infected by a
single bacteriophage will lyse and release progeny bacteriophages, which infect adjacent bacteria. Several cycles of infection result
in a plaque, a circular cleared area containing millions of genetically identical bacteriophages.
4. Counting bacteriophages by serial dilution. A small sample of a concentrated solution of bacteriophages is transferred to a test
tube containing fresh medium, and a small sample of this dilution is transferred to another tube of fresh medium. Successive repeats of this
process increase the degree of dilution. A sample of the final dilution, when mixed with bacteria in molten agar, yields a countable number
of plaques from which it is possible to extrapolate the number of bacteriophages in the starting solution. The original 1 ml of solution in this
7
illustration contained roughly 2.5 × 10 bacteriophages.
−
(b) Phenotypic properties of rII (b.1) (b.2)
mutants of bacteriophage T4
−
1. rII mutants, when plated on rII + T4 strain E. coli strain
E. coli B cells, produce plaques that rII – B K( )
are larger and more distinct (with
sharper edges) than plaques formed – Large, distinct No plaques
+
by rII wild-type phages. rII
−
2. rII mutants are particularly
useful for finding rare recombination
events because they have an altered rII + rII + Small, fuzzy Small, fuzzy
+
host range. In contrast to rII wild-type
−
phages, rII mutants cannot form
plaques in lawns of E. coli strain K(λ) b.1: © Seymour Benzer (Continued)
243