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9.1 Fragmenting DNA 319

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Serendipity in Science: The Discovery of Restriction Enzymes
Most of the tools and techniques for cloning and analyzing DNA Figure A Operation of the restriction enzyme/
fragments emerged from studies of bacteria and the viruses that modification system in nature. (1) E. coli strain C does not
infect them. Molecular biologists had observed, for example, that have a restriction enzyme/modification system and is susceptible
viruses able to grow abundantly on one strain of bacteria grew to infection by the lambda phage. (2) In contrast, E. coli strain
poorly on a closely related strain. While examining reasons for K12 generally resists infection by viral particles produced by a
this discrepancy, these scientists discovered restriction enzymes. previous infection of E. coli C. Cells of E. coli K12 make the
To follow the story, one must know that researchers com- EcoRI restriction enzyme, which cuts the lambda DNA molecule
pare rates of viral proliferation in terms of plating efficiency: the before its genes can be expressed. (3) In rare K12 cells,
fraction of viral particles that enter and replicate inside host modification enzymes add methyl groups (me) to lambda DNA,
protecting it from the restriction enzymes. Modified lambda DNA
bacterial cells, causing the cells to lyse and release viral prog- can now replicate, and as the DNA methylation marks are
eny. These progeny go on to infect neighboring cells, which in copied during DNA replication, progeny viruses that readily form
turn lyse and release further virus particles. When a petri dish is plaques on K12 bacteria are generated.
coated with a continuous lawn of bacterial cells, an active viral Lambda E. coli C
infection forms as a visibly cleared spot, or plaque, where bac- (1) virus
teria have been eliminated (see Fig. 7.24). The plating efficiency particle
of lambda virus grown on the E. coli C strain is nearly 1.0 (Fig. A.1).
This means that 100 original virus particles will cause close to
100 plaques on a lawn of E. coli C bacteria.
The plating efficiency of the same virus grown on E. coli Replication
4
K12 is only 1 in 10 , or 0.0001. The ability of a bacterial strain
to prevent the replication of an infecting virus, in this case the
growth of lambda on E. coli K12, is called restriction. Lysis—bacterium dies
Restriction is rarely absolute. Although lambda virus grown
on E. coli K12 produces almost no progeny (the viruses infect
cells but can’t replicate inside them), a few viral particles inside
a few cells do manage to proliferate. If their progeny viruses are (2) (3)
then tested on E. coli K12, the plating efficiency is nearly 1.0. E. coli K12— E. coli K12—rare cell
The phenomenon in which growth on a restricting host modifies most cells
a virus so that succeeding generations grow more efficiently on
that same host is called modification.
What mechanisms account for restriction and modification?
Studies following viral DNA after bacterial infection found that DNA restriction DNA modification
during restriction, the viral DNA is broken into pieces and de- me me
graded (Fig. A.2). The enzyme responsible for the initial break- me me
age was found to be an endonuclease, an enzyme that breaks Replication
phosphodiester bonds, usually making double-strand cuts at
specific locations in the viral chromosome. Because this break-
age restricts the biological activity of the viral DNA, researchers Bacterium lives. No Lysis—bacterium dies
called the enzymes that accomplish it restriction enzymes. viruses produced
Subsequent studies showed that the small percentage of
viral DNA that escapes digestion and goes on to generate new
viral particles has been modified by the addition of methyl groups
during its replication in the host cell (Fig. A.3). Researchers Researchers did not set out to find restriction enzymes; they
named the enzymes that add methyl groups to specific DNA se- could not have known these enzymes would be one of their
quences modification enzymes. finds. Rather, they sought to understand the mechanisms by
Biologists have identified complementary restriction- which viruses infect and proliferate in bacteria. Along the way,
modification systems in a wide variety of bacterial strains. they discovered restriction enzymes and how they work. The
Purification of the systems has yielded a mainstay of recombinant politicians and administrators in charge of allocating funds often
DNA technology: the battery of restriction enzymes used to cut want to direct research spending to urgent health or agricultural
DNA in vitro for cloning, mapping, and ligation (see Table 9.1). problems, while scientists often call for a broad distribution of
This example of serendipity in science sheds light on the funds to all projects investigating interesting biological phe-
debate between administrators who distribute and oversee re- nomena. The validity of both views suggests the need for a
search funding and scientists who carry out the research. balanced approach to the funding of research activities.