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Describe how Benzer used genetic mapping and complementation testing to elucidate
the fine structure of the rII region of bacteriophage T4 (2005)
The application of genetics has been utilized by humans for thousands of years. Yet until the 1950s, our
understanding of the physical nature of genes, the units of hereditary, were severely limited. The
distribution of genes on a chromosome was envisioned to be alike to a string of beads on a string:
indivisible units of structure with unique chromosomal loci. Crossing over could only occur in between
separate genes. These 'beads' would produce phenotypes in a Mendelian manner when crossed.
However this theory was challenged by Seymour Benzer's work with T4 bacteriophages and Escherichia
coli in 1955, following Watson and Crick's paper on the double helix nature of DNA in 1953.
The absence of a conventional sexual cycle in prokaryotes meant Benzer could not perform normal
Mendelian crosses, this posed an issue when attempting to use genetic mapping. Instead, he took
advantage of horizontal gene transmission between bacteriophages and E.coli cells. T4 Phages are
comprised of a protective protein coat surrounding a chromosome of DNA (168,000 bp coding around
50 genes) and a delivery system. This delivery system is an array of protein fibers which bind to protein
receptors on the E.coli cell walls. The phage chromosome is then injected into the host, a rapid process
shown to last no longer than 1.5s. Next, the chromosome is replicated and phage proteins synthesized
by the host's own polymerases, meanwhile the host's own DNA is broken down. Self assembly of the
parts of the phage then occurs and eventually around 100 new phages are released during lysis of the
host cell. Phages can be detected and counted by mixing a bacteriophage suspension with E.coli cells in
molten agar. The mixture is then immediately poured onto an agar plate to create a bacterial lawn. Over
time, plaques will form on the lawn which represent where a single phage was successfully infected a
host, replicated and lysed many E.coli cells. The number of plaques is equal to the number of phages in
the original phage suspension.
Benzer studied the rII (rapid lysis) locus, phages with mutations at this locus are able to infect E.coli B
strain cells and produce larger plaques than wild type T4 phages. This is brought about by the mutants
blocking the expression of lysis inhibition (LIN) which can inhibit the lysis of host cells for several hours if
related phages are nearby or infecting the same cell. The rII mutants do not carry out lysis inhibtion and
so phages are released from the host cells faster, meaning more host cells can be infected in a shorter
time period. Thus, the mutants produce larger plaques on E.coli B strain bacterial lawns than wildtype
phages. However, if E.coli KII(lambda) is used instead, the rII mutants cannot successfully lyse the cells
and so no plaques form, whereas the wildtype phages do form plaques with the KII(Lambda) strain.
Benzer used these observations to form the genetic map of the rII region. He used two rII mutants with
different mutations to double infect a single E.coli B strain cell. The DNA of the two mutants were able
to recombine to produce one double mutant and one wildtype phage. The progeny could then be
counted and the resulting phages were then replated on the E.coli KII (lambda) strain and the plaques
counted to give the number of wildtype progeny and by extension the number of recombination events.
This meant Benzer could find the map distances between the two mutations in the original 2 mutants by
calculating the percentage of the progeny which had undergone recombination events. By analysing
over 2000 independent mutants, he was able to create a detailed genetic map of the rII region. He
found that although his theoretical sensitivity of the distances was 0.0001%, the minimum mapping
distance he found was 0.01%. Benzer concluded this must be the distance between adjacent mutations,
this is actually the distance between base pairs in DNA.
The genetic mapping of the rII region was a leap forward in our understanding of genetics, however the
problem still remained that it was not known how many genes were contained in the region or where
their boundaries were. Benzer overcame this issue using complementation tests with more T4
bacteriophages. Complementation occurs when two organisms with mutations on different genes which
have the same mutated phenotype produce wildtype offspring (with no mutations). More than one
gene can control a single phenotype in several ways, for example by coding for different sub-units of the
same protein, or by multiple proteins controlling the same phenotype. Benzer found that the rII region
was infact two separate genes by using the following method: infecting an E.coli KII (lambda) cell with
two rII mutants which had mutation in different parts of the rII region. He then incubated the agar
plates, plaques would only be formed if the mutations were in different genes. Complementation would
then occur, meaning the progeny of the two phages would have a functioning version of all the genes
required for a wildtype strain. Benzer showed the rII mutations fell into two complementary groups (or
genes) rIIA and rIIB. Also, the method could be used to find the boundary between the two genes on the
genetic map he had already produced. By using a huge number of combinations of the two mutations,
he could show where the boundary must be to have the two mutations on separate genes. He went on
further to describe the nature of the mutations, he reasoned that most of the mutations that would
complement each other must be point mutations, single base substitutions. This way, only that part of
the genetic code would change and the rest of the code remain unaffected.
Seymour Benzer's findings revolutionized our understanding of genetics in many ways, the gene was no
longer seen as a bead upon a string but more a stretch of code which could be mutated or recombined
in any position along it's stretch. This also solidified the work of Watson and Crick on the double helix
nature of DNA and set the way for the huge increase in the study of genetics for the decades to come.
Interestingly, his findings are still being used today to question our previously accepted ideas about
genetics. The genetic map of the rII region shows two major 'mutation hotspots' where the rate of
mutation was much higher than in the rest of the region. Benzer's finding could suggest that mutations
preferentially form around existing mutations, making us question whether or not mutations are truly
random as they are usually described. Either way, Benzer's thorough use of genetic mapping and
complementation tests have created one of the most detailed fine structures of any genetic region
studied thus far.