Genetic Fine Structure;

Benzer's work with rII mutants of bacteriophage T4

Non-mutant, rII+ FT4 make small fuzzy plaques on lawns of E. coli strains
B & K. Rapid lysis or "r" mutants in general make large plaques; however
rII mutants make large plaques on lawns of E. coli strain B, but cannot
reproduce on E. coli K cells, so make no plaques at all on this host.

Benzer collected thousands of independently isolated rII mutants from

mutagenesis experiments and from friends who happened to discover
them. Stocks of each mutant were made by growth on E. coli B. Each
mutant was verified to have the phenotypes described above.

Benzer devised a "complementation test" to test for functional allelism by

simultaneously infecting E. coli K with two independently isolated rII
mutant phage (the "trans-test"). Complete lysis of the lawn of K cells
meant that the mutations affected different functions, that is, that one
mutant provided what the other was lacking. Inability to lyse K suggested
that the two mutations were in the same gene. Benzer realized that in
theory rII mutants might be "dominant", and not result in lysis of K cells
even if the two co-infecting viruses were in different genes. He used "cis"
tests to prove that the mutations were recessive to wild type. For these, a
double mutant had to be created with both mutations on the same viral
DNA. The double mutant would then be used in a mixed infection with a
wild type T4 pahge to verify that lysis occured. Since making and
confirming double mutants requires considerable work, relativley few of
these were actually done.

He found that all the mutants could be put into either of two functional
groups that he called the A and B "cistrons".
TRANS-TESTS;
E. coli K co-infected with two Phage T4 rII mutants

X
ppB made, no A

Both mutations in the same cistron (functional gene)

gives no complementation; no growth on E. coli K

TRANS TESTS ctd

ppA and ppB made

One mutation in the A cistron, the other in B
gives complementation; plaque formed on E. coli K

CIS TEST

X X

ppA and ppB made

When both mutations are in the same virus (either both A,

both B or one A and one B) and growth of E. coli K occurs in
the presence of a coinfecting wild type virus, the mutations are
recessive to wild type.

In order to see if recombination could occur between rII mutants and to

map the mutations in the A and B cistrons, Benzer made pairwise crosses
on E. coli B. the progeny were collected and placed on lawns of E. coli K
where only rII+ progeny would grow. Reversion rates of point mutations
were determined by self-crosses, and were generally in the order of 10-7
or less. Recombination could be detected at a significantly higher rate,
even when the two mutations were in the same cistron.

Benzer also found some mutant strains that did not revert. He assumed
these were contained deletions and reasoned that crosses betweeen two
different deletions could not give wild type progeny if the deletions
overlapped. This allowed him to establish a series of deletion mutants to
quickly locate new point mutations.

The example below shows how crosses to just 3 deletions could define 5
different map regions for the point mutations.

rII+

Del 1

Del 2

Del 3

(Deleted regions shown by thick lines)

The maps showed that all of the rIIA mutants mapped at one end of the
cluster and that the rII B muatants mapped at the other.

It also showed that some sites had very frequent mutations (hot-spots)
while aothers were rare.

Benzers work showed that:

a) Recombination could occur within as well as between genes.
b) Multiple alleles should be expected rather than rare occurences. .

It also established the "unit of function" as the best way to describe a

gene and showed that base pairs, not whole genes are the "unit of
mutation".