BENZER’S WORK AND COMPLEMENTATION TEST

Fine Structure of Gene

✓ A gene expresses itself through a series of steps involved in a sequential synthesis of a product and,
therefore, may have one or more functional units.
✓ There can be several sites in a gene, each capable of being independently involved in mutational and
recombinational events.
✓ A gene thus is neither a functional, nor a mutational or a recombinational unit, but is a complex
locus, whose fine structure should be studied.
✓ Such fine structure has been studied in a number of cases using higher resolving power of
recombination technique.
✓ The most refined analysis of a single gene ever conducted is the one undertaken by Seymour Benzer
for a locus in T4 bacteriophage infecting E. coli.
✓ This locus is known as rII locus and a mutant at this locus is responsible for the formation of rough
plaques or colonies.

Figure 1: A mottled plaque showing rII mutant among a large number of normal plaques.

Benzer’s Experiment on rII locus in T4 phage

✓ Bacteriophages or phages in short are the viruses which infect bacteria. These are of two types with
respect to their infective life style.
(1) Virulent: always kill and lyse the infected cell example T even-T2, T4 and T odd- T1 and
T3.
(2) Temperate: choose one of the two alternative modes of development lytic and lysogenic.
✓ In lytic they kill and lyse the infected cells while in lysogenic these phages integrate into host
chromosome and form prophage, the resulting cells carrying prophage called lysogen, once in a while
prophage can turn virulent.
✓ Phages are indentified by ‘Plaques’. When individual phage particle fall on a lawn of bacteria they
undergo several cycles of infection releasing hundreds of progeny phages during every cycle.
✓ This process causes zone of clearing on bacterial lawn around the sites when first infection took place.
✓ They appear as hole on dense lawn of unlysed cells.
✓ In bacteriophages genetics mutation of a gene is designated as r, r stands for rapid lysis.
✓ Gene r has three different gene loci rI, rII and rIII, mutation in any one produces a rapid lysis phenotype.
✓ But there may be many mutations found in each of these.
Fine Structure of rII locus in T4 phage
✓ This locus had largest number of rapid lysing (r) mutants, and is called rII locus.
✓ It can be distinguished from other r loci, by the inability of rll mutants to produce plaques on
lysogenic 'K' strain of E. coli.
✓ The rII mutants, may though infect 'K' strain, but cannot cause lysis and are, therefore, unable to
produce any plaques.
✓ In contrast, these rII mutants make large sharp plaques on E. coli, strain B.
✓ The wild type phage T4 (rII+) will make small and fuzzy plaques, both on B and K strains.

Figure 2: Pattern of Bacteriophage infection on strains of E.coli

✓ Further, when 'K' was infected simultaneously by rII+ and rII, large plaques were formed, since rII+
helps in lysis so that rII may express.
✓ These distinguishing features enabled Benzer to identify mutants and wild type phages with high
efficiency.

Benzer wanted to findout, could wild type viruses be formed by recombination between
mutation within the same gene?
✓ Wild types phages T4 gives plaques that are 1.0-1.5 mm in diameter and have a fuzzy ring at periphery.
✓ This is called the wild type or ‘r+’ morphology.
✓ Genetic variants rI, rII and r III show rapid lysis phenotypic resulting in different plaques morphology
called ‘r’ type plaques.
✓ These are larger 2.0-2.5 mm in diameter with the help of sharp periphery. r+ and r plaques can be
distinguished just by looking at plate containing large number of plaques.
✓ The ‘r’ types of plaques arise at a frequency of approximately one in a million as a result of mutation
in r locus.
✓ Among the r mutants, the r II mutants have been a boon to molecular biology because of their
characteristic phenotype, which has proved useful in many ways.
✓ These r II mutants can infect the B strain of E.coli and give rise to typical r type plaques.
✓ But these rII mutants don’t form plaques on K strain of E coli K.
✓ This strain is lysogenic for phage λ which is denoted as E coli K 12 (λ).
✓ In his experiment Benzer took strain B in liquid culture and infect with two mutants to be tested
(designated here as rx and ry).
• strain B- supports the growth of all viruses thus giving the total number of viruses liberated.
• strain K- on which only wild-type viruses can grow.
Recombination Frequency Calculation

The recombination frequency between any pair of mutations is calculated as

Recombination Frequency = 2 × number of wild-type plaques (strain K plaques) ÷ total

number of plaques (on strain B).

You have to double the number found on strain K because you only see one-half the recombinants —
the other half consists of double mutants. Using this technique, Benzer eventually found some 2000
different mutations in the rII gene. The recombination frequency between some pairs of these was as
low as 0.02.

• The T4 genome has 160,000 base pairs of DNA extending over ~1,600 centimorgans (cM).
• So 1 cM ≅ 100 base pairs
• So 0.02 cM represents a pair of adjacent nucleotides.
• From these data, Benzer concluded that the
o smallest unit of mutation and
o the smallest unit of recombination

was a single base pair of DNA.

Benzer’s Work-
✓ The gene is a unit of function, each gene specifies one function. Benzer designed genetic experiment
to determine whether this classic view is true for the rII region.
✓ To find out whether two different rII mutants belonged to same gene (unit of function).
✓ Benzer adapted cis-trans test or complementation test which was earlier adapted by Edward Lewis
to study the nature of functional unit of gene in Drosophila.
✓ In this case rII region actually consists two genes (unit of function) rII A and rII B.
✓ A mutation anywhere in either gene produces the rII plaque morphology phenotype.
✓ In other words rII A and rII B each specify a different product needed for growth in E.coli K 12 (λ).
✓ In Benzer’s work with rII mutants, non-permissive strain K12 (λ) was infected with a pair of rII mutants
phages to see whether the two mutants, each unable by itself to grow in strain K12 (λ), could work
together to produce progeny phages.
✓ Although in most cases, this does not result into lysis and plaque formation, in some cases it does
lead to plaque formation.
✓ If the phages do produce progeny, the two mutants are said to complement each other, meaning
that the two mutations must be in different genes (units of function) that encodes different products,
these two products work together to allow progeny to be produced.
✓ If two mutants did not form plaques on mixed infection, they were placed in the same group, but
if plaques are produced, the two mutants involved in mixed infection were placed in two different
groups.

✓ Therefore, mutant alleles present in the same gene do not show complementation, while those
located in different genes show complementation.
✓ The basis of complementation test may be simply described as follows.
“A gene produces its effect primarily by directing the production of an active enzyme or
polypeptide. On the other hand, a mutant allele of this gene directs the production of an inactive form
of the enzyme as a result of which it produces the mutant phenotype”.

Figure 4: Complementation Test

Figure 5: Results of mixed infection by (a) a double mutant (having two rII mutations A1 and A2
belonging to same cistron A) and the wild type strain, phage; (b) two of T4 phage mutant
strains, having different rll mutations (A1 and A2) belonging to same cistron A.

✓ Since groups A and B are distinguished on the basis of cis-trans test, these were termed as cistron A
and cistron B.
✓ Mutants belonging to the same cistron i.e. A or B would exibit cis- trans phenomenon, meaning that
they would give wild type only in cis configuration and not in trans configuration.
✓ Two mutants from different cistrons (A and B) would give wild type (plaque formation) even in trans
configuration, which in other words is called complementation.
✓ From the complementation test, it is obvious that in rII region, two cistrons A and B are independent
functionally and must be responsible for sequential synthesis of two separate products, which
presumably are polypeptide chains.
✓ Therefore, all mutants belonging to one cistron share a common deficiency, which is different from
the deficiency due to mutants belonging to the second cistron.
✓ When two mutants belong to same cistron, both are deficient for same product and therefore, they
cannot complement, but when two mutants belong to two different cistrons, they, being deficient for
different products, can complement, and may express wild phenotype i.e. lysis and plaque formation.