Lecture 7:

Benzer and the rII locus

I. Mutations in bacteriophages
II. Mapping genes in bacteriophages
III. Benzer and the rII locus
IV. Deletion Mapping
V. Complementation and Recombination analysis
Assigned Readings and Problems:
3
rd
Ed: Chapter 7, pg. 224-232 and problem 16-17, 18a, 19-23
4
th
Ed: Chapter 7, pg. 216-224, problems 18-19, 20a, 21-25
Adapted from F.A. Laski and J. Ribaya
I. Mutations in bacteriophages
Spread E. coli cells on agar growth
medium in a sterile petrie dish and
grow at 37 C overnight.
Phage Plaques
-Eventually all of the bacteria in the vicinity of the original phage
particle will be lysed yielding a clear spot (hole) in the lawn of bacteria,
called a plaque.
-Each overnight plaque contains ~1 x 10
8
bacteriophage.
-Formation of lawn of bacteria on
the surface of the medium.
If you add a single bacteriophage
(T4 or T2), it will infect one
bacterium, lyse it, and release 300
progeny phage about 25 minutes
laterinfecting neighboring bacteria
(cycle repeats)
Most phage phenotypes are visualized as plaques on lawns of
bacteria
Plaques can vary in morphology:

- Large or small plaques are determined by how fast lysis occurs
- Host range is a reflection of what strains of bacteria the phage can
bind to and lyse
Observing Phage
Wild type T4 phage produce small plaques with fuzzy margins,
while rapid lysis (r) mutants produce large plaques with sharp
margins.
Rapid Lysis mutant
(rapid lysis mutant)
(wildtype)
-When E.coli are infected with wild-type T4 (r
+
), lysis is delayed for
up to 2 hours--a process called lysis inhibition ! fuzzy margins of
wild-type plaques.

-Lysis inhibition does not occur in bacteria infected with r mutants
all cells infected with r mutants all lyse rapidly (sharp-dened edges).
Usually bacteriophage infect a single strain of bacteria.
-Some bacteriophage have acquired the ability to infect different host
strainsthese phages are known as host range mutants (h).
Host range mutants
T2 wild-type and T2h mutants can be distinguished by growing
them on a mixed lawn of E.coli R and E.coli S cells.
E.coli S
T2
E.coli R
T2
X
T2h
T2h
Host range mutants
Mutant (h) virus ! clear plaques because they infect and lyse all
host cells--whether E.coli S or E.coli R.

Wild-type (h
+
) virus ! produce turbid plaques because they infect
only S cells not the R cellsR cells are not lysed and can still grow
in the region of the plaque.
II. Mapping Genes in Bacteriophage
Phage-phage Recombination
Hershey experiment:
-Two different phage genotypes can be recombined by
simultaneously infecting host bacteria with two different types of
phage and then screening progeny phage for recombinant genotypes.
-Denes recombination frequency in phage.

Powerful experimental tool - one can measure very rare events
Cross: h
-
r
+
x h
+
r
-

E.coli S
High multiplicity of infection (m.o.i.):
High concentration of phage.
Can Phage Genomes Recombine?
Alfred Hershey and Max Delbrck, 1947:
Traits: r
+
- small plaques
r
-
- large plaques
h
+
- infects E. coli S strain only
h
-
- infects both E. coli S and R strain
h- r+
h+ r-
(S)
Genetic Recombination in Phage
Hershey simultaneously infected (at high m.o.i.) E.coli S cells
with two different strains of bacteriophage T2.
Because of the large amount of
bacteriophage progeny that will
result from this cross (10
10
phage /
mL)one must first make serial
dilutions of progeny phage before
plating
Genetic Recombination in Phage
S
S
R
R
S
Phage progeny: Four possible
genotypes are produced by this
recombination event:

Parental: h
-
r
+
and h
+
r
-
Recombinant: h
+
r
+
and h
-
r
-

10
7
cells/ml
E. coli S
(permissive host:
h
-
and h
+
phage
can both infect)
10
8
h
-
r
+
phage
10
8
h
+
r
-
phage
Use centrifugation to
separate cells from
progeny phage.
Plate 100 L on mixture of E.
coli S and R
Serial Dilutions
Use serial dilutions to dilute phage
from 10
10
phage/ml to 10
3
phage/ml.
1mL
10 L 10 L
10
10
10
8
10
6
10
3
990 L 999 L 990 L
1 L
When plated on a mixed lawn of E.coli R and E.coli S, each
genotype produces a plaque with a distinct phenotype.
Genetic Recombination in Phage
Parental:

h
-
r
+
! small clear plaques with fuzzy
edges

h
+
r
-
! large turbid plaques with
sharp edges
Recombinant:

h
+
r
+
! small turbid plaques with
fuzzy edges

h
-
r
-
! large clear plaques with sharp
edges
What are the phenotypes of each genotype?
genotypes
h
-
r
+

h
+
r
-

h
+
r
+

h
-
r
-
phenotypes #
clear, small 42
cloudy, large 34
cloudy, small 12
clear, large 12
Results: Phage can Recombine
Parental
Recombinant
Distance between h and r genes can be determined by calculating
the recombination frequency between the two genes:

Recombination Frequency:
RF = # recombinants/total

RF = (12 + 12) /100 = .24 or 24 mu
Recombinant phage were observed:
III. Benzer and the rII locus
What is a gene? Approaches to a ne scale
analysis of gene structure.
A
a
b
B
X
Old model of gene organization - Bead Theory

The gene is the fundamental unit of structure. Recombination takes
place between but not within genes.
The gene is the fundamental unit of change. One allele is converted
to another at the whole gene level. Alleles are completely different
from one another.

The gene is the fundamental unit of function. Genes only function
as whole units, no partial function.
Seymore Benzer and the rII genes of T4 phage
A physicist in the 1940s
-His experiments with semiconductors helped lead to
the invention of the transistor.

In the 1950s he decided to dabble into the field of
biology
-His studies and discovery of the numerous
mutations in the rII genes of the T4 phage lead to the
understanding of the relationship between genes and
proteins.
Benzers Experiments Addressed Three
Basic Questions:
What is the basic element of structure?
What is the basic element of change?
What is the basic element of function?
Why did Benzer choose to work on rII locus
in T4 bacteriophage?
2. Easy to generate a lot of mutants with the bacteriophage system
-can screen large number of phages (> 10
9
) in
short period of time (15 hours, overnight culture).
3. Easy assays
-use selective agents to detect the presence of very small
portion of recombinants within a large proportion of
parentals (conditional mutant)
1. Mutants breed true
What is a conditional mutant?
Conditional mutations
Mutations cause certain phenotype only in certain environment.
Restrictive condition mutant phenotype
(non-permissive)

Permissive condition wild type
a. auxotrophs
b. temperature sensitive
c. host range !
Examples:
rII (Rapid Lysis) Mutant Plaques
rII (conditional lethal) affects lysis and host range of T4 phage:

rII
+
! produces small plaques in both strains B and K

rII ! only grows on E. coli strain B (permissive host).
! cannot grow in E. coli strain K (non-permissive host).
! produces large plaques in strain B
There are three rapid lysis mutations in T4 phage:
rI, rII, rIII
rII
-
rII
+
E.coli B E.coli B or K
rII mutants grow on E. coli B but not on E. coli K
-Note: rII can infect E. coli K but cannot lyse it.
Large plaques
Wild type plaques
r
+

r
No growth
Wild type plaques
r
+

Bacteria Strain Phage
rII
E. coli B E. coli K
rII
+

rII (Rapid Lysis) Mutant Plaques
Recombination within a gene
rare w.t. recombinants
E.coli B
E.coli B
double mutant
recombinant
wild-type
recombinant
Whats an easier way to screen for wild-type recombinants?
Very rare recombinants!
double mutant
recombinant
wild-type
recombinant
Screening made easy!
Mix of mutant
and w.t. phage
(total # of phage)
w.t. phage only
(1/2 # of total
recombinants)
Conclusion: Using conditional lethality is much easier than
looking at plaque morphology.
Benzer isolated thousands of rII mutants.
rII-1 rII-2
E. coli B
Benzer: Mapping his mutants

rII-a rII-b
rII-g
rII-c rII-d rII-e rII-f
He constructed a fine-structure genetic map of the rII locus by
crossing these mutants two-by-two to calculate map distance.
rII-1 rII-2
E. coli B
lysate
B
K
titer
1. High MOI (multiplicity of infection) phage :
bacteria

-To be sure that bacteria are infected by both
mutants
2. Infect permissive E. coli strain B (recombination
only occurs under permissive conditionBOTH
bacteriophages must be able to replicate their
genomes.)
Procedure for calculating the distance between two mutants:
Mapping rII mutations by 2-factor crosses
3. Plate lysate (after serial dilutions) on plates
containing B and K.
Before we start: How did Benzer isolate his thousands of rII
mutants from a mixture of w.t., rI, rII, and rIII bacteriophages?
Mapping rII mutations by 2-factor crosses
Plaques on
E. coli B
Co-infect B cells with T4 phages carrying different rII mutations
Phage will
replicate their
genomes.
Recombination
can occur...
(within a gene)
Plate progeny phage
on plates with either
E.coli B or K
Mapping rII mutations by 2-factor crosses
B B
B
Summary/Calculating R.f.
Recombination freq. = 2 x (# wild type plaques on K)
total phage (# of plaques on B)
Serial dilution ! number of phages
To calculate the total number of progeny phage produced per unit
volume or the total number of wild-type recombinants per unit
volumeyou will have to dilute the initial lysate before you plate on
E.coli B or E.coli K, respectively.
Calculating Total Phage Progeny
For exampleif 250 plaques (on E.coli B) are counted in a sample
diluted 10
6
-fold, then the original sample contained 2.5 x 10
8
phage
per unit volume (250 x 10
6
).
-If 150 wild-type plaques (E.coli K) are present in a 10
4
dilution of
the lysate, then the original sample contained 1.5 x 10
6
recombinant
wild-type phage per unit volume (150 x 10
4
).
Recombination freq. = 2 x (# wild type plaques on K)
total phage (# of plaques on B)
The recombination frequency between the two mutant sites is
3 x 10
6
/2.5 x 10
8
= 0.012 or 1.2 mu
X
X
X
#1
+
+
+
+
+
+
+ +
+
+ +
+
#1
#1
#1
#2
#3
#2
#2 #2
#3
#3
#3
R.F. =
0.2% = 0.2 m.u.
1% = 1 m. u.
1.2% = 1.2 m.u.
rII-2 rII-1
rII-3
0.2m.u.
1 m.u.
Mapping using 2-Factor Crosses
IV. Deletion Mapping
Deletion mutations
Initially he mapped 60 independent rII mutants using two factor
crosses.

Most could spontaneously revert to wt at a frequency of 1/10
7

What type of mutations do you think these are?
Some would never revert

What type of mutations do you think these are?
Deletion mutations!
Mutations
rII-
Point mutation: single nucleotide change
m
-Results in amino acid substitution or possibly a nonsense
mutation.
rII!
Deletion mutation: one or more nucleotide missing
-Removing coding DNA.
-Can cause a frameshift mutation if the deletion is not in
a multiple of 3.
Deletion Mapping
-To map all 2400 of his mutations via 2-factor crosses would
have taken many years.
Benzer isolated 2400 independent mutations within the rII locus
-As a short-cut, Benzer used his deletion mutants to conduct
deletion mapping.
Recombination only occurs at homologous sequences
Overlapping and Non-overlapping mutations
If these mutations do not overlap, recombination can occur between
a) two deletion mutations or
b) between a point mutation and a deletion mutation
Do the two rII mutations overlap??? Yes or No
D1
D2
rII!1 rII
-
rII!2 rII
-
Wild type
Double Mutant
D2 D1
rII
+
rII
-
Non-overlapping mutations
Take home message: If two rII mutations do not overlap,
recombination can occur to produce wild-type phage.
Plated on B ! # of total: (Deletion mutant 1 and 2 + wt + double mutant)
Plated on K ! # of wt recombinants
RF =
#recombinants
#total
# K x 2
# B
=
2 x wt
total
=
D1
D3
If deletions do overlap No wt recombinants
rII!1 rII
-
rII!3 rII
-
Is there any way you can make a wild type chromosome
from these two mutant chromosomes?
Take home message: A phage that carries a deletion cannot recombine
with another phage that has a mutation in the region removed by the
deletionneither has the w.t. nucleotide sequence in this region of the
gene.
Overlapping mutations
Deletion Mapping
Before he could use deletions in his short-cut mapping procedure,
Benzer first had to:
1) Determine the sizes of the deleted regions relative
2) Determine their relative positions to one another along
the rII locus.

He decided to cross the different rII deletions with each other (2-
factor crosses):
!1
!2
!3
!4
Cross
w.t. recombinant?
(growth on K)
D1
D1
D1
D2
D2
D2
D3
D3
D4
D4
X
X
X
X
X
yes
no
yes
yes
no
Sizing up his deletions
rII
-

No plaques
rII1 rII
-

rII
+
plaques
D1
rII!1 rII
-
rII2 rII
-
Plated on K
Deletion Mapping
He also characterized the deletions by crossing them to the 60 point
mutations he previously mapped by standard two- and three- factor
crosses.
The Big Seven
Seven large deletions that were missing overlapping segments of
the rII locus were used to map each new point mutation to one of
seven intervals of the locus.
The Big Seven and more
Benzer also characterized many smaller deletions that defined 47
intervals within the rII region.
Benzer mapped 2400 rII mutations to 308 distinct sites by
determining the RF between the mutations.
Frequencies
of rII
+
recombinants
from crosses between
two rII mutants
Mapping of rII mutants
The smallest recombination frequency observed was 0.02% ! 2.3 bp

-Suggested (later proved) that the basic unit of recombination is not the
gene but the nucleotide.
What is the basic unit of
recombination?
The rII mutants were not randomly distributed over the the 308 sites.
Some rII sites were mutated more than other sites (hot spots).
each tick mark represents a
specific mutation
mutlple marks at the same site means mutations are
not separable by recombination
distribution of mutations
is not randomhot spots
Fine Genetic Map of rII locus
What is the basic unit of mutation?
Mutation can occur at the nucleotide level; individual mutations
can occur at the resolution of a single base pair.
The distribution of mutations need not be random within the
nucleotide sequence, some nucleotides may be more susceptible to
mutation than others. (hot spots).
V. Complementation and Recombination
Analysis
How many genes dene the rII locus?
Benzer isolated many rII mutants and generated a genetic map.
Do all these mutants belong to one gene?
Do they all mutate the same polypeptide?
-Test used to determine if mutations are in the same or in different
genes.
Method: complementation test
Note: This experiment is not mapping the gene location
Complementation analysis does NOT involve recombination between
gene products

-It is testing for gene function.
When do you normally conduct a complementation test?
A B
wt
Example: Benzers T4 bacteriophageFunctional products from A
and B genes are required for T4 phage to grow on E. coli K.
Two mutations can complement each other if they provide different
functions in the cell (are in different genes).

Mutations that failed to complement each other are in the same
complementation group (same gene).
Complementation Testing
E. coli K
A B
wt
A B
mut1
A B
mut3
A B
mut2
wt mut1 mut3 mut2
growth no growth no growth no growth
Complementation Testing
A B
mut1
E. coli K
mut1 mut3
no growth no growth
mut3
growth
mut1
Conclusion:
-mut1 and mut3 can complement each other.
-mut1 and mut3 are in different genes and affect different gene
products.
A B
mut3
Complementation Testing: Cond. lethal x Cond. lethal
A B
mut1
E. coli K
mut1 mut2
no growth no growth
mut2
no growth
mut1
Conclusion:
-mut1 and mut2 cannot complement each other.
-mut1 and mut2 are in the same gene and affect the same gene
product.

A B
mut2
Complementation Testing
Recombination v.s. Complementation
Recombination: tests gene location.
Complementation: tests gene function.
gene A
gene B
rII-2 rII-1 rII-3
0.2 m.u.
1 m.u.
R.F.=
DO NOT CONFUSE COMPLEMENTATION WITH
RECOMBINATION!!!
Bacteria Strain Phage
rII
E. coli B E. coli K
Large plaques
No growth
rII
+

Wild type plaques
Wild type plaques
Recombination (between two different mutants of the same gene)
only occurs under permissive conditions.
E.coli B is a permissive host for rII mutants ! recombination
E.coli K is a non-permissive host for rII mutants ! no recombination
How do we know that the phage growing on E.coli K come
from complementation rather than recombination?
Recombination v.s. Complementation
How do we distinguish complementation and
recombination assays?
Recombination: test gene location.
Complementation: test gene function.
Exchange genetic material
Supplement gene products
rII-1 rII-2
E. coli ?
lysate
E. coli
Which bacteria host are you infecting at high m.o.i.?
Recombination v.s. Complementation
Recombination test
You want to make sure that recombination occurs

1. Two mutants have to co-exist in the same bacteria
2. Because recombination only occurs during replication,
you have to use the permissive strain, E. coli B.
rII-1 rII-2
E. coli B
lysate
E. coli B E. coli K
In this experiment, you want to ask if two mutants can provide each other with
the required proteins.

-You do not want them to exchange genetic materials. Therefore, you have to use
the non-permissive strain, E. coli K.
Complementation test
rII-1 rII-2
E. coli K
lysate
E. coli B
Recombination
Complementation
Phage (rII)
Permissive bacteria
(E. coli B strain)
Phage (rII)
Non-permissive bacteria
(E. coli K strain)
liquid culture
plate
B
B (plaque morphology)
K
test gene location
test gene function
Recombination v.s. Complementation
Temporary
Diploid
The rII locus contains two genes
The rII locus contains two genes
These experiments allowed Benzer to group rII mutants, into two
classes, A and B which form two separate clusters in the rII locus
This showed that the rII locus is made up of two functionally
independent genes.

The basic unit of function is the gene.
The rII locus contains two genes: rIIA and B
MUTANTS 1 2 3 4 5 6

1 - + + + + +
2 - + + - +
3 - - + -
4 - + -
5 - +
6 -

Mutant 1 can complement all other mutants; it is a separate gene .
Mutant 2 does not complement mutant 5; they are in the same gene.
Mutants 3, 4 and 6 do not complement each other; they are in the same gene.
3 complementation groups3 separate genes
Determining Complementation Groups
COMPLEMENTATION RECOMBINATION
12 3 4 5 1 2 3 4 5
1 - - + + - 1 - - + + +
2 - - - - 2 - - - +
3 - - + 3 - + +
4 - + 4 - +
5 - 5 -
2 complementation groups:
Group A: 1, 5
Group B: 3, 4
2, fails to complement with
1,5 (gene A) and 3, 4 (gene B) => large deletion
2
1
5 (3,4)
Problem involving complementation and recombination
Six Neurospora strains auxotrophic for proline were crossed to each other to determine
complementation and linkage relationships. First growth of the heterokaryons on
minimal media was recorded for each pair (Table 1). Then heterokaryons for each
combination were grown on rich medium, and allowed to go through meiosis &
sporulate. 1000 haploid spores from each cross were then germinated on minimal media.
The # of spores that grew into colonies is shown in Table 2.
Table 1 proA proB proC proD proE proF
proA - + + + + +
proB - + + - +
proC - + + +
proD - + -
proE - +
proF -
Table 2 proA proB proC proD proE proF
proA 0 250 100 50 250 50
proB 0 150 200 0 200
proC 0 50 150 50
proD 0 200 0
proE 0 200
proF 0
a) How many complementation groups are found in these mutants? Write the
complementation groups and which alleles belong to each.
b) Draw a map of the complementation groups with the map distances between adjacent
genes.
Problem involving complementation and recombination
a) Four = (A), (B and E), (C), (D and F)
b) A----10mu---D/F----10mu----C------------30mu----------B/E
Summary of Benzers Discovery
Identified the deletion as a mutation

Identified the point mutation (Defined mutation at the level of a
nucleotide)

Developed deletion mapping

Proved linearity of a gene

Clarified the basis of complementation

Defined recombination at the level of the nucleotide

Identified mutational hotspots

Defined the structural basis of genes