Вы находитесь на странице: 1из 68

Lecture 7:

Benzer and the rII locus

I.

Mutations in bacteriophages

II.

Mapping genes in bacteriophages

III. Benzer and the rII locus

IV.

V.

Deletion Mapping

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

Phage Plaques

Spread E. coli cells on agar growth

medium in a sterile petrie dish and grow at 37º C overnight. -Formation of lawnof 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 later…infecting neighboring bacteria (cycle repeats)

later…infecting neighboring bacteria (cycle repeats) -Eventually all of the bacteria in the vicinity of the

-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.

Observing Phage

• Most phage phenotypes are visualized as plaques on lawns of bacteria

phenotypes are visualized as plaques on lawns of bacteria • Plaques can vary in morphology: -

• 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

Rapid Lysis mutant

• Wild type T4 phage produce small plaques with fuzzy margins, while rapid lysis (r) mutants produce large plaques with sharp margins.

(r) mutants produce large plaques with sharp margins. (rapid lysis mutant) (wildtype) -When E.coli are infected

(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-defined edges).

Host range mutants

• Usually bacteriophage infect a single strain of bacteria. -Some bacteriophage have acquired the ability to infect different host strains…these phages are known as host range mutants (h).

T2

T2h

E.coli S
E.coli S
T2 T2h X E.coli R
T2
T2h
X
E.coli R

• 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.

Host range mutants

Host range mutants • Mutant (h) virus → clear plaques because they infect and lyse all

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 cells…R cells are not lysed and can still grow

in the region of the plaque.

II. Mapping Genes in Bacteriophage

II. Mapping Genes in Bacteriophage

Phage-phage Recombination

Phage-phage Recombination • Hershey experiment : -Two different phage genotypes can be recombined by simultaneously

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. -Defines recombination frequency in phage.

• Powerful experimental tool - one can measure very rare events

Can Phage Genomes Recombine?

• Alfred Hershey and Max Delbrück, 1947:

Traits:

r + - small plaques r - - large plaques

h

+ - infects E. coli S strain only

h

- - infects both E. coli S and R strain

Cross:

h - r +

x

h + r -

High multiplicity of infection (m.o.i.):

High concentration of phage.

E.coli S

Genetic Recombination in Phage

h- r+ h+ r- (S)
h-
r+
h+
r-
(S)

• Hershey simultaneously infected (at high m.o.i.) E.coli S cells with two different strains of bacteriophage T2.

Genetic Recombination in Phage

Phage progeny: Four possible genotypes are produced by this recombination event:

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

• 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…

S S R R S
S
S R
R
S

Serial Dilutions

Use serial dilutions to dilute phage from 10 10 phage/ml to 10 3 phage/ml.

10

10

8 h - r + phage

8 h + r - phage

10 µ L 10 µ L 1 µ L 990 µ L 990 µ L
10 µ L
10 µ L
1 µ L
990 µ L
990 µ L
999 µ L
10 10
10 8
10 6
10 3
µ L 990 µ L 990 µ L 999 µ L 10 10 10 8 10
µ L 990 µ L 990 µ L 999 µ L 10 10 10 8 10
µ L 990 µ L 990 µ L 999 µ L 10 10 10 8 10
µ L 990 µ L 990 µ L 999 µ L 10 10 10 8 10
µ L 990 µ L 990 µ L 999 µ L 10 10 10 8 10

Use centrifugation to separate cells from progeny phage.

3 Use centrifugation to separate cells from progeny phage. 1mL 10 7 cells/ml E. coli S

1mL

10 7 cells/ml

E. coli S (permissive host:

h - and h + phage

can both infect)

Plate 100 µ L on mixture of E. coli S and R

Genetic Recombination in Phage

• When plated on a mixed lawn of E.coli R and E.coli S, each genotype produces a plaque with a distinct phenotype.

What are the phenotypes of each genotype?

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

large clear plaques with sharp edges

h

- r -

: h + r + → small turbid plaques with fuzzy edges → large clear plaques

Results: Phage can Recombine

• Recombinant phage were observed:

Parental

Recombinant

genotypes

h + - h + + h - r + r r - r -
h
+
-
h
+
+
h
- r +
r
r
- r -
h

phenotypes

#

clear, small

42

cloudy, large

34

cloudy, small

12

clear, large

12

• 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

III. Benzer and the rII locus

III. Benzer and the rII locus

What is a gene? Approaches to a fine scale analysis of gene structure.

Old model of gene organization - Bead Theory

The gene is the fundamental unit of structure. Recombination takes place between but not within genes.

A
A
a
a
b
b
B
B

X

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.

mutations in the rII genes of the T4 phage lead to the understanding of the relationship

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?

1. Mutants breed true

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)

What is a conditional mutant?

Conditional mutations

• Mutations cause certain phenotype only in certain environment.

Restrictive condition (non-permissive)

Permissive condition

mutant phenotypeRestrictive condition (non-permissive) Permissive condition wild type

wild typeRestrictive condition (non-permissive) Permissive condition mutant phenotype

• Examples:

a. auxotrophs

b. temperature sensitive

c. host range

rII (Rapid Lysis) Mutant Plaques

• There are three rapid lysis mutations in T4 phage:

rI, rII, rIII

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

rII -
rII -

E.coli B

rII +
rII +

E.coli B or K

rII (Rapid Lysis) Mutant Plaques

• rII mutants grow on E. coli B but not on E. coli K -Note: rII can infect E. coli K but cannot lyse it.

Phage

Bacteria Strain

 

E. coli B

E. coli K

rII +

Wild type plaques

Wild type plaques

rII

Large plaques

No growth

r

+

B E. coli K rII + Wild type plaques Wild type plaques rII Large plaques No

r

r

+

B E. coli K rII + Wild type plaques Wild type plaques rII Large plaques No

Recombination within a gene

Recombination within a gene double mutant recombinant E.coli B E.coli B wild-type recombinant rare w.t. recombinants
double mutant recombinant
double mutant
recombinant
E.coli B E.coli B
E.coli B
E.coli B

wild-type

recombinant

mutant recombinant E.coli B E.coli B wild-type recombinant rare w.t. recombinants What ’ s an easier

rare w.t. recombinants Whats an easier way to screen for wild-type recombinants?

Screening made easy!

Screening made easy! double mutant recombinant Mix of mutant and w.t. phage (total # of phage)

double mutant recombinant

Screening made easy! double mutant recombinant Mix of mutant and w.t. phage (total # of phage)

Mix of mutant and w.t. phage (total # of phage)

recombinant Mix of mutant and w.t. phage (total # of phage) wild-type recombinant w.t. phage only

wild-type

recombinant

and w.t. phage (total # of phage) wild-type recombinant w.t. phage only (1/2 # of total

w.t. phage only (1/2 # of total recombinants)

Conclusion: Using conditional lethality is much easier than

looking at plaque morphology.

Benzer: Mapping his mutants

• Benzer isolated thousands of rII mutants.

rII-1
rII-1
rII-2
rII-2
E. coli B
E. coli B
E. coli B

E. coli B

E. coli B
E. coli B

• He constructed a fine-structure genetic map of the rII locus by crossing these mutants two-by-two to calculate map distance.

rII-a

rII-b

rII-c

rII-d

rII-e

rII-f

rII-g

locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f
locus by crossing these mutants two-by-two to calculate map distance. rII-a rII-b rII-c rII-d rII-e rII-f

Mapping rII mutations by 2-factor crosses

Procedure for calculating the distance between two mutants:

rII-2
rII-2
rII-1
rII-1

1. High MOI (multiplicity of infection) phage :

bacteria

E. coli B
E. coli B
E. coli B
E. coli B

E. coli B

E. coli B

-To be sure that bacteria are infected by both mutants

lysate

be sure that bacteria are infected by both mutants lysate B K titer 2. Infect permissive

B

K

titer

2. Infect permissive E. coli strain B (recombination only occurs under permissive condition…BOTH bacteriophages must be able to replicate their genomes.)

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

• Co-infect B cells with T4 phages carrying different rII mutations

by 2-factor crosses • Co-infect B cells with T4 phages carrying different rII mutations Plaques on

Plaques on E. coli B

Mapping rII mutations by 2-factor crosses

B B
B
B

Phage will replicate their genomes….

Recombination can occur

(within a gene)

B
B
their genomes…. Recombination can occur (within a gene) B Plate progeny phage on plates with either
their genomes…. Recombination can occur (within a gene) B Plate progeny phage on plates with either
their genomes…. Recombination can occur (within a gene) B Plate progeny phage on plates with either

Plate progeny phage on plates with either E.coli B or K

Summary/Calculating R.f.

Summary/Calculating R.f. Recombination freq. = 2 x (# wild type plaques on K) total phage (#

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 volume…you will have to dilute the initial lysate before you plate on E.coli B or E.coli K, respectively.

per unit volume…you will have to dilute the initial lysate before you plate on E.coli B

Calculating Total Phage Progeny

• For example…if 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

Mapping using 2-Factor Crosses

#1

+

X

+

#2

#1

+

Mapping using 2-Factor Crosses #1 + X + #2 #1 + X + #3 #2 +

X

+

#3

#2

+

Mapping using 2-Factor Crosses #1 + X + #2 #1 + X + #3 #2 +

X

+

#3

rII-2

2-Factor Crosses #1 + X + #2 #1 + X + #3 #2 + X +

rII-1

#1

#2

+

+

#1

#3

+

+

#2

#3

+

+

R.F. = 0.2% = 0.2 m.u.

1% = 1 m. u.

1.2% = 1.2 m.u.

rII-3

+ + #2 #3 + + R.F. = 0.2% = 0.2 m.u. 1% = 1 m.
+ + #2 #3 + + R.F. = 0.2% = 0.2 m.u. 1% = 1 m.
+ + #2 #3 + + R.F. = 0.2% = 0.2 m.u. 1% = 1 m.
+ + #2 #3 + + R.F. = 0.2% = 0.2 m.u. 1% = 1 m.

0.2m.u.

1 m.u.

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

Point mutation: single nucleotide change

rII-

m

-Results in amino acid substitution or possibly a nonsense mutation.

Deletion mutation: one or more nucleotide missing

rII

-Removing coding DNA. -Can cause a frameshift mutation if the deletion is not in a multiple of 3.

Deletion Mapping
Deletion Mapping

• Benzer isolated 2400 independent mutations within the rII locus

-To map all 2400 of his mutations via 2-factor crosses would have taken many years.

-As a short-cut, Benzer used his deletion mutants to conduct deletion mapping.

Overlapping and Non-overlapping mutations

• Recombination only occurs at homologous sequences Do the two rII mutations overlap??? Yes or No • If these mutations do not overlap, recombination can occur between… a) two deletion mutations or b) between a point mutation and a deletion mutation

can occur between… a)   two deletion mutations or b)   between a point mutation and
Non-overlapping mutations D1 •   rIIΔ1 •   rIIΔ2 rII - rII - D2
Non-overlapping mutations
D1
•   rIIΔ1
•   rIIΔ2
rII -
rII -
D2
rII + rII - D1 D2
rII +
rII -
D1
D2

Wild type

Double Mutant

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

#recombinants

2 x wt total
2 x wt
total

RF =

=

 

=

#total

# K x 2

#

B

Overlapping mutations D1 rII ∆1 rII - rII ∆3 rII - D3
Overlapping mutations
D1
rII ∆1
rII
-
rII ∆3
rII -
D3

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 deletion…neither has the w.t. nucleotide sequence in this region of the gene.

If deletions do overlap

deletion…neither has the w.t. nucleotide sequence in this region of the gene. If deletions do overlap

No wt recombinants

Deletion Mapping
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):

another along the rII locus. He decided to cross the different rII deletions with each other
another along the rII locus. He decided to cross the different rII deletions with each other

Sizing up his deletions

Δ1

Δ2

Δ3

Δ4

Cross

D1

X

D2

D1

X

D3

D1

X

D4

D2

X

D3

D2

X

D4

w.t. recombinant? (growth on K)

no

yes

yes

yes

no

Deletion Mapping
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.

rII1

rII -

rII1

rII -

rII2

rII -

Plated on K

- •   rII ∆ 1 rII - •   rII2 rII - Plated on K
D1
D1
D1
D1

D1

D1
D1
D1
D1
D1
- •   rII ∆ 1 rII - •   rII2 rII - Plated on K
- •   rII ∆ 1 rII - •   rII2 rII - Plated on K

plaques

rII +

rII -

No plaques

“The Big Seven”
“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.

overlapping segments of the rII locus were used to map each new point mutation to one
The Big Seven and more…
The Big Seven and more…

• Benzer also characterized many smaller deletions that defined 47 intervals within the rII region.

Seven and more… • Benzer also characterized many smaller deletions that defined 47 intervals within the

Mapping of rII mutants

• 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

by determining the RF between the mutations. Frequencies of rII + recombinants from crosses between two

What is the basic unit of recombination?

• 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.

• 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

each tick mark represents a specific mutation Fine Genetic Map of rII locus mutlple marks at

Fine Genetic Map of rII locus

a specific mutation Fine Genetic Map of rII locus mutlple marks at the same site means

mutlple marks at the same site means mutations are not separable by recombination

at the same site means mutations are not separable by recombination distribution of mutations is not
at the same site means mutations are not separable by recombination distribution of mutations is not

distribution of mutations is not random…hot spots

What is the basic unit of mutation?

What is the basic unit of mutation? • Mutation can occur at the nucleotide level ;

• 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 define 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?

• Method: complementation test

-Test used to determine if mutations are in the same or in different genes.

When do you normally conduct a 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.

Complementation Testing

• 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).

Example: Benzers T4 bacteriophage…Functional products from A and B genes are required for T4 phage to grow on E. coli K.

wt

A B
A
B

Complementation Testing

wt

mut1

mut1
mut1

A

B

A

B

Complementation Testing wt mut1 mut1 A B A B mut3 A B wt mut2 A B
Complementation Testing wt mut1 mut1 A B A B mut3 A B wt mut2 A B

mut3

A B
A
B
wt
wt

mut2

Testing wt mut1 mut1 A B A B mut3 A B wt mut2 A B mut3

A

B

mut3 mut2
mut3
mut2

E. coli K

growth

no growth

no growth

no growth

Complementation Testing: Cond. lethal x Cond. lethal

mut1

mut3

Testing: Cond. lethal x Cond. lethal mut1 mut3 B mut1 mut3 A B A mut1 mut3
B mut1 mut3
B
mut1
mut3

A

B

A

Cond. lethal x Cond. lethal mut1 mut3 B mut1 mut3 A B A mut1 mut3 E.
mut1
mut1
mut3
mut3

E. coli K

Conclusion:

no growth

no growth

growth

-mut1 and mut3 can complement each other. -mut1 and mut3 are in different genes and affect different gene products.

Complementation Testing

Complementation Testing A E. coli K Conclusion: mut1 B A mut1 mut2 no growth no growth

A

Complementation Testing A E. coli K Conclusion: mut1 B A mut1 mut2 no growth no growth

E. coli K

Conclusion:

mut1

B

A

mut1 mut2
mut1
mut2

no growth

no growth

mut2 B mut1 mut2
mut2
B
mut1
mut2

no growth

-mut1 and mut2 cannot complement each other. -mut1 and mut2 are in the same gene and affect the same gene product.

Recombination v.s. Complementation

DO NOT CONFUSE COMPLEMENTATION WITH RECOMBINATION!!!

Recombination: tests gene location. Complementation: tests gene function.

rII-2

rII-1

rII-3

tests gene location. Complementation: tests gene function. rII-2 rII-1 rII-3 R.F.= gene A 0.2 m.u. gene

R.F.=

gene A

0.2 m.u.

gene B

1 m.u.

Recombination v.s. Complementation

Phage

Bacteria Strain

 

E. coli B

E. coli K

rII +

Wild type plaques

Wild type plaques

rII

Large plaques

No growth

How do we know that the phage growing on E.coli K come from complementation rather than recombination?

• 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

Recombination v.s. Complementation

rII-1
rII-1
rII-2
rII-2
E. coli ?
E. coli ?
E. coli ?
E. coli ?
E. coli ?

E. coli ?

lysate

E. coli

Recombination: test gene location.

Exchange genetic material

Complementation: test gene function.

Supplement gene products

How do we distinguish complementation and recombination assays?

Which bacteria host are you infecting at high m.o.i.?

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-1
rII-2
rII-2
E. coli B
E. coli B
E. coli B
E. coli B
E. coli B

E. coli B

lysate

you have to use the “ permissive strain ” , E. coli B. rII-1 rII-2 E.

E. coli B

E. coli K

Complementation test

• In this experiment, you want to ask if two mutants can provide each other with the requiredproteins.

-You do not want them to exchange genetic materials. Therefore, you have to use the non-permissivestrain, E. coli K.

rII-1
rII-1
rII-2
rII-2
E. coli K
E. coli K
E. coli K
E. coli K
E. coli K

E. coli K

lysate

E. coli B

Recombination v.s. Complementation

Recombination

Phage (rII)

Permissive bacteria (E. coli B strain)

Complementation

Phage (rII)

Non-permissive bacteria (E. coli K strain)

B K
B
K

liquid culture

plate

B (plaque morphology)

test gene location

test gene function

The rII locus contains two genes

Temporary Diploid
Temporary
Diploid

The rII locus contains two genes

The rII locus contains two genes

The rII locus contains two genes: rIIA and B

• These experiments allowed Benzer to group rII mutants, into two classes, A and B which form two separate clusters in the rII locus

A and B which form two separate clusters in the rII locus • This showed that

• This showed that the rII locus is made up of two functionally independent genes.

• The basic unit of function is the gene.

Determining Complementation Groups

MUTANTS

1

1

-

2

3

4

5

6

2 3 4 5 6 + + + + + - + + - +
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 groups…3 separate genes

Problem involving complementation and recombination

COMPLEMENTATION

12 3 4 5

RECOMBINATION

1 2 3 4 5

1

- - + + - - - - + - + -

-

- -

 

1

- - + + + -

-

- +

- + + - + -

2

3

2

3

4

4

5

5

2 complementation groups:

 

Group A: 1, 5 Group B: 3, 4

 

5

1

(3,4)

   

2

2, fails to complement with 1,5 (gene A) and 3, 4 (gene B) => large deletion

Problem involving complementation and recombination

Six Neurospo ra strains auxo trophic 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 ge rminated 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 group s and wh ich alleles belong to each.

b) Draw a map of the complementation groups with the map distances between adjacent

genes.

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