GENE FINE STRUCTURE ANALYSIS

IN BACTERIA
 What is a gene?
• In1940, C. Oliver showed evidence that recombination can occur
within a gene (working with Drosophila melanogaster).

Lozenge locus

Izs = spectacle eye; Izg = glassy eyes; Lz+ = lozenge eyes

 What is a gene?
Recombination between adjacent nucleotide pairs
 RECOMBINATION TESTS
Recombination test (uses Hfr bacteria)
If two mutants of a gene in recombination experiments:

- yield a wild type the test is said to be positive, and indicates that the two
mutations are different.

- yield only mutant types the test is said to be negative and indicates that
the two mutations are the same.

Positive recombination tests also allows calculation of the distance between

the two mutations.

Recombination test: +ve - mutations are different; -ve - mutations are the
same.
COMPLEMENTATION TESTS (cis-trans test)

Recombination tests cannot tell whether two mutations affecting a

certain phenotype are on the same gene or not.

Concept of complementation

The ability of two recessive mutations to restore the wild type

phenotype in the cis and trans positions is referred to as
complementation.
COMPLEMENTATION TESTS (cis trans test)

Cis test is a control!!!

COMPLEMENTATION TESTS (cis trans test)

-ve trans= mutation in same gene

+ve trans = mutation in different genes
 COMPLEMENTATION TESTS ( cis-trans test)

Complementation test (uses F΄ strains of bacteria)

This test brings together two mutations in the cis and trans
configuration in a single cell system.

The cis test is a control and should always result in a wild type
phenotype

The trans test:

- if it results in a wild type (+ve test) indicates that the two
mutations are in different genes.

- if it results in a mutant type (-ve test) indicates that the two

mutations are in the same gene.
COMPLEMENTATION TESTS (cis-trans test)

Can you work this out?

Recombination test with mutations, m1 and m2 affecting arginine
production were positive.
Complementation test with m1 and m2 was also positive.

m1 m2 m1 +

+ + + m2

cis trans
What can you say about the two mutations? (mutations in different
genes)
COMPLEMENTATION TESTS (cis trans test)

How its done?

In bacteria:
Two mutations can be brought together into a single cell system by
sexduction (complementation uses F΄ strains)

Creates a partial diploid situation for the transferred bacterial

chromosomal segment on the F΄ plasmid.
 COMPLEMENTATION TESTS
An example
E.g. Fine structure of the histidine locus

The process involves:

- Induction of mutations that affect histidine production

- Recombination tests between mutations to determine which

ones are different

- Complementation test between mutations to determine which

mutations belong to which genes.

- Fine structure map of the histidine locus – one locus can have
many genes.
COMPLEMENTATION TESTS (cis trans test)

E.g. There are 5 mutations in the histidine locus. The results of the cis-
trans test are provided below: + = complementation; - = no
complementation. Draw a complementation map for the histidine locus.
How many genes are at the histidine locus?
CD16 245 261 D-566 1438
CD16 - - - - -
245 - + + +
261 - + +
D-566 - -
1438 -
COMPLEMENTATION TESTS
An example
CD16 245 261 D-566 1438
CD16 - - - - -
245 - + + +
261 - + +
D-566 - -
1438 -

Complementation map
Histidine locus has three genes
245 261 D-566 & 1438

CD16: Deletion spanning all three genes

Limitations to cis-trans test.

1. Cannot be used for dominant mutations.

m1 m2 m1 +
Mutant type

+ + + m2

cis trans

- Mutant types are produced in cis and trans configuration regardless of

whether the mutations are in the same gene or different genes.

- This is why a control cis test is necessary!!!

Limitations to cis-trans test.

2. Mutations that exhibit intragenic complementation

m1 m2 m1 + m1 +

+ + + m2 m2 +
Cis (wild) Trans (wild) Trans (wild)

m1
m2

In oligomeric proteins, the defect in one polypeptide can be overcome by the other.
Limitations to cis-trans test.

3. Polar mutations in one of the coordinately controlled genes

m1 m2 m1 + m1 +

+ + + m2 m2 +
Cis (wild type) Trans (mutant) Trans (mutant)

Promoter m1

gene1 gene2 gene3

Consider m1 to be a chain-terminating mutant

Limitations to cis-trans test.

4. Mutations on cis-acting elements of coordinately controlled genes

m1 m2 m1 + m1 +

+ + + m2 m2 +
Cis (wild type) Trans (mutant) Trans (mutant)

Promoter

m1 gene1 gene2 gene3

Consider m1 to be a mutation in the promoter that prevents the RNA polymerase

from binding to it. Then there will be no transcription of any downstream genes.
Limitations to cis-trans test.

5. Conditions that allow leakage of recombination.

m1 m2 m1 + m1 +

+ + + m2 m2 +
Cis (wild type) Trans (wild type) Trans (wild type)
m1 + m1 +

+ m2 m2 +
COMPLEMENTATION TESTS (cis-trans test)

Complementation test
Whether two mutations affecting a character are on the same gene
or not. (allelic or not)

- All mutations that give a negative cist-trans test are referred as

belonging to a complementation group or a cistron.

- Cistron is considered to be synonymous to a gene

- Complementation test provided the first functional definition of a

gene.
Recombination test and Complementation tests
- differences
• . Recombination test
1. Creation of new combinations of
mutations through cross overs
2 Progeny are produced with has new
combination of genes.
3. Tells whether mutations are same or
different and if different the distance
between them.
4. Provides the definition of a mutational
unit, the smallest unit non-divisible by
Mutations. Unit of structure of a gene.
Complementation test
New combinations of mutations are not
formed by cross over; rather the products
of new gene combinations in a single cell
system mix and complement
New progeny are not produced, nor is
there a change in genotype.
Tells us if mutations are allelic or non-allelic
(ie on the same gene or different genes.
Functional definition of a gene.
GENE FINE STRUCTURE ANALYSIS
IN BACTERIOPHAGES
COMPLEMENTATION TESTS (cis trans test)

How its done?

In bacteriophages:
Two viral mutations can be brought together into a single bacterial cell
system by transduction.
COMPLEMENTATION TESTS (cis trans test) rII
locus
h+ = ability to infect TWO distinct E. coli strains- strains B and K12λ
h- = ability to infect only ONE E. coli strain- strain B

r+ = small turbid plaques with fuzzy margin = normal

r- = large clear plaques = mutant

h-r+ = turbid, small plaques on B

h+r- = large clear plaques on B and K12λ
h+r+ = turbid, small plaques on B and K12λ
h-r- = large clear plaques on B.
Complementation test using the BACTERIOPHAGE T4
rII REGION

Bacteriophage T4 rII mutants have a distinct plaque morphology

phenotype and distinct host range properties:

PLAQUE MORPHOLOGY

E. coli infected with wildtype T4 (r+) phage result in the formation of small
turbid plaques with fuzzy borders

E. coli infected with rII mutants result in the formation of large clear
plaques
Recombination tests
Bacteriophage T4 rII mutants have a distinct plaque morphology
phenotype and distinct host range properties:

HOST RANGE PROPERTIES

Wild-type T4 (r+) phage has the ability to infect and lyse both E. coli strains
B and K12λ

Mutant rII T4 phage only has the ability to infect and lyse E. coli strain B; it
cannot lyse K12λ

E. coli strain B is referred to as the permissive host for rII mutants

E. coli strain K12λ is referred to as the non-permissive host for rII mutants
Recombination tests

= Phage cross
 COMPLEMENTATION TESTS (cis trans test)
Here we perform a complementation spot test.

1. The TWO mutants are spotted together on a E. coli K12λ lawn at a conc.
that allows double infection and incubated at 37oC to determine if plaques
form.
Double infection brings two different phages into one bacterial system.

2. If plaque forms, then +ve complementation- mutations in different

genes.

If no plaque forms, then –ve complementation- mutations are on the same

gene.

NB: If two mutations occur at exactly the same site, they are called
homoallelic mutations.
E. coli B
Benzer’s deletion mapping technique

Most of the rII mutants isolated were point mutations resulting from the
alteration of a single base pair

However, some of the mutants did not produce recombinants when

crossed with other mutants known to be at a different location

These mutants were deletion mutants and had lost a segment of DNA

Deletion mutants can be used to help map the location of point mutations
Benzer’s deletion mapping technique

Benzer established several reference mutations at the rII locus.

A negative recombination test between an unknown point mutation and

a standard deletion mutation indicates that the point mutation is located
within that particular segment of deleted DNA

A positive recombination test between an unknown point mutation and a

standard deletion mutation indicates that the point mutation is not
located within that particular segment of deleted DNA