BIOL 102

Lecture 11
Gene Function and Mutational Analysis
Reading pages: 219-226
Problems: Ch6 15-17

Mutational Analysis
Powerful tool for studying gene function
forward genetics: identification of mutants and
description of their heritable phenotypes precedes
molecular analysis of products
reverse genetics: based on genome sequences,
gene of potential interest is mutated and the
phenotype of mutated gene is studied
In classical genetics, mutagens were widely used
In a neo-classical approach, insertional mutagens
(e.g., transgenic elements) both disrupt gene and tag
it for isolation
Modern genetics- RNA interference (RNAi)

Forward and Reverse Mutations

Unrelated to forward and reverse genetics
Forward mutation: change away from wildtype allele
a+ a
D+ D
Reverse mutation: change back toward wildtype allele (reversion)
a a+
D D+

Genetic Screens (1)

Can be applied to any problem, depending upon
ingenuity and resources
Morphological mutations
change in shape or form
Biochemical mutations
screening for auxotrophs from mutagenized
prototrophs by supplying various substrates
required for growth
Prototroph= an organism that can grow on
minimal growth media
Auxotroph= a mutant strain that cannot synthesize
molecules required for growth and needs a
special substrate(s) to grow

Genetic Screens (2)

Lethal mutations
premature death
recessive lethals are more useful than
dominant lethals that are difficult to maintain
Conditional mutations
display wild-type under permissive
(nonrestrictive) conditions
display mutant phenotype under restrictive
conditions
e.g., temperature sensitive mutations

Mutational Analyses
Mutagenize seeds
with purple color
flower. Self the plants
from the mutagenized
seeds and screen
progeny

Look for any new

phenotype or a
specific phenotype
2

Mutational Analyses
To determine the nature of the mutations, cross each
mutant with the wild-type parent
Mutant 1
x
wild-type
white
blue
F1
F2

all blue
3/4 blue and 1/4 white

- Similar results were obtained when mutant 2 was

crossed to the wild-type, as well as when mutant 3
was crossed to wild-type.
- This indicates that all three mutants have recessive
mutations in a single gene.

Complementation Test
Cross the
homozygous
recessive
mutants to
each other

The mutations in
mutant 1 and mutant
2 are in the same
gene
No functional
copy of w1 gene

No wild-type blue color

flower was recovered

w2
gene

Complementation Test
1

Cross
homozygous
recessive
mutants to
each other
The mutations in
mutant 1 and mutant
2 are in the same
gene
No functional
copy of w1 gene

Complementation Test
1

Cross the
homozygous
recessive
mutants to
each other

The mutations in
mutant 2 and mutant 3
are in different genes

Wild-type blue color

flower was recovered

One functional
copy of w2 gene

One functional
copy of w1 gene
w1
gene

w2
gene

Complementation Test
2

Cross
homozygous
recessive
mutants to
each other

The mutations in mutant

2 and mutant 3 are in
different genes
One functional
copy of w1 gene

One functional
copy of w2 gene

Biochemical Mutations
Mutation alters gene function by altering
structure/function in a product
wild-type: normal allele
designated by plus (+) sign
example: arg+ or just +
mutation: change in nucleotide sequence
sometimes designated by minus () sign
example: met- or just met
Nutritional mutants
prototroph: wild-type, synthesizes nutrients
auxotroph: mutant, fails to make essential
nutrient(s). (Example, an amino acid)

Biochemical Pathways
George Beadle and Edward Tatum performed the first genetic
dissection of a biochemical pathway - genes specify
enzymes and catalyze specific steps leading to a product.
Nobel Prize 1958
Experiment
1. Irradiate Neurospora crassa (haploid) to induce mutations.
Cross with opposite mating type and collect progeny spores.
2. Identify auxotrophs that can not grow on minimal media (MM)
3. Grow auxotrophs on:
- complete nutrient media- positive control
- MM (negative control)
- MM + all Vitamins (to identify vitamin auxotrophs)
- MM + all amino acids (a.a.) (to identify amino acid
auxotrophs)

i.e. an auxotroph

Amino acid (a.a.) auxotroph,

requires a.a.

Methionine

Tryptopan

Amino acid (a.a.) auxotroph,

requires a.a.

Methionine auxotroph (met mutant),

requires methionine

Biochemical Pathway

Experiment
4. Identified multiple a.a. auxotrophs
5. You are interested in methionine biosynthetic mutants only
6. We know that methionine (met) biosynthesis involves certain
intermediates. Grow the mutants on the intermediate
substrates.

Cystathionine

homocystine

methionine

Growth Response (+ = growth; - = no growth) on Different Media

Strain Complete MM MM+aa
WT
1
2
3
4

+
+
+
+
+

+
-

+
+
+
+
+

MM+cystath MM+homocys

+
-

+
+
-

MM+ met

+
+
+
+

Biochemical Pathway

Experiment
4. Identified multiple a.a. auxotrophs
5. You are interested in methionine biosynthetic mutants only
- Check which mutant grows when MM is supplemented with
methionine

Cystathionine

homocystine

methionine

Growth Response (+ = growth; - = no growth) on Different Media

Strain Complete MM MM+aa
WT
1
2
3
4

+
+
+
+
+

+
-

+
+
+
+
+

MM+cystath MM+homocys

+
-

+
+
-

MM+ met

+
+
+
+

Mutant 1 is not a met mutant; Mutants 2,3 and 4 are met mutants

Biochemical Pathway
Experiment
6. Identify mutations in methionine pathway and in specific
branches of the pathway.Work backward from methionine.
Assume that each mutant is defective in only a single gene.

Cystathionine

homocystine

methionine

Growth Response (+ = growth; - = no growth) on Different Media

Strain Complete MM MM+aa

MM+cystath MM+homocys

MM+ met

WT
+
+
+
+
+
+
1
+
+
2
+
+
+
+
3
+
+
+
4
+
+
+
Supplementing homocystine to MM restores growth of Mutant 2
but not mutant 3 or mutant 4

Biochemical Pathway
Experiment
6. Identify mutations in methionine pathway and in specific
branches of the pathway-work backward from methionine.
Assume that each mutant is defective in only a single gene.

Cystathionine

homocystine

methionine

Growth Response (+ = growth; - = no growth) on Different Media

Strain Complete MM MM+aa
WT
1
2
3
4

+
+
+
+
+

+
-

+
+
+
+
+

MM+cystath MM+homocys

+
-

Mutants 3 and 4 require methionine to grow

+
+
-

MM+ met

+
+
+
+

Biochemical Pathway
Cystathionine

homocystine

methionine

Summary of the mutants

Mutant 1 is not a met mutant
Mutants 2, 3 and 4 are met mutants or met
auxotrophs
Mutant 2 is defective in converting cystathionine to
homocystine; requires homocystine to growth
Mutants 3 and 4 are defective in converting
homocystine to methionine; both require methionine to
grow

Biochemical Pathway
mutant 2
Enzyme 2

mutant 3 and mutant 4

Enzyme 3 and enzyme 4
or both have mutation in
the same gene?

Biochemical Pathway
Are mutants 3 and 4 mutated in the same or
different genes?
Complementation Test
- In Neurospora complementation test is done by
generating a heterokaryon - fusing cells from 2
distinct mutants to give rise to the heterokaryon.
- Use the heterokaryon to test growth on complete
media and media without methionine (or minimal
media)

Testing complementation by using a

heterokaryon

Biochemical Pathway

Will be solved in class

Complementation Test: Generate heterokaryon by

fusing the cells from the 2 mutants.
If the heterokaryon does not grow on the media
without methionine, then the mutations is in the
same gene. i.e. mutant alleles of the same gene.

Biochemical Pathway

mutant 2
Enzyme 2

mutant 3 and mutant 4

Mutations in the same
gene which encodes a
single enzyme

Biochemical Pathway
Complementation Test: Generate heterokaryon by
fusing the cells from the 2 mutants
If the heterokaryon does grow on the media
without methionine, then the mutations are in two
different genes. Two possible segregations.
Will be solved in class

1. Two unlinked genes

Biochemical Pathway
Complementation Test:
If the heterokaryon does grow on the media
without methionine, then the mutations are in
different genes. Two possible segregations.

Will be solved in class

2. Two linked genes

Biochemical Pathway

mutant 2

mutant 3 and mutant 4

Enzyme 2

Enzyme 3 and Enzyme 4

Biochemical Pathway
Complementation test: heterokaryon growth
Strain
WT
Mutant 1
Mutant 2
Mutant 3
Mutant 4

WT
+

Mutant 1
+
-

Mutant 2
+
+
-

Mutant 3

Mutant 4

+
+
+
-

+
+
+
-

Conclusions
Mutant 1 differs from mutant 2, 3, and 4
Mutant 2 differs from mutant 1, 3, and 4
Mutant 3 differs from mutant 1 and 2 but not from mutant 4
Mutant 3 and 4 do not complement, therefore encode the same
gene product
Mutant 3 and 4 are alleles of the same genes

Biochemical Pathway
mutant 2
Enzyme 2

mutant 3
Enzyme 3

mutant 4
Enzyme 3

Each step in a biochemical pathway is catalyzed by a specific

enzyme
One gene one polypeptide- some enzymes are made of more
than one polypeptide
Mutations that result in loss of enzyme activity lead to
accumulation of precursors which may be toxic

Biochemical Pathway
We considered a linear pathway for our answer. Other
interpretations of the pathway is also possible.
Branched pathways
Mutant 2

Cystathio

Mutant 3 & 4

homocys

Mutant 1

Precursor

homoser
threonine

If enzyme 1 is absent, the mutant would require

addition of met and threonine for growth

met

Biochemical Pathway
Two or more pathways leading to the same product.
These examples are unrelated to the Neurospora
mutations we studied
Gene A
Precursor 1

Enzyme A
Product

Precursor 2

Gene B
Enzyme B

Either enzyme A or enzyme B are required to produce

the final product

Biochemical Pathway
Two or more products (polypeptides) required to
form a single enzyme (multisubunit enzyme)
Gene A

Gene B

Enzyme Subunits A + B
Precursor

Product

Mutation in genes encoding subunits A or B results

in no product