1. How can you determine the function of an isolated gene?
You can over-express the gene by putting it under control
of a stronger promoter
Promoter A
= Weak expression
Coding Sequence A
= Strong expression
Promoter B
Coding Sequence A
2) Strong over expression may lead to a detectable change in
phenotype

2. How can you determine the function of an isolated gene?
You can “knockout” the gene by mutation, and then determine
the phenotypic effect of this knockout.
Mutations can be either in the promoter or the coding sequence
PromoterA
Coding sequence A
= Normal
= Mutated

3. How can you determine the function of an isolated gene?
You can link the promoter of the gene whose function you
want to determine to a coding sequence which produces a
protein which can be easily assayed.
The resultant construct is called a
Promoter – reporter fusion
The promoter-reporter fusion is then introduced into an
organism - Bacterium, Plant, Animal, Fungus, Insect
Expression of the reporter gene is controlled by the promoter
and detection of expression can give clues about gene function.

4. Reporter genes are useful because:
They can indicate where the gene promoter is active,
and what developmental signals control activation
2) They can be used to study gene expression from
individual members of a gene family
3) They can be used to separate transcriptional from
post-transcriptional control.
4) They can be used to identify specific controlling
promoter motifs
5) They can be used in “Reverse Genetics”

5. Reporter enzymes should be:
1) Easy to quantify (How much)
2) Product should be detectable histochemically so that
it can be localised in specific cells and tissues (Where)
3)Reaction performed to detect the product should not interfere
with normal cellular metabolism
4)Substrates for the assays should be relatively cheap
5) Reporter enzyme should NOT be present in transformed
organisms

6. Reporter genes whose products can be easily assayed include:
b – Glucuronidase
Luciferase
Green Fluorescent Protein
Promoter A – b-Glucuronidase
Promoter A – Luciferase
Promoter A – Green Fluorescent Protein.

7. b-glucuronidase is an E. coli enzyme which is NOT
present in animals and plants
b-glucuronidase is a hydrolase which cleaves b-glucuronide.
b-glucuronidase is stable, and tolerates varying ionic conditions
Artificial substrates have been developed which allow both
qualitative and quantitative assays.

8. Histochemical analyses are performed using the substrate
5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
b-glucuronidase (GUS)
Indoxyl derivative
Oxidative dimerisation
Deep blue, insoluble product (highly visible)

9. To determine the expression patterns of the extA plant extensin
cell wall protein gene, a extA- GUS promoter – reporter fusion
was made and introduced into Nicotiana tabacum
extA promoter extA coding sequence
extA promoter b-glucuronidase coding sequence

10. Developmental expression of the atExt1 – GUS fusion construct.
a) Arabidopsis seedling …
10 days
4 days
Germinating seed

11. Expression of the extA – GUS promoter –reporter fusion at the
nodes and internodes of transgenic Nicotiana tabacum.
TS internode
TS Node
LS NODE

12. These expression patterns support the idea that the extA
extensin gene codes for a structural protein whose function
is to provide strength and support to the plant at regions
where the plant experiences mechanical stresses.

13. Quantitative analyses are performed using a fluorescent
substrate – 4- methyl umbelliferyl glucuronide
Transgenic tissues expressing the GUS gene are used to extract
GUS enzyme.
4- methyl umbelliferyl glucuronide
b-glucuronidase
4-methyl umbelliferone (Fluorescent)
Spectrophotometer

14. Promoter deletions are used to discover promoter elements
regulating quantitative and qualitative expression.

15. +ve and –ve
controlling elements
have been
identified.

16. Does GUS have any disadvantages as a reporter system ?
It can be used for both quantitative and qualitative assays
BUT
The assay is destructive, and cannot be used on live tissues
so in-vivo experiments are not possible.
A relatively recent reporter system which is widely used for
in-vivo analysis of gene expression is the GFP system
Green Fluorescent Protein.

17. Jellyfish Aequorea aequorea
Blue Green luminescence

18. Light organs of Aequorea contain 2 kinds
of protein
Aequorin + Green Fluorescent Protein
Fluorescent compounds absorb light at
one wavelength and re-emit it at another.
Luminescent compounds emit light
in the presence of co-factors

19. Aequorin Blue Light 460nm (Luminescence) Touch, stress, dev. Signals
Ca 2+
Aequorin
Blue Light 460nm
(Luminescence)
GFP
Excitation
Fluoresces
509nm

20. GFP mutant variants have been produced

21. Transformation of mice using an actin – GFP construct:

22. Transformation of mice using an actin – GFP construct:

24. How do you find genes controlling interesting phenotypes?
Conventional Approach 1
Study phenotype
Isolate protein possibly coding for (1)
Sequence protein
Deduce DNA sequence
Make a cDNA library
Screen library with deduced sequence
Isolate specific cDNA clone
Use cDNA to screen genomic library
Characterise gene (s)

26. Promoter traps can locate interesting genes!
1) Screen for plants where the
trap is off
2) Inoculate plants with virus
3) Allow infection to proceed
4) See if trap has come on !
5) Clone out the gene
Courtesy: J.I.I. Norwich, U.K.