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

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

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.

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”

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

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.

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.

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)

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

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

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

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.

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

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

+ve and –ve controlling elements have been identified.

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.

Jellyfish Aequorea aequorea Blue Green luminescence

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

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

GFP mutant variants have been produced

Transformation of mice using an actin – GFP construct:

Transformation of mice using an actin – GFP construct:

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)

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.