REGULATION OF GENE EXPRESSION

Objective: to understand how genes are regulated in prokaryotes (lower organisms) and eukaryotes (higher organisms)

To achieve the objective: study the expression of reporter genes in transgenic organisms

What are reporter genes?

What are transgenic organisms?

What are reporter genes?Reporter genes are protein-coding genes whose expression in thecell can be quantified by the techniques of protein detection.

What does this mean?

Thus, the blue color “reports” to you the presence of an intact lacZ gene. In other words, lacZ is a reporter gene.

Agar plate containingLB + kan + Xgal

TOPO

Polylinker

Intact lacZ gene

ßgal + Xgal Blue color

lacZ gene

Other reporter genes include:

gene in photorhabdus luminescens, emits light

gene in jellyfish, expression patterns turn green

gene in E. coli, expression patterns turn blue

luxE

gfp

gusA

What are transgenic organisms?Organisms carrying any piece of foreign DNA that researchershave inserted into the genome through the manipulation of germcells (gametes) or early embryonic stages, are called transgenicorganisms.

In today’s experiment, Arabidopsis seedlings are transgenic becausethey carry the gusA gene of E. coli origin.

The concept of operon

In bacterial cells, operon is a cluster of structural genes (coding region) alongwith the adjacent regulatory region that controls the transcription of those genes.

Lac I gene Lac Z gene Lac Y gene Lac A genePromoter Operator

Regulatory region Structural region

Promoter

RNA polymerase

Codes for repressor protein

The lactose operon is an operon required for the metabolism of lactose in E. coli. Its structure is shown below.

How is the metabolism of lactose regulated in E. coli cells?

1. Gene Regulation in Prokaryotes

E. coli is able to grow in media containing salt (including a nitrogensource) and a carbon source such as glucose.

These compounds provide molecules that can be manipulated by thecell’s enzymatic machinery to produce everything the cell needs togrow and reproduce, such as nucleic acids, proteins, and lipids.

The energy for these biochemical reactions comes from themetabolism of glucose.

If lactose, instead of glucose, is provided to E. coli as a carbonsource, three enzymes are rapidly synthesized: ß-galactosidase(aka ß-gal), permease, and transacetylase. These enzymes areneeded for the metabolism of lactose.

The genetic mechanism of the production of these enzymes isillustrated below.

Lactose absent

Lactose present

The lux operonLuminescence (or emission of light) by certain bacterial species is acommon phenomenon in nature.

Example: Photorhabdus luminescens emits light which can be seenin the dark.

The biochemical substance capable of luminescence is calledluciferin (= light bringing).

undergoes oxidation in the

Luciferin light is emittedpresence of luciferases

In Ph. luminescens, the genes responsible for the production ofluciferin and luciferases are the component of the lux operon.

Pr Op E C D B A

luciferin luciferases

Demonstration of lux expression in E. coli1. The lux operon has been cloned into the TOPO vector.

2. The cloned fragment has been genetically manipulated:

The lux operon is controlledby the promoter of the lacoperon.

lux

TOPO

Pr Op E C D B A Wild-type lux

Pr E C D B A Mutant: deletion of Opresults in the constitutiveproduction of luciferases

Pr E C D B A Op Z Y APr

Mutant lux Wild-type lac

Pr E C D B A

Mutant lux operon carrying lac promoter

3. Check agar plates in the dark, you will see the emission oflight from E. coli colonies.

TOPO

lux

E. coli

E. coli colonies glow in the dark!

Demonstration of gfp expression in E. coli1. From jellyfish (Aequorea Victoria) a protein has been isolated and

called green fluorescent protein, it emits very bright greenish fluorescence under the UV light. The gene responsible for the green fluorescent protein is called gfp (= green fluorescent protein). This gene has been isolated and cloned.

2. A mutated version of the gfp genehas been developed and cloned into a plasmid called pGreen. The structure of the plasmid is shownon the right.

3. This plasmid has been introduced intoE. coli cells and plated on LB agar medium containing ampicillin.

4. Check the agar plates; the colonies show green color under ceiling light.

Ori

mutant gfp

pGREEN4528 bp

ampr

BamHI

HindIII

EcoRI

SacII

2. Gene Regulation in Eukaryotes

Eukaryotic and prokaryotic cells have many features of gene regulationin common, but they differ in several ways including:1. specialized cell-types 2. lack of operon3. in most cases lack of polycistronic mRNA 4. structural genes have their own promoter and are transcribed

separately5. the presence of nuclear membrane in eukaryotic cells separates

transcription and translation in time and space

In eukaryotes despite the fact that almost all cells contain the same DNA, in each cell-type different sets of genes are active (“on”, are expressed). This different pattern of gene expression causes cell-types to have different sets of proteins, making each cell-type uniquely specialized for a specific function.

Example: Pancreas is an organ in abdomen that produces enzymes for the break down of food. One of its important function is the production of insulin which regulates body’s glucose level. Insulin is produced only when the INS gene is expressed (turns on) in pancreatic cells. The neurons in brain cells have nothing to do with the insulin production,therefore the INS gene is “off” in brain cells.

The gene regulation is achieved through signals originating within the cell itself (e.g. repressor proteins) or in response to external conditions (e.g. heat, cold, humidity, chemical signals….).

Eukaryotic gene expression is regulated at many stages:1. chromatin accessibility2. transcriptional level3. RNA processing4. translational level

A view of eukaryotic gene expression at many stages is shown below.

Reporter genes are excellent tools for viewing gene expression in organisms.

Example of a reporter gene in eukaryotes:

The gusA gene encodes an enzyme called β-glucuronidase (GUS).

i. β-glucuronidase + Xgluc (chromogenic substrate)glucuronic acid + chloro-bromo-indigo

ii. Chloro-bromo-indigo 5,5’dibromo-4,4’-dichloro-indigo (blue color)

E. coli

E. colichromosome

gusA

dimerization

i. β-glucuronidase + Xgluc (chromogenic susbtrate)----glucuronic acid + chloro-bromo-indigo

dimer

ii. Chloro-bromo-indigo ---- 5,5’dibromo-4,4’-dichloro-indigo (blue color)

In order to view the expression pattern of the genes of interest inplants, the gusA gene is used.

Promoter of the gene of interest

Coding region of the gene of interest

The original promoter of the reporter gene is removed and replaced by the promoter of the gene of interest.

Promoter of the gusA gene

Coding region of the gusA gene

GusA gene

Translational chimeric gene

Gene ofinterest

Procedure

1. Remove a seedling from soil.

2. Rinse in water to remove soil attached to it.

3. Place the seedling in the well of a microwell dish.

4. Fill the well with GUS staining solution.

5. Loosely close the lid of the microwell dish, place the dish in a tupperware with a wet paper towel in the bottom, tightly close the lid of the tupperware, and place it in a 37ºC incubator overnight.

6. In the next lab session, inspect the seedling for blue precipitate.

7. Locate the tissues which show the GUS activity.