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 the cell can be quantified by the techniques of protein detection.

What does this mean? Polylinker

lacZ gene

TOPO

Intact lacZ gene

Agar plate containing ßgal + Xgal Blue color LB + kan + Xgal

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

luxE gene in photorhabdus luminescens, emits light

gfp gene in jellyfish, expression patterns turn green

gusA gene in E. coli, expression patterns turn blue What are transgenic organisms? Organisms carrying any piece of foreign DNA that researchers have inserted into the genome through the manipulation of germ cells (gametes) or early embryonic stages, are called transgenic organisms.

In today’s experiment, Arabidopsis seedlings are transgenic because they carry the gusA gene of E. coli origin. The concept of operon

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

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

Regulatory region Structural region RNA polymerase

Promoter Lac I gene Promoter Operator Lac Z gene Lac Y gene Lac A gene

Codes for repressor protein

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 nitrogen source) and a carbon source such as glucose.

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

The energy for these biochemical reactions comes from the metabolism of glucose.

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

The genetic mechanism of the production of these enzymes is illustrated below. Lactose absent

Lactose present The lux operon Luminescence (or emission of light) by certain bacterial species is a common phenomenon in nature.

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

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

undergoes oxidation in the Luciferin light is emitted presence of luciferases

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

Pr Op E C D B A

luciferin luciferases Demonstration of lux expression in E. coli 1. The lux operon has been cloned into the TOPO vector.

lux

TOPO

2. The cloned fragment has been genetically manipulated:

Pr Op E C D B A Wild-type lux

Pr E C D B A Mutant: deletion of Op results in the constitutive production of luciferases

Mutant lux Wild-type lac

Pr E C D B A Pr Op Z Y A

The lux operon is controlled Mutant lux operon carrying lac promoter by the promoter of the lac Pr E C D B A operon. 3. Check agar plates in the dark, you will see the emission of light from E. coli colonies.

lux

E. coli

TOPO

E. coli colonies glow in the dark! Demonstration of gfp expression in E. coli 1. 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. EcoRI SacII 2. A mutated version of the gfp gene has been developed and cloned into BamHI

a plasmid called pGreen. The ampr pGREEN 4528 bp mutant structure of the plasmid is shown gfp on the right.

Ori HindIII 3. This plasmid has been introduced into E. coli cells and plated on LB agar medium containing ampicillin.

4. Check the agar plates; the colonies show green color under ceiling light. 2. Gene Regulation in Eukaryotes

Eukaryotic and prokaryotic cells have many features of gene regulation in common, but they differ in several ways including: 1. specialized cell-types 2. lack of operon 3. in most cases lack of polycistronic mRNA 4. structural genes have their own promoter and are transcribed separately 5. 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 accessibility 2. transcriptional level 3. RNA processing 4. 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:

gusA E. coli

E. coli chromosome

The gusA gene encodes an enzyme called β-glucuronidase (GUS). i. β-glucuronidase + Xgluc (chromogenic substrate) glucuronic acid + chloro-bromo-indigo

dimerization ii. Chloro-bromo-indigo  5,5’dibromo-4,4’-dichloro-indigo (blue color) 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 in plants, the gusA gene is used.

Promoter of Coding region of the the gene of gene of interest interest Gene of interest

Promoter of Coding region of the the gusA gusA gene gene GusA gene

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

Translational chimeric gene 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.