Gene Regulation: What it is, and how to detect it

By Jordan, Jennifer, and Brian

What is the Promoter Region of a Gene?

•Upstream region of a gene, which regulates when/where the gene is to be transcribed.

• Essentially the promoter region acts as the “on switch” for the gene.

How Can the Gene Promoter Region be Located and Isolated?

Location:• The vital region of the

promoter is somewhere upstream from the gene of interest, and downstream from the “stop” codon directly before the gene of interest.

• Knowing that the region of interest is somewhere between these two points is all that is necessary!

Isolation• If the entire genome of the

organisms has been sequenced (as is the case with arabidopsis), then the exact nucleotide sequences of these areas is known.

• From the sequence information primers can be designed and the gene promoter fragment can be amplified by the polymerase chain reaction (PCR).

How can an isolated promoter region show where a gene is active?

• A previously amplified promoter region (as described in the slide prior) can be fused to a GFP reporter gene.

• The fused region can then be transformed back into the original organism’s genomic DNA.

• Observing where the green fluorescence is present will visualize where the promoter region is active in the original organisms.

G.F.P. (Green Fluorescent Protein)

• The GFP is a protein derived from the jellyfish Aequorea victoria (actual protein name is aequiron), which fluoresces green when exposed to blue light.

• Because other factors are not required for fluorescence, GFP expression can be used to monitor gene expression in living organisms.

Isolate RNA from Plant

AAAA

AAAA

AAAA

Inflorescence

Silique

Leaf

mRNA

mRNA

mRNA

Synthesize cDNA Using RT

AAAAAAA

Reverse Transcriptase

mRNA

cDNA

RV

FW

Amplify cDNA Using Primers

Polymerase Chain Reaction

Visualize with Gel ElectrophoresismRNA Present

mRNA Absent

Positive Control

Negative Control

Presence of mRNA

• Starting point for determining expression

• Expression of gene determined by observing proteins

• Can only determine transcription

GeneChip Analysis

• Using cDNA created from the mRNA isolated from various organs, we can analyze the mRNA accumulation levels for all genes.

• This is done by creating the complementary strands of the known gene sequences and assembling them on a chip.

• The sequences are tagged with flourescent tags that glow a certain color when in contact with the complementary strand.

This is what you get as a resulting image:

Red means active

Green means not as active

No color means no activity

We can then translate the data into graphs like

This is an example of GeneChip data accumulated for one gene across many organs of the plant