Genetics
- Predator and prey perspective
In this topic I would like to focus on the relation between the genome of predator and species. The organisms set as example are various species of Cobra, Mongoose and rat.
Like other serpents Cobras have venom which it uses to kill/ paralyze its prey.
Cobras have highly neurotoxin and causes death by paralysis. The evolved neurotoxins of Cobras are α BTX which attacks the ACh receptors in synapse.
α-BTX is highly toxic to humans, thus cobras are the most feared and hated of all the snakes. A small vial of α BTX can kill over thousand people. Though the amount of venom injected by the cobra is controlled voluntarily small amount is injected into the body when cobras fight for the territory etc. the α BTX doesn’t kill the cobra, its ineffective. This led the curiosity of naturalist into a genetic discovery.
Naja ophiophagus, Naja spectalecobra, O. Hannah, Hemachatetus hemachateus, Naja nigricollis. Were captured from the wild habits
The blood was collected from the heart for sequencing.
The protein which acts as the binding site of α-BTX was obtained by affinity chromatography. The western blot test is preformed to bind the protein to α-BTX
The protocol is as follows:
Protocol 1
(1)Transfer buffer (1 litre): Dissolve 5.82 g of Tris base, 2.93 g of protein, 3.75 ml of 10% SDS in 800 ml water. Make it to 1 litre with 200 ml of Methanol
(2) TBST (1 litre): Dissolve 8.8 g of Tris base, 1.2 g of Sodium Chloride and 500μl of Tween 20 in 750 ml of water. Adjust the pH of the solution to 7.5 and finally bring the volume to 1 liter
(3) Blocking solution: 5 g of fat free milk in 100 ml of TBST. (Carnation milk works well)
(4) Stripping buffer (1 litre): To 125 ml of 0.5M Tris (pH 6.8) buffer add 100 ml of 10% SDS and 8ml of 2-Mercaptoethanol. Bring the volume to 1 litre with water
Protocol 2
1) After the gel run, transfer into the transfer buffer.
(2) Soak the filter paper in the transfer buffer and lay flat.
(3) Soak the nitrocellulose membrane and lay it on top of the filter paper.
(4) Place the gel on top of the nitrocellulose membrane.
(5) Layer the gel again with a filer paper soaked with transfer buffer (Fig. B.5).
(6) Roll a pipette so as to remove trapped air bubbles.
(7) Transfer the sandwich directly to a transfer apparatus incase of semi-dry apparatus, or to a cast in case of wet transfer.
(8) Place the gels with the membrane on the anode or positive electrode (usually Red) and the gel on the cathode or negative electrode (usually Black).
(9) Cover the apparatus and transfer as per the specification of the instrument as stated in the manufacturer’s instructions.
(10) After the transfer, mark the blot and then wash with TBST buffer.
(11) Block the membrane in blocking solution for at least 1 hr at RT or overnight at 4◦C.
(12) After blocking, incubate the blot with primary antibody at an appropriate dilution in 10ml of blocking solution.)
(13) Incubate on rocking, shake for at least 2 hr at RT or overnight at 4◦C.
(14) Wash the blot 3 times for 5 min each with TBST.
(15) Add 15 ml of blocking solution with appropriate amount of secondary antibody (Either Alkaline phosphatase or HRP conjugated antibody).
(16) Incubate the blot on the rocker for 2 hr at RT.
(17) Wash the blot 3 times for 5 min each with TBST.
(18) Develop the blotwith the respective developer depending on the secondary antibody.
Blot development
When using Alkaline Phosphatase based secondary antibody:
• Add the commercially available substrate solution containing
5-Bromo-4-chloro-3-indolyl Phosphate (BCIP)/Nitroblue
Tetrazolium (NBT) (SIGMA) to the blot till it is just submerged.
• Incubate the blot in the substrate solution for 10–30 minutes until the development of bluish purple color.
• Stop the reaction by washing the strips in several changes of water.
• Air-dry the strip and photograph or scan it for records.
When using the HRP conjugated secondary antibody:
• Just before developing, prepare the substrate solution by mixing equal parts of reagents 1 and 2 (Commercially available like Pierce ECL Western Blotting Substrate).
• Incubate the blot with gentle shaking in the substrate for 1min at RT.
• Using a forceps, lift the blot from the substrate and drain off the excess solution by placing the tip of the blot on a filter paper.
• Wrap the blot in a saran wrap.
• Place the blot in a film cassette.
32 _ Protein Analysis
• Take the cassette to the developing room without lights except for the safelight; place an X-ray film on the top of the blot.
• Expose the film to the blot for various time points.
• Develop the film.
The receptor protein and the toxin binds thus giving purple color a positive test.
The experiment concluded that the binder protein is not modified in the snake. The next step was comparing the sequence of nucleic acids of snake and rat.
Following is the sequence of mouse protein which is a neuroreceptor for Ach YRGWKHWVFYSCCPTTPYLDIT
YRGWKHWVFYSCCPTTPYLDIT
The amino acids shown red in the peptide are the binding site for the neurotoxin.
On comparing the sequencing from .510-.560 position on Cobra DNA and 180-170 in mouse
.510 | .530 | |||||||||
DNA | AGT | AAC | TAC | ATG | CGA | AGT | GGA | GAA | TGG | AGA |
PRTN | S | N | Y | M | Q | S | G | E | W | T |
PRTN | F | E | V | |||||||
.170 |
.540 | .550 | . | .560 | ||||||
TTG | AAA | GAT | TAC | CGA | GGT | TTT | TGG | CAC | TCG |
L | K | D | Y | R | G | F | W | H | S |
I | E | A | W | K | |||||
.180 |
GTG | ACC | ||||||||
V | N | ||||||||
F |
The experiments showed that the Histidine Serine Valine sequence in peptide binds specifically to –GlcNAc-(Fucα1, 6)- GlucNac- (GlucNac ß 1,4 ) –(man – R1) – man (r2 (r3)) a carbohydrate. This shields the vulnerable protein from α BTX.
Mongoose is a predator that preys on cobras and other snake. In its evolution it has developed the resistance to α BTX in the similar fashion as cobras. This has laid foundation to the future of anitvenom.
