Clathrin & Primer Selection with the NCBI Database

A+K gel (edit)

Sometimes you need to find a primer. Not just any primer but a specific one. This is a small post concerning the development and acquisition of a specific primer for a portion of the clathrin protein. Clathrin is a protein that plays a key role in the process of endocytosis. It’s main function being to coat the inward bound lipid bilayer as it enters inwards into the cell. Its triskelion shape enables it to have the flexibility and reach needed for this specific function.

The overall structure of clathrin is composed of three heavy chains (~190 kDa). These interact at their c-terminal ends, and each of these heavy chains is intercalated with a smaller ‘light’ chain (`25 kDa). When these proteins connect as they encircle a vesicle they form a multi-sided polyhedral lattice structure, which is illustrated to the right, (Figure 1). This protein, by playing the gatekeeper to the cell, becomes a significant cellular component due in part to the overwhelming abundance by which our cells must depend on it for proper communication between the intracellular and extracellular environment.

Each clathrin ‘limb’ is broken down into simplistic anatomical terms, with a foot, ankle, and shaft portion. The mechanism by which the triskelions bind together is housed in the foot portion of the protein. Due to this there is a constraint on the size that is allowable in regards to clathrin cage formation around incoming vesicles. Should the vesicle be too small the feet of each limb will struggle to adhere to one another and this will therefore inhibit proper cage formation. It should be noted also, that clathrin as a whole, does not itself interact directly with the incoming vesicle’s membrane, but must rely on adapter proteins for this process.

Since there are two sub-units, there will be at least two genes coding for the two different proteins. There are some exceptions, for example in the case of the light chain, which is present in two forms, α and β. The main heavy chain gene, heavy chain 1, in human tissue is located on the 17th chromosome, loci q23.1-qter, an exception made for muscle tissue which houses heavy chain 2 on chromosome 22 q11.21, and the light chains are, in sequence, at loci in chromosome 9 q13 and chromosome 5 q35.

To properly utilize the Polymerase Chain Reaction (PCR) we need to know the primers needed to replicate the given genetic sequence, or in the case of a protein, reverse transcription polymerase chain reaction (RT-PCR) to find out the specific genetic expression by way of creating complimentary DNA (cDNA) and assessing the template vs genetic strands. Through the NCBI database e-PCR we can find the needed Primers and run them to ensure they will give us the results we are looking for. Below is the acquired heavy chain data.

NCBI primer data.jpg

24 APR 2015

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