You are new to the lab and your job is to test a new antibody made against the l

Question

You are new to the lab and your job is to test a new antibody made against the lab’s favorite protein,

guerkin. You run a series of Western blots using cellular extracts from a variety of different mouse

tissues to see if the antibody recognizes the 50 kD guerkin protein. In almost all of the extracts, you

observe a strong band at 50 kD. However, in extracts from skeletal muscle, you always see a 70 kD

band. You’re pretty sure this band is specific to the guerkin protein because you don’t observe it in

skeletal muscle samples from mice lacking the guerkin gene. Your advisor thinks this is really interesting

and wants you to look at the genomic structure of the guerkin gene to see if you can find an explanation

for this observation.

Genomic organization of guerkin

The open boxes indicate exons and the filled boxes are introns. Nucleotide number is shown.

A) Provide an explanation for the generation of 50 kD and 70 kD proteins that react with the anti-guerkin

antibodies. Be certain to illustrate the molecular differences between the two proteins. [Hint: the average

molecular weight of an amino acid is 110 Daltons]

B) In what region of the two proteins would you predict the antibodies to bind in order to recognize both?

Explanation / Answer

NOT ABLE TO SEE GUERKIN GENOMIC ORGANIZATION IN THE QUESTION PROVIDED BUT STILL EXPLANATION IS PROVIDED ACCORDING TO MY VIEWPOINT

A)

As antibody is specific to Guerkin protein (G), recognition of 50 kD (G50) and 70 kD (G70) Guerkin proteins in western blot clearly depict that these are isoforms of Guerkin protein. Four basic mechanisms (and combinations of them) can give rise to more that one mRNA from a single gene. First, several primary transcripts can be initiated at alternative promoters. Second, differential termination or 3’ post-transcriptional processing. Third, alternative splicing of the same primary transcript. Fourth, after re- combination of genomic sequences, such as for the immunoglobulin genes.

Out of these four mechansims, majorily alternative promoters mediate tissue specific expression of protein isoforms in eukaryotes. If the alternative leader exons produced by alternative promoter usage contain coding sequences, the use of alternative promoters can result in alterations of the primary structure in the amino terminus of the protein that may affect the function of the protein or its subcellular localization.

Note, G70 protein expression is visible only in skeletal muscle, so it represents tissue specific expression. Thus clearly showing that Alternative promoters are involved in our study. The use of alternative promoters result in two mRNA isoforms with a different primary structure in their 5’-region.

From, molecular weight of proteins it is quite evident G70 protein is formed by addition of extra amino acids which will be towards N-terminus. So, the molecular differences between these two proteins will be:

At protein level: G70 has extra 182 amino acids towards N-terminus than G50. (1 amino acid = 110 Da).

At mRNA level: G70 has additonal 546 bases towards 5'-end than G50. (1 amino acid = 3 bases)

B)

In case, the protein isoforms are formed by alternative promoters, the c-terminus of the protein will be recognized by antibody which is conserved, as n-terminus is altered by addition of extra amino acids.

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