Analyze the data 10-2: Alternative Splicing of a Protein Kinase A specific prote

Question

Analyze the data 10-2: Alternative Splicing of a Protein Kinase

A specific protein kinase (PK) gene is speculated to be differentially spliced in muscle tissue. This gene comprises three exons and two intron sequences and in fibroblast cells encodes a 38.5-kD protein. Investigators have transfected various portions of a genomic or cDNA copy of the PK gene into both muscle and fibroblast cells (see the constructs in part (a) of the figure). The expression system utilizes a promoter active in both cell types and a C-terminal fusion to a small epitope tag called V5, which contributes ~5.5 kD to the fusion protein. Part (b) of the figure shows the results of an immunoprecipitation experiment designed to analyze the expression products of the transfected cells. A negative control (Neg) of untransfected muscle cells is included. Molecular weight markers in kD are indicated on the left. Immunoprecipitated proteins as shown in part (b) were then placed in protein kinase assays with two different substrates, A and B, to discern activity of the expressed proteins.

a.) What can be concluded about differential splicing of the PK gene in fibroblast versus muscle cells? Are there other experiments that could confirm these results?

Explanation / Answer

The 45 kD band is the complete PK protein coded by the three exons plus the 6.5 kD epitope tag V5. Hence, the P1 construct that is translated to 45kD fusion protein is detected in fibroblast (Lane 1 of Part b). The P2 construct antibody detected a 25 kD band in Lane 2 in fibroblast. Similarly, P3 construct antibody detected a 29 kD protein in Lane 3 for fibroblast. The 25 kD protein is due to exon 1 and exon2 while 29 kD protein is coded by exon 1 and exon 3.

In the muscle, the P1 probe/ antibody shows a 29 kD band. Hence, the full length PK protein is absent in muscle. The P2 and P3 probes (Antibodies) gives similar proteins (25 and 29 kD respectively) for muscle. As the 45 kD protein is never detected by any of the antibodies, it is not expressed in muscle. Hence, muscle tissue two isoforms of PK- one that lacks exon 2 and the other that lacks exon 3. Muscle PK gene has does not express full length protein and the transcript is always alternatively spliced. Fibroblast, on the other hand show normal alternate splicing. It has the full length transcript, a transcript lacking exon 2 and an transcript lacking exon 3.

Northern blot analysis can also be used to detect alternate splicing. The presence of different sizes of mRNA transcript on the gel will indicate alternate splicing. Real time PCR/ Semi-quantitative PCR can also be used to detect alternate splicing. A primer in alternative exon and an primer to the constitutive exon is used to detect the alternate transcripts formed.

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