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?

b.)What sequence or sequences contribute to regulation of alternative splicing?

**************ANSWER PART B please************

Answer A if possible

Explanation / Answer

Please find the answers below:

Answer A: According to the information, the original size of the fragment was 38.5 kDa and after addition of an epitope of 5.5 kDa, the final size would become 44 kDa. The fact that one of the experimental group of fibroblasts shows a band at 44 kDa suggests that the construct A was unspliced in these cells. Secondarily, the observation of two bands at more than 30 and 25 kDa shows that the construct B and C underwent direct splicing in the fibroblasts.

Similarly, the muscle cells showed only spliced fragments of all the constructs showing that the final construct of 44 kDa was not stable in these cells, only spliced fragments were stablized in the muscle cells secondary to alternative splicing.

Answer B: As the exon sequence 1 is common in both constructs B and C, it is very likely that this sequence located neary the promoter and start site regulates the mode of alternative splicing in both fibroblasts and muscle cells. Thus, the localization and orientation of the sequence 1 will determine the mode and extent of alternative splicing in these cell lines.

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