Analyze the data 10-2: Alternative Splicing of a Protein Kinase A specific prote
ID: 3167115 • Letter: A
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?
c.) How does the presence or absence of exon 3 alter the catalytic activity of the encoded PK protein? Which data support your conclusions?
**************ANSWER PART C in detail please************
Answer A and B if possible
3 V5 2 = 300 bpExplanation / Answer
Please find the answers below:
Answer a: According to the information, the main gene in this experiment encodes for a protein of molecule weight 38.5 kDa whereas after addition of the 5.5 kDa tag, the total weight would become 44 kDa. Thus, the fibroblast having construct 1 shows no splicing as the complete sequence with tag is observed as a result. However, alternative splicing does take place in the constructs 2 and 3 as the molecular bands observed in these cases are much smaller as compared to the master band. This observation of alternative splicing holds true for both fibroblasts having construct 2 adn 3 as well as muscle cells having all the 3 constructs. Experimentally, this construct is favorable for the cells as the substrate reaction rate is much higher for the spliced sequences as compared to the master sequence construct.
Answer b: The illustrations and the graphical data present in the information suggest that the sequence 1 remains common in both cell lines during the cases of alternative splicing. Thus, the sequence 1 in all the construct seems to manage and control the alternative splicing in these constructs.
Answer c: The presence of exon 3 causes a significant enhancement in the catalytic activity of the encoded PK protein in both fibroblasts and the muscle cells. This can be seen clearly in the graphical data that both the cell lines using construct 1 and 3 show very high catalytic activity in both cell lines that too in presence of both the substrates.