2. Loss of Factor LX (F9) function leads to the blood clotting disorder hemophil

Question

2. Loss of Factor LX (F9) function leads to the blood clotting disorder hemophilia. A research group from the Netherlands found a T-> A mutation 20 base pairs upstream of the transcription initiation site of the F9 gene in a hemophilia patient. They noticed that the normal base was part of a sequence with similarity to the consensus recognition site (TGNACNTT) of a transcriptional activator protein called HNF-4. Based on the knowledge that HNF-4 is expressed in the cells that normally produce F9, they proposed the following hypothesis: The patient's mutation disrupts the binding of HNF-4 to the F9 promoter, which results in loss of F9 expression As a first test of their hypothesis, they created a radiolabeled dsDNA fragment containing the WT sequence from this region (WT"). They also created unlabeled dsDNA fragments with the same WT sequence (WT or with the patient's mutation (Mut as cold competitors. They incubated HNF-4 protein with: (1) WT" alone; (2) WT" 50-fold excess of WTCC; or (3) WT" 50-fold excess Mutc, and then ran the samples on a native gel, which is shown below dsDNA fragment sequences with highlighted mutation: WT *5 CTC.AGCTTGTACTTIGGTACAA CTA-3 CC 5' CTC AGCTTGTAC TTTGGTACA ACTA-3 WT CC 5' -CTCAGCTTGTACTTAGGTACAACTA-3 Mut. position of WT with no HNF-4 (1) (2) (3)

Explanation / Answer

The hypothesis is correct.

In all three lanes hot WT was present. In the first lane there is no competitor for it. So, hot WT could bind to purified HNF4 protein. So, we are seeing the band in lane 1.

In lane 2, there is no band. Here, hot WT has a cold WT as a competitor. In addition cold WT is 50 fold high in concentration compared to hot WT. WT can bind to HNF-4 protein. So, cold WT which is in excess could almost replace hot WT from the protein. So, we did not see the band in lane 2.

In lane 3, there is a band. Here, hot WT has a cold MUT as a competitor. As cold Mut sequence carries a mutation which will inhibit binding to HNF-4, It will not compete with hot WT in binding to HNF-4. So, hot Wt could bind to HNF-4. Therefore we are seeing the band in lane 3.

Absence of F9 causes haemophilia. Research group found a mutation in 5’ UTR of F9. Therefore, they know that it will not change the sequence of protein. If it is associated with disease, it may likely interfere with transcription of the gene.

Mutation testing is usually done on DNA isolated from peripheral blood of the patients. We cannot genetically engineer/manipulate the patient’s cells to study the regulation of the gene, in vivo in patients. So, an in-vitro experiment is the best way to conduct.

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