23. An individual suffers from a genetic disease. You isolate and identify the p

Question

23. An individual suffers from a genetic disease. You isolate and identify the protein normally encoded by the gene. Utilizing an in vitro assay, you find the protein is a phosphofructokinase catalyzing the addition of a phosphate group from ATP to fructose (phosphorylation). Analysis of the protein from both affected and unaffected individuals indicates that the polypeptides are found at identical levels in the affected and unaffected cells and the molecular weight of the polypeptides determined by gel electrophoresis appear to be identical in both affected and unaffected individuals. In addition, the N-terminal amino acids are methionine in both the affected and unaffected individuals.

A) Is the mutation more or less likely to be in the promoter, 5’UTR, exons, introns, 3 ‘UTR of the gene? Why or why not.

B) Is the mutation likely to be a silent, missense or nonsense mutation? Why or why not.

C) The protein has a molecular weight of approximately 58,000 daltons. The average amino acid has a molecular weight of 110 da. Approximately, how many amino acids are in the primary sequence of the polypeptide?

D) What would be the number of nucleotides in the ORF found in the mRNA assuming there was no posttranslational processing of the polypeptide?

E) You determine the primary amino acid sequence of the polypeptide and find an internal fragment with the sequence NH3+-Met-Met-Trp -Trp-Met -Trp- COO-terminal. What are the chances that this peptide sequence will occur randomly?

F) Design a nucleotide sequence that would encode the peptide sequence given in E indicating 5’ and 3’ ends of the polynucleotide.

G) From the start codon to the stop codon of the ORF would suspect there be more or less nucleotides in the genomic DNA or in the mRNA sequence? Why?

H) You isolate all the mRNA from the affected cells and create cDNA. Why is this done and how is this done?

I) Your cDNA library contains ORFs for thousands of polypeptide sequences that where being transcribed and processed in the cell when the mRNA was isolated. How could you use the answer in E to find the cDNA of interest?

J) You isolate the cDNA for the ORF for the polypeptide in the affected individual. You determine the sequence of nucleotides in the ORF and find it is 1611 nucleotides in length. Would you suspect that the polypeptide postranslationally modified? If so how, and at what end of the polypeptide chain would the modification occur?

K) You find three point mutations in the ORF of the affected individual when compared to that of the ORF in an unaffected individual. These mutations do not affect the reading frame. You do know that point mutations are fairly common in human genomes (polymorphisms) and not prima facie evidence of causality. The mutations you find are GCG to GTG, and GGC to GGT and AAA to GAA. Reasoning from the properties of the particular amino acid R groups which mutation is most likely to result in the largest change in the physiochemical properties of the polypeptide and result in the observed disease phenotype?

L) You clone the open reading frame for the affected gene into a prokaryotic specific plasmid and express the encoded polypeptide. The polypeptide synthesized has a molecular weight of 59,000 daltons. Why is it different from that produced in the eukaryotic host?

M) You change the altered codons found in the affected individual to those found in the unaffected individual one at a time by site-directed mutagenesis. You find that only the alteration of GTG to GCG results in restoration of activity of the protein in vitro. Does this alter your initial hypothesis? If so, generate a new hypothesis explaining how the mutation is causing a lack of function.

N) You alter the codon that causes inactivation of the protein to all remain 18 possible amino acid by placing the associated codons at this position one at a time. Only the substitution of alanine or glycine at this position results in activity. Does this support your new hypothesis?

Explanation / Answer

A) Mutation may be in 3-UTR or introns because if there will be mutation in promoter or exon there will be no normal protein formation . And as methionine is common in both proteins so 5-UTR also will be intact.

B) It may be silent mutation.

C) Approximately 537 amino acids.

D) As 527 amino acids are there, so there will be 1584 nucleotides ( three for each amino acid and one stop codon).

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