Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified.
DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.
Explain how the DNA sequence change results in a Ser-to-Thr missense mutation.

Question

Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified.
Restriction map showing BamHI sites in wild-type and mutant alleles, indicating a Ser-to-Thr missense mutation.

Restriction map showing BamHI sites in wild-type and mutant alleles, indicating a Ser-to-Thr missense mutation.

DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.
Southern blot results showing DNA bands for mother (M), father (F), and children (C1, C2) after BamHI digestion.

Southern blot results showing DNA bands for mother (M), father (F), and children (C1, C2) after BamHI digestion.

Explain how the DNA sequence change results in a Ser-to-Thr missense mutation.

Restriction map showing BamHI sites in wild-type and mutant alleles, indicating a Ser-to-Thr missense mutation.

DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.

Southern blot results showing DNA bands for mother (M), father (F), and children (C1, C2) after BamHI digestion.

Explain how the DNA sequence change results in a Ser-to-Thr missense mutation.

Accepted Answer

Hi, everyone. Welcome back. Let's look at our next question. Which of the following options correctly describes the DNA alteration responsible for the sea to three miss sense mutation in the B mh, one restriction site of the mutant H A O homogenic acid oxidase gene that causes al CAPP auria, an autism resistive disorder in humans. And our answer choices describe four different changes to the sequence and the effect they have on the wild type sequence for the B MH one restriction enzyme to the mutant sequence. So choice A says a deletion of two nucleotides and then you see two nucleotide deleted from that B mh one site choice B is a substitution of one nucleotide. Choice C is an insertion of one nucleotide. And choice D is a substitution of two nucleotides. But we know we have a miss sense mutation which means that our change to our DNA sequence has changed one amino acid to another. This is usually a point mutation although it could be a mutation of 12 or three base pairs, but the point is, it only affects a single amino acid in our protein sequence. There is no frame shift that occurs with a miss sense mutation. So in a frame shift mutation, you have a disruption of the reading frame of your enzyme. So something like a deletion or an insertion of base pairs, that means that not only do you change the amino acid where the mutation occurs, but you can affect every single amino acid downstream of that if you throw off the reading frame. So we know that in this case, our change is amino acid Cine amino acid thine. And as we look through our answer, choices here, we can actually eliminate two of them right away because in choice A, we have a deletion of two nucleotides. But that would result in a frame shift since we would throw off our reading frame, we deleted two out of three nucleotides. So choice A cannot be our correct answer. And that's the same in the case of choice C which has an insertion of one nucleotide. Again, that would affect our reading frame and be a frame shift mutation. So let's look at choices B and C which involves substitution of one nucleotide and choice B substitution of two nucleotides. And choice D. Well, the more likely case would be substitution of one nucleotide. That's the more common occurrence. So let's evaluate that. First, we're given the original wall type sequence, which would be that B mh one restriction enzyme site and it is G G A T C C. And then we see that the substitution gave us the mutant sequence of G G A AC C. We've changed that T to an A. When we look at the codons for amino acids, we get U C U U C C U C G and U C A with that wobble in the third position. So we see that our T C C sequence will correspond to a UCC code on. And as expected code for searing, then we look at our mutation, we have instead an AC C code on. And indeed, when we look at the codons for the amino acid three, we have a cuac C AC G and AC A. So we do have the AC C code on there coding 43 and E. So we had a single substitution of a nucleotide leading to the replacement of the amino acids by the amino acid. And that's going to disrupt the gene for homogenic acid oxidase which disrupt, disrupts the tyrosine degradation pathway and causes the byproducts there to build up in the body. So it's looking B is looking like our answer here, but we do need to look briefly at choice D here. Choice D says a substitution of two nucleotides. But when we look at how it changes our sequence, it says it changes the sequence from G G A T C C wild type to G G C T C C. But that is not a substitution of two nucleotides. That is only a substitution of one nucleotide, the A for the C. So that is not even a correct description of the mutation they show us. So choice D is not our correct answer and we can definitely choose choice B that's the correct description of the DNA alteration that occurs in al captain area that causes ay to three and mis sense mutation is a substitution of one nucleotide that changes the sequence from G G A T C C in the wild type to G G A AC C and the mutant. See you in the next video.

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Key Concepts

Missense Mutation

Restriction Enzymes and Sites

Southern Blotting