Gene segments in the heteroduplex formed between mRNA and DNA


What is heteroduplex

A heteroduplex is formed of the base pairing of DNA to mRNA from the same gene. A paper by Davis et al. describes a simple experiment, but highly creative using heteroduplex analysis whether or duplex ich reconstructs the lineage of the heavy chain locus DNA from mouse myeloma cells. In my example we similarly determine which segments are represented by the double stranded DNA molecules Shown below is a crude drawing of the heteroduplex formed from the messenger mRNA of an IgH heavy chain and the antisense DNA strand of the germline DNA. We can use the size of the mRNA strands that do bind the anti-sense DNA.
Before we can identify the gene segments we need to identify what protein segments form the IgH heavy chain. So for this example we need to know the IgH heavy chain is composed of a variable region, a D region, and a J region. Additionally the gene contains a leader region and a constant region. We can deduce the any bubbled out region represents an intron where the sequence has been spliced out. We can also deduce because the number of the single stranded DNA molecules (bubbles) the IgH chain must come from a cell that has undergone VDJ rearrangement.  Therefore we can just need to determine what is the 5′ and 3′ ends, however I have already done so in my image above. My first piece of evidence is the size of region 5 and the missing complement from the anti-sense DNA (bottom). I know from the conventional outline of an antibody the constant region is always much greater than the variable region. This is the most difficult part of the problem because it requires you to either have complete understanding of the finished gene product and the exons that encode it or you can look it up in a resource.
From here the problem becomes simple just by noting that Region 1 must encode the leader sequence. The Leader is followed by a small intron and region 2 must encode the V region. Region 3 would then encode a D region. Region 4 would incoporate the J region, and here  is where the annealing of DNA to mRNA stops.
Davis et al. An immunoglobulin heavy-chain gene is formed by at least two recombinational events. Nature 283:733 (1980)



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