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free120/Block 1/Question#9 (34.7 difficulty score)
During an experiment, a Southern blot ...
Gene rearrangement🔍
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 +6 
submitted by waterloo(71),

You can look at this question strategically.

  1. Southern blot --> so DNA.
  2. they are using an endonuclease to transfer bits, this doesn't really mean anything to you yet.
  3. cDNA is probing for single immunoglobulin-constant region.

That's important, because now you know they are looking at the constant region of immunoglobulin aka Fc of the heavy chain.

So why is this cDNA that encodes Fc, lighting up only once in other tissues, but the same cDNA lights up multiple times in lane 2 (bone marrow)? This is because, at the bone marrow random recombination of light or heavy chain occurs, and our cDNA is present in different random combinations with light chains. That specific cDNA sequence they used didn't change between the tissues or even within the bone marrow, and it will only bind to the one gene sequence. The fact it's lighting up multiple times tells you that the same sequence is present, your question is why so many times.

Also a side note, southern blots are time consuming, so labs use other methods to do the same thing (like PCR), but southern blots are still the best when it comes to checking immunoglobulin and T cell receptor gene rearrangement.




 +5 
submitted by sugaplum(324),
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varunmehru  in the question stem, they are asking about a constant region. VDJ rearrangement is for the variable. It doesn't make sense :( +3  
sallz  Both the constant (heavy chains) and the light chains undergo gene rearrangement. The heavy chain undergoes V(D)J random recombinations, while the light chain undergo VJ random recombinations. So gene rearrangement could work for both regions. +6  
azibird  The constant region does not undergo recombination. That's why it's called constant. It's just right next to the variable region though, so they get expressed together as one protein. That's why the constant-labeled DNA region is variable length here. +1  



 +2 
submitted by em_goldman(22),

I believe (pls correct me if I'm wrong) you would have similar Southern blot results seen in B cells undergoing somatic hypermutation, but that takes place in the secondary lymphoid tissue, not in the bone marrow.




 +1 
submitted by azibird(158),

I read this question as analyzing immunoglobulins in the bone marrow vs out circulating peripherally. I now realize this is wrong, and they are just analyzing the DNA of different cell population, like a hepatocyte for example, not immunoglobulins present in the liver.

With this cleared up, I can see how V(D)J recombination could make the original DNA section shorter. But how could V(D)J recombination possibly make the DNA section longer? There are bands on the gel that are higher molecular weight in the bone marrow. Recombination only shortens the DNA, someone please correct me.

Also why is there no band in the bone marrow that is the same size as peripheral bands? Surely the HSCs have not undergone recombination, where is their DNA???

azibird  Here is the figure from Kaplan Immunology. See how recombination only makes the sequence shorter? https://imgur.com/a/fI2jHfu Kaplan also says: "While heavy chain gene segments are undergoing recombination, the enzyme terminal deoxyribonucleotidyl transferase (Tdt) randomly inserts bases (without a template on the complementary strand) at the junctions of V, D, and J segments (N-nucleotide addition). The random addition of the nucleo- tide generates junctional diversity." However, this would not account for the equal stepwise lengthening of DNA, which is clearly from these recombinable units. +  



 +0 
submitted by benwhite_dotcom(465),
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ali  I still don’t understand this one. Could you provide a better explanation? +  
benwhite_dotcom  The cDNA tag is tagging a constant region common to immunoglobulins, so it normally only finds the one band corresponding to that particular gene (the bands travel different amounts due to their differing size/weight). In the bone marrow sample, that gene has rearranged itself, so the cDNA clone instead tags multiple different genes that are of different sizes on the gel (each one has that same constant region the cDNA is tagging, but with different stuff around it such that the restriction enzyme has cut it up differently). I’d be happy for someone to step in and do a better job on that explanation. +11  
em_goldman  A Southern blot starts by cutting DNA strands at a particular (short) site and running them through gel electrophoresis, so identical DNA sequences get cut at the same site and thus are the same length, so they are at the same place on the gel. If there's lots of different sequences, the restriction endonuclease (the scissors) cut the DNA at different places, leading to strands that were the same length originally but are now lots of different lengths -> different places on the gel. But how do you know this is the same gene, just with different mutations? The Southern blot uses a probe to look for a more specific (long) region of DNA that you know is in the target gene. So even though there are mutations causing the less-specific endonuclease to cut the DNA at different parts, the overall architecture of the gene is similar enough that the probe can bind, thus we know it's the same gene. (And in bone marrow WBCs, the mechanism here is genetic rearrangement.) +1  



 +0 
submitted by hello(302),

Echoing @waterloo's comment.

This Q is essentially asking "What is the function of Southern blots?"

Answer: The function of Souther blots is to check for BCR or TCR gene rearrangements.

See @waterloo's explanation for what is specifically going on as far as why there are different bands at different places. Again, the answer to this is "gene rearrangement" but @waterloo explains in with more detail.