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DNA was isolated from wild type (Gal+) and mutant (Gal-) E. coli cells and separated by density gradient centrifugation technique. DNA from Gal- strain acquired a lower position. This indicates that the mutation is caused by:

(a) deletion (b) insertion (c) mis-sense mutation (d) point mutation

Me and my friends had an extended discussion on this question and we all agreed that since the Gal- strain acquired a lower position, it was obviously more dense than Gal+ strain. Now, one of them claimed that if the volume were to remain constant, then mass is directly proportional to density hence the insertion mutation should cause increase in mass corresponding to a similar increase in density. I am not convinced by this explanation. They also said that, "Keeping the volume constant is a necessity since the experiment would lose its meaning otherwise". I am curious to know how far these assertions are correct.

After doing some research on this topic on the internet, I found a paper related to this question :

Mutations caused by the insertion of genetic material into the galactose operon of Escherichia coli

The answer given by the organizing committee of the 2nd Indian National Biology Olympiad was (b) insertion.

What could be the possible explanation for such an occurrence? Me and my friends are still unable to grasp the logic behind it even after going through different papers and books.

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    $\begingroup$ Volume is not kept constant as adding an insertion will definitely increase volume. In fact the volume of the object does not even matter much in density gradient centrifugation. sigmaaldrich.com/technical-documents/articles/biofiles/…. Look at isopycnic centrifugation in this sheet. $\endgroup$ – Roni Saiba Oct 28 '20 at 6:17
  • $\begingroup$ @RoniSaiba Yes. I get your point. It makes sense. $\endgroup$ – Shishir Maharana Oct 28 '20 at 6:18
  • $\begingroup$ Would you want to win a competition run by people who are stupid enough to set this question? It’s complete and utter nonsense to imagine that the increase in mass of DNA caused by an insertion in a gene could have a significant effect on the sedimentation of bacterial DNA of 4 million base pairs, even if it is partially fragmented. $\endgroup$ – David Oct 28 '20 at 23:26
  • $\begingroup$ @David Sure, but the question is right: Four gal negative mutations, which affect the expression of the gal operon severely as described in the preceding paper (Saedler et al.), are characterized as insertions of DNA by CsCl density gradient centrifugation... from link.springer.com/article/10.1007/BF00268890 $\endgroup$ – Bipasha Nov 3 '20 at 16:38
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    $\begingroup$ @David Right, I understand, but please keep an open mind while looking at INBO questions- they are set by professors at HBCSE- the premier institution in the country for research in science. They can make mistakes, sure, but they are definitely not stupid. $\endgroup$ – Bipasha Nov 4 '20 at 1:54
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Now, one of them claimed that if the volume were to remain constant, then mass is directly proportional to density hence the insertion mutation should cause increase in mass corresponding to a similar increase in density.

I don't think that's quite how it works. You don't worry about the volumes being used. In those experiments (I don't know that anyone does them anymore) the DNA is in a solution of really salty water. Larger molecules get pushed more to the bottom of the tube, so the mutant DNA is larger, since it was close to the bottom.

https://embryo.asu.edu/pages/equilibrium-density-gradient-centrifugation-cesium-chloride-solutions-developed-matthew#:~:text=Density%20gradient%20centrifugation%20enables%20scientists,molecules%20based%20on%20density%20alone.

Thus, centrifugal force pushes the larger components of a mixture farther from the rotor and closer to the bottom of the tube.

Larger DNA means an insertion. Point mutations are not detectable with this method.

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