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These DNAs were all stored in the fridge (4 °C) in aqueous solution (10 mM Tris 1 mM EDTA pH 8.0) for two months before this gel was run. The DNAs in the 'ss' lanes were heated to 95°C for 10 min before loading to separate the strands and reveal the effects of any single-strand breaks.
These DNAs show no sign of degradation; compare to the original photo here. In particular, the DNA fragments from cells grown with limiting phosphate and 40 mM arsenate are actually slightly longer than the fragments from cells grown with limiting phosphate and no arsenate. (I don't think this difference is significant; the important point is that the fragments aren't any shorter.)
Because these large fragments typically migrate at the resolving limit of the gel, all I can say with confidence is that the fragments in all four preps are all significantly larger than 30 kb. This is the size range we expect for chromosomal DNA in a normal DNA prep. I don't have size standards for single-stranded DNA (I should have heated the lambda fragments but forgot to) so all I can say about the length distribution of single strands is that the four preps are all very similar.
This result tells is that DNAs from arsenate-grown cells are not undergoing degradation in storage due to slow hydrolysis of arsenate diester bonds in the DNA backbone, as suggested by an earlier anonymous commenter.
Have you done restriction cuts and run the results on a gel? That would be one of the first things that I would do.
ReplyDeleteWhy would you do that?
ReplyDeleteThis is genomic DNA, and a restriction digest would typically produce about 1000 poorly resolved fragments. And cutting the DNA into shorter fragments would reduce the ability to detect random breakage.
I really meant that it was one of the first things that I would do if were going to claim that As had replaced P more or less completely. If it were As-DNA, it might not cut (other nucleases could be tried as well; nuclease resistance would be worth noting). If it did cut, As-DNA might run differently than regular DNA (we can't tell from the gel in the original paper because it lacks restriction digests). The second point could be further pursued with Southern blots.
ReplyDeleteSince there are so many other lines of evidence showing that As has not replaced most of the P in the DNA backbone, a restriction digest didn't seem worth the trouble.
DeleteConsider it a belated comment on the original paper (and one which the reviewers should have made, since it's an afternoon's worth of work).
DeleteSure, there's no reason to believe that it's mostly As, but does it really matter whether it's zero or 1 in 1000? Either way, it's not a means of alleviating or significantly reducing the need for P.