APPENDIX B - Letter for sixth discussion
What definitive experiments can be done to test the basic predictions of the model?
The model suggests three experiments.
- Since the substrate for the micrococcal nuclease [or, equivalently, 1, 10-phenanthroline copper (I)] is the high-energy beta-structural element within the center of a premelton, it follows that the probability a premelton exists within a given DNA region determines its level of sensitivity to enzymatic hydrolysis.
Although, in principle, premeltons should be able to form within every region of DNA, the existence of multiple micrococcal nuclease hypersensitive sites along DNA suggests they have an enhanced probability to exist at particular regions of DNA.
Since ethidium “pins” the high-energy beta-structural element within the center of the premelton, it is expected to interfere with the ability of the micrococcal nuclease to bind to and to cleave these sites. Hypersensitive sites, in particular, should be extremely sensitive to the presence of ethidium, disappearing readily with increasing concentrations of ethidium.
Ethidium should therefore be used to probe the structure and dynamics of the premelton as a competitive inhibitor of the reaction.
[Note: Since the high- and low-energy beta-structural elements alternate within the centers of premeltons, a similar effect is predicted to occur with irehdiamine A and other related steroidal diamines].
- The model predicts micrococcal nuclease hypersensitive sites to be present in long chain DNA molecules, but absent in shorter chain oligonucleotide duplexes.
Again, the histone gene cluster in D. melanogaster would be an ideal model system to study this. The general plan would be to synthesize a series of DNA molecules containing the micrococcal nuclease hypersensitive site (located at, say, the 5' end of the H4 gene), surrounded by increasing lengths of the histone gene cluster sequence on either side.
Should a gradual enhancement in sensitivity to cleavage by the micrococcal nuclease occur, this can be detected by gel electrophoresis. Ideally, two new fragments will appear. These can be purified, and their nucleotide sequence determined.
Such an experiment would provide crucial evidence to understand whether a nuclease hypersensitive site requires the presence of a more extended structure on either side. A premelton is expected to span a minimum of about fifty base pairs from end to end. To observe a micrococcal nuclease hypersensitive site, it may be necessary to create a DNA fragment one hundred or more base pairs long.
- Finally, the existence of the high-energy beta-DNA form “pinned” by ethidium can be established by determining the structure of an ethidium-oligonucleotide complex by X-ray crystallography. A similar structure determination of the low-energy beta-DNA form “pinned” by irehdiamine should also be determined.
As many of you realize, the Encyclopedia of Nonlinear Science (Routledge, 2005) has appeared this past year. The volume is a major reference source in this growing interdisciplinary area. As Al has edited this volume, I have asked him to comment about the current discussion. All of you are invited to join in on the more general discussion, which follows.
|<< Previous ¦