Search

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene of Xenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 +/- 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 +/- 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.

We report the use of hydroxyl radical footprinting to analyze the interaction of distamycin and actinomycin with the 5S ribosomal RNA genes of Xenopus. There is a qualitative difference in the hydroxyl radical footprints of the two drugs. Distamycin gives a conventional (albeit high-resolution) footprint, while actinomycin does not protect DNA from hydroxyl radical attack, but instead induces discrete sites of hyperreactivity. We find concentration-dependent changes in the locations of distamycin binding sites on the somatic 5S gene of Xenopus borealis. A high-affinity site, containing a G.C base pair, is replaced at higher levels of bound drug by a periodic array of different lower affinity sites that coincide with the places where the minor groove of the DNA would face in toward a nucleosome core that is known to bind to the same sequence. These results suggest that distamycin recognizes potential binding sites more by the shape of the DNA than by the specific sequence that is contained in the site and that structures of many sequences are deformable to a shape that allows drug binding. We discuss the utility of hydroxyl radical footprinting of distamycin for investigating the underlying structure of DNA.

We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.

Bolus intravenous injection of an alpha-agonist is a widely accepted method used for studying baroreceptor function. However, the method is invasive, multiple baroreceptor regions are stimulated, and there are diverse direct effects of these pharmacologic agents, e.g., direct effects on the carotid sinus region. A recently described noninvasive neck suction technique may be highly specific for assessing the carotid sinus to sinoatrial node baroreflex. We compared neck suction-derived baroslopes with those obtained from the standard, invasive phenylephrine infusion method. These techniques were applied to 15 adult volunteers while awake and during 1.34 and 2% isoflurane anesthesia. The correlation coefficients between the two methods were 0.74 (P = 0.002) in awake subjects and 0.75 (P = 0.001) overall. The neck suction method of repetitive, ramped carotid stimuli yielded results that were qualitatively and quantitatively similar to those of the standard phenylephrine method. The neck suction method is simple, noninvasive, and can be repeated at frequent intervals. This method may be highly specific for determining carotid baroreceptor-cardiac reflex physiology in humans.