Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

Crystallization of membrane proteins: bovine rhodopsin.

The conditions for crystallization of the integral membrane protein bovine rhodopsin were determined. Crystals form over a wide range of protein concentration (0.4-15 mg ml-1) and pH (5.5-7.5) in detergent solutions of octyl-polyoxyethylene (o-POE) that contain inorganic salts as precipitants. Crystallization occurs in the detergent-rich phase under conditions of phase separation. This corroborates the results of experiments on the phase behavior of the detergent solution and on the distribution of rhodopsin between the salt-rich and detergent-rich phases formed at high salt concentrations. Crystals were needle shaped, and the best crystals were obtained by vapour diffusion, at room temperature, of a 2.5 mg ml-1 protein solution in a 1.5% (v/v) o-POE solution containing 1.5 M ammonium sulphate (pH 7.0) against unbuffered 2.85 M ammonium sulphate. The crystals thus produced had dimensions of approximately 70 microns x 70 microns x 1000 microns. These crystals were too small to allow X-ray diffraction studies of rhodopsin structure, but electron microscopic studies of negatively stained thin crystals allowed the definition of the projection belonging to the two-sided plane group p21, and showed unit cell parameters alpha (50 A), b (72 A), and gamma (90 degrees).