Biomedical subjects

D L Mielke

Publications and source records attributed to D L Mielke.

2 recordsLinked to original sources

PubMed1992

A modified TnphoA useful for single-stranded DNA sequencing.

The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.

Alkaline Phosphatase↗

PubMed1988

Secondary structural analyses of the nicotinic acetylcholine receptor as a test of molecular models.

Circular dichroism (CD) spectroscopy was used to determine that the secondary structure of purified nicotinic acetylcholine receptor (AChR) of Torpedo californica in both reconstituted vesicles and a cholate-solubilized state is, on average, 23% alpha-helix, 43% beta-sheet, 6% beta-turn, and 28% random coil. These data can serve to test models by placing limits on the particular types of secondary structural motifs which make up the receptor. A number of models proposed for the AChR are discussed in relation to the experimental data presented. Additionally, the protein in both vesicle and solubilized environments was exposed to an agonist, carbamylcholine, or a competitive antagonist, hexamethonium, to monitor net conformational changes upon ligand binding. The protein secondary structure was not changed upon solubilization, nor was any large net conformational change observed upon ligand binding, although small local or compensatory changes cannot be ruled out.

Animals↗