Biomedical subjects

D M John

Publications and source records attributed to D M John.

3 recordsLinked to original sources

PubMed2000

Tagging DNA mismatches by selective 2'-amine acylation.

BACKGROUND: Widespread characterization of genetic variation and disease at the gene-sequence level has inaugurated a new era in human biology. Techniques for the molecular analysis of these variations and their linkage with measurable phenotypes will profoundly affect diverse fields of biological chemistry and biology. RESULTS: A chemical tagging method has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligodeoxynucleotide probes in which one 2'-ribose position (-H) is substituted with an amine (-NH(2)) group. 2'-Amine-substituted nucleotides are specifically acylated by succinimidyl esters to form a 2'-amide product. The mutation detection method exploits our observation that 2'-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base-paired nucleotides. 2'-Amine acylation is governed primarily by local, rather than global, differences in nucleotide dynamics, such that site-specific tagging of DNA mismatches does not require discriminatory hybridization conditions to be determined. CONCLUSIONS: 2'-Amine mismatch tagging offers an approach for chemically interrogating the base-paired state of individual nucleotides in a hybridized duplex and for quantifying nucleicacid hybridization with single-base specificity.

Acylation↗

PubMed2000

van't Hoff enthalpies without baselines.

Analysis of thermal melting curves represents one important approach for evaluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differential melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (Tm) and van't Hoff enthalpy (deltaHvH). Calculated Tm and deltaHvH values are insensitive to the choice of pre- and post-transition baselines. Consequently, the method accurately computes Tm and deltaHvH for extremely truncated data sets, in the complete absence of baseline information, and for proteins with low melting temperatures, where the traditional direct approach routinely fails. Moreover, agreement between deltaHvH values obtained using points derived from pre- vs. post-transition data provide an independent method for detecting some classes of non-two-state transitions. Finally, fitting of the differential denaturation curve should prove useful for analysis of abbreviated data sets obtained from high throughput array analysis of protein stability.

Cytochrome c Group↗

PubMed1998

Mechanism for biotransformation of nonylphenol polyethoxylates to Xenoestrogens in Pseudomonas putida.

A strain of Pseudomonas putida isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEOx, where x is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEOav6, NPEOav9, and NPEOav20 (mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO2). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEOx. Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEOx mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEOx-degrading activity was inducible by substrate, and cell extracts of NPEOav9-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO8), producing sequentially, under either aerobic or anaerobic conditions, AEO7, AEO6, AEO5, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEOav9 or AEO8 under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEOx and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEOx-1, which then undergoes further cycles of the same reaction until x = 2.

Biotransformation↗