Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

The binding of nitrate to the human anion exchange protein (AE1) studied with 14N nuclear magnetic resonance

The binding of [14N]nitrate to the human erythrocyte anion transport protein, AE1, was studied using 14N nuclear magnetic resonance spectroscopy (14N-NMR). The line-width at half-height of the 14NO3- resonance increased in direct proportion to the concentration of erythrocyte ghost protein. Addition of the AE1 specific inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate markedly reduced this line-broadening, indicating that the broadening was predominantly due to a specific interaction between nitrate and AE1. The dependence of the AE1 specific line-broadening on nitrate concentration had a first-order dissociation constant KD of 6.9 +/- 0.9 mM. In contrast, Cl- interaction with AE1 studied by 35Cl-NMR showed a chloride concentration-dependent line-broadening with a KD of 74 +/- 10 mM, indicating that AE1 has a higher affinity for nitrate than for chloride. Bicarbonate and chloride were found to be competitive inhibitors of the AE1 specific 14NO3- line-broadening (94 +/- 6% and 101 +/- 3% inhibition, respectively). Based on the concentration dependence of inhibition and using a model of competitive inhibition, the KD of bicarbonate binding to AE1 was estimated to be 5.4 +/- 1.3 mM. Nitrate is a structural analog of bicarbonate, making the interaction of nitrate with AE1 a good model for the bicarbonate-AE1 interaction. The 14N-NMR nitrate binding assay, along with the 35Cl-NMR binding assay now in use, will provide a powerful tool for studying the structure of the AE1 binding site for both physiologic substrates, bicarbonate and chloride.