Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.     

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.