Asymmetry in reproductive isolation and its effect on directional mitochondrial introgression in the parapatric ground beetles <Emphasis Type="Italic">Carabus yamato</Emphasis> and <Emphasis Type="Italic">C. albrechti</Emphasis>

Speciation studies seek to clarify the origin of reproductive isolation, the various mechanisms working from mate recognition through postzygotic stages. Asymmetric effects of isolating barriers can result in asymmetrical gene introgression during interspecific hybridization. The flightless ground beetles Carabus yamato and C. albrechti are distributed parapatrically in Japan, showing repeated asymmetrical introgression of mitochondria from C. albrechti to C. yamato. This pattern suggests that reproductive isolation between these species is strong, but incomplete and asymmetric (i.e., weaker for the cross between a C. albrechti female and a C. yamato male). To test this hypothesis, we conducted interspecific mating experiments in the laboratory. The estimates of total reproductive isolation, which occurred mainly at the premating and postmating/prezygotic stages, were high (isolation index = 0.964 for C. yamato female × C. albrechti male and 0.886 for the reciprocal cross), supporting the hypothesis of strong, but incomplete isolation. However, the observed difference between the reciprocal crosses was not sufficiently large to conclude that it caused directional introgression of mitochondria. Instead, we found asymmetry in individual isolating barriers in the postmating/prezygotic stages that coincided with the prediction, perhaps resulting from morphological mismatch of heterospecific genitalia. Although this asymmetry was compensated for by an inverse asymmetry of isolation in the postzygotic stage, the contribution of these individual barriers to total isolation may change for our expectation when considering females mating with multiple heterospecific males.     

Evidence for an essential histidyl residue at the active site of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

Pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) from rabbit liver is inactivated by diethylpyrocarbonate in an all-or-none fashion with first order kinetics with respect to modifier concentration. The rate of inactivation increases with pH and reflects a group with a pKa of 7.5. Inactivated enzyme is in the holo form with intact FMN. Four histidyls and a cysteinyl residue are modified by excess reagent. The restoration of enzymatic activity by hydroxylamine, the spectrophotometric and colorimetric amino acid analyses, and our previous studies on cysteine modification (Tsuge, H., and McCormick, D.B. (1979) in Flavins and Flavoproteins (Yamano, T., and Yagi, K., eds) Japan Scientific Societies Press, Tokyo, in press) all suggest that inactivation occurs solely by modification of histidine. Analyses by kinetic and statistical methods indicate that three histidines are modified slowly and are not critical for activity, while one histidine is modified nine times more rapidly and accounts for the observed inactivation. Inactivated enzyme shows no significant perturbations in structure, as evidenced by absorption, CD, fluorescence, and gel filtration, but is unable to bind the product, pyridoxal 5'-phosphate. Furthermore, the substrate-competitive inhibitor, pyridoxal 5'-phosphate oxime, protects from inactivation. Hence, diethylpyrocarbonate inactivates this enzyme by modifying a crucial histidyl residue at the substrate/product-binding site.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

Interaction between D-amino acid oxidase and small molecules.

1. From the standpoint of monomer-dimer equilibrium of hog kidney D-amino acid oxidase [EC 1.4.3.3] and the interaction between the enzyme and small molecules, the effect of pH on the binding of p-aminobenzoate to the monomer and dimer of the enzyme was studied by kinetic methods and spectrophotometric titration. 2. The maximum binding number of p-aminobenzoate to the dimer is two molecules, and there is no interaction between the two active sites of the dimer (i.e., no cooperativity) over the range of pH from 6.5 to 10. 3. The affinity of the dimer for p-aminobenzoate is several times higher than that of the monomer at pH 6.5-10, and consequently p-aminobenzoate induces dimerization in the equilibrium state of D-amino acid oxidase. The interaction energy of two subunits of the dimer is stabilized by the binding of p-aminobenzoate by 1-2 kcal/mole over the pH range studied. 4. The binding sites of the quasi-substrate, p-aminobenzoate, in the dimer and the intersubunit binding site of the dimer are clearly different, because p-aminobenzoate induces dimerization of the enzyme. 5. The pK values of ionizing groups in the free monomer and the free dimer which participate in the binding of the competitive inhibitor, p-aminobenzoate, are approximately the same, 8.7, as determined from the pH dependence of the affinity of the inhibitor for the enzyme. Furthermore, no pK for the enzyme-inhibitor complex in the pH range 6.5-10 was observed. 6. There is no interaction between the two ionizing groups of the dimer during protonation-deprotonation, because a theoretical equation involving no cooperativity between the two ionizing groups in the dimer explains the results well.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.     

Latitudinal variation and coevolutionary diversification of sexually dimorphic traits in the false blister beetle Oedemera sexualis

Sexual traits are subject to evolutionary forces that maximize reproductive benefits and minimize survival costs, both of which can depend on environmental conditions. Latitude explains substantial variation in environmental conditions. However, little is known about the relationship between sexual trait variation and latitude, although body size often correlates with latitude. We examined latitudinal variation in male and female sexual traits in 22 populations of the false blister beetle Oedemera sexualis in the Japanese Archipelago. Males possess massive hind legs that function as a female‐grasping apparatus, while females possess slender hind legs that are used to dislodge mounting males. Morphometric analyses revealed that male and female body size (elytron length), length and width of the hind femur and tibia, and allometric slopes of these four hind leg dimensions differed significantly among populations. Of these, three traits showed latitudinal variation, namely, male hind femur was stouter; female hind tibia was slenderer, and female body was smaller at lower latitudes than at higher latitudes. Hind leg sizes and shapes, as measured by principal component analysis of these four hind leg dimensions in each sex, covaried significantly between sexes, suggesting coevolutionary diversification in sexual traits. Covariation between sexes was weaker when variation in these traits with latitude was removed. These results suggest that coevolutionary diversification between male and female sexual traits is mediated by environmental conditions that vary with latitude.     

Mechanical reproductive isolation via divergent genital morphology between Carabus insulicola and C. esakii with implications in species coexistence

The mechanical isolation hypothesis predicts that physical incompatibility between divergent reproductive morphologies hinders hybridization between populations. However, evidence for this hypothesis remains scarce. We examined this hypothesis using two parapatric carabid beetles, Carabus insulicola and C. esakii, which are of the subgenus Ohomopterus and exhibit a species-specific genital lock-and-key system. Our interspecific crossing experiment revealed that incompatibility of genital morphologies served as a strong postmating-prezygotic isolation barrier. This isolation was asymmetric: a decrease in female fitness was more costly in the cross with greater genitalic incompatibility between a C. esakii female and a C. insulicola male. These two species share a limited sympatric area, but the mechanism responsible for their coexistence is unclear given no evidence of premating isolation via male mate choice. A comparison of the present results with those of previous studies that quantified reproductive isolation between Ohomopterus species suggest that strong mechanical isolation via genitalic incompatibility plays a major role in species isolation, but that it may be less important in species coexistence.     

HGT-Gen: a tool for generating a phylogenetic tree with horizontal gene transfer

Horizontal gene transfer (HGT) is a common event in prokaryotic evolution. Therefore, it is very important to consider HGT in the study of molecular evolution of prokaryotes. This is true also for conducting computer simulations of their molecular phylogeny because HGT is known to be a serious disturbing factor for estimating their correct phylogeny. To the best of our knowledge, no existing computer program has generated a phylogenetic tree with HGT from an original phylogenetic tree. We developed a program called HGT-Gen that generates a phylogenetic tree with HGT on the basis of an original phylogenetic tree of a protein or gene. HGT-Gen converts an operational taxonomic unit or a clade from one place to another in a given phylogenetic tree. We have also devised an algorithm to compute the average length between any pair of branches in the tree. It defines and computes the relative evolutionary time to normalize evolutionary time for each lineage. The algorithm can generate an HGT between a pair of donor and acceptor lineages at the same evolutionary time. HGT-Gen is used with a sequence-generating program to evaluate the influence of HGT on the molecular phylogeny of prokaryotes in a computer simulation study.

Availability

The database is available for free at http://www.grl.shizuoka.ac.jp/˜thoriike/HGT-Gen.html