Genetic composition and diploid hybrid speciation of a high mountain pine, Pinus densata, native to the Tibetan plateau

Pinus densata has been suggested to have originated from hybridization events involving P. tabulaeformis and P. yunnanensis. In this study, allozyme differentiation at 12 loci was studied in 14 populations of P. tabulaeformis, P. densata, and P. yunnanensis from China. The observed genetic composition of P. densata supported the hybrid hypothesis and showed varying degrees of contribution from P. yunnanensis and P. tabulaeformis among its populations. These data, together with previous chloroplast DNA results, indicated different evolutionary histories among P. densata populations. To examine the possibility of ongoing hybridization among the three species, we analyzed patterns of linkage disequilibria between allozyme loci in ovule, pollen, and zygote pools. None of these tests suggested that there is significant ongoing gene exchange, implying that populations of P. densata have a stabilized hybrid nature. The normal fertility and high fecundity of P. densata indicate that this hybrid is maintained through sexual reproduction. P. densata represents an example of diploid hybrid speciation in an extreme ecological habitat that is both spatially and ecologically separated from that of its parents.     

Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

In order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1-3 interface, and in combination with a histidine conversion in the 1-2 interface (Trp110). The single replacements Met96His and Val115His in the 1-3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1-2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein-ligand complex formation were pH-sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin-biotin technology.     

Rational design of an active avidin monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.     

Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences

To study the inter- and infrafamilial phylogenetic relationships in the order Caryophyllales sensu lato (s.l.), ～930 base pairs of the matK plastid gene have been sequenced and analyzed for 127 taxa. In addition, these sequences have been combined with the rbcL plastid gene for 53 taxa and with the rbcL and atpB plastid genes as well as the nuclear 18S rDNA for 26 taxa to provide increased support for deeper branches. The red pigments of Corbichonia, Lophiocarpus, and Sarcobatus have been tested and shown to belong to the betacyanin class of compounds. Most taxa of the order are clearly grouped into two main clades (i.e., "core" and "noncore" Caryophyllales) which are, in turn, divided into well-defined subunits. Phytolaccaceae and Molluginaceae are polyphyletic, and Portulacaceae are paraphyletic, whereas Agdestidaceae, Barbeuiaceae, Petiveriaceae, and Sarcobataceae should be given familial recognition. Two additional lineages are potentially appropriate to be elevated to the family level in the future: the genera Lophiocarpus and Corbichonia form a well-supported clade on the basis of molecular and chemical evidence, and Limeum appears to be separated from other Molluginaceae based on both molecular and ultrastructural data.     

Solution properties of chitin in alkali

The solution properties of alpha-chitin dissolved in 2.77 M NaOH are discussed. Chitin samples in the weight-average molecular weight range 0.1 x 10(6) g/mol to 1.2 x 10(6) g/mol were prepared by heterogeneous acid hydrolysis of chitin. Dilute solution properties were measured by viscometry and light scattering. From dynamic light scattering data, relative similar size distributions of the chitin samples were obtained, except for the most degraded sample, which contained aggregates. Second virial coefficients in the range 1 to 2 x 10(-3) mL.mol.g(-2) indicated that 2.77 M NaOH is a good solvent to chitin. The Mark-Houwink-Sakurada equation and the relationship between the z-average radius of gyration (Rg) and the weight-average molecular weight (Mw) were determined to be [eta] = 0.10Mw0.68 (mL.g(-1)) and Rg = 0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm.     

Genetic basis of climatic adaptation in scots pine by bayesian quantitative trait locus analysis

We examined the genetic basis of large adaptive differences in timing of bud set and frost hardiness between natural populations of Scots pine. As a mapping population, we considered an "open-pollinated backcross" progeny by collecting seeds of a single F(1) tree (cross between trees from southern and northern Finland) growing in southern Finland. Due to the special features of the design (no marker information available on grandparents or the father), we applied a Bayesian quantitative trait locus (QTL) mapping method developed previously for outcrossed offspring. We found four potential QTL for timing of bud set and seven for frost hardiness. Bayesian analyses detected more QTL than ANOVA for frost hardiness, but the opposite was true for bud set. These QTL included alleles with rather large effects, and additionally smaller QTL were supported. The largest QTL for bud set date accounted for about a fourth of the mean difference between populations. Thus, natural selection during adaptation has resulted in selection of at least some alleles of rather large effect.     

Phylogenetics of flowering plants based on combined analysis of plastid atpB and rbcL gene sequences

Following (1) the large-scale molecular phylogeny of seed plants based on plastid rbcL gene sequences (published in 1993 by Chase et al., Ann. Missouri Bot. Gard. 80:528-580) and (2) the 18S nuclear phylogeny of flowering plants (published in 1997 by Soltis et al., Ann. Missouri Bot. Gard. 84:1-49), we present a phylogenetic analysis of flowering plants based on a second plastid gene, atpB, analyzed separately and in combination with rbcL sequences for 357 taxa. Despite some discrepancies, the atpB-based phylogenetic trees were highly congruent with those derived from the analysis of rbcL and 18S rDNA, and the combination of atpB and rbcL DNA sequences (comprising approximately 3000 base pairs) produced increased bootstrap support for many major sets of taxa. The angiosperms are divided into two major groups: noneudicots with inaperturate or uniaperturate pollen (monocots plus Laurales, Magnoliales, Piperales, Ceratophyllales, and Amborellaceae-Nymphaeaceae-Illiciaceae) and the eudicots with triaperturate pollen (particularly asterids and rosids). Based on rbcL alone and atpB/rbcL combined, the noneudicots (excluding Ceratophyllum) are monophyletic, whereas in the atpB trees they form a grade. Ceratophyllum is sister to the rest of angiosperms with rbcL alone and in the combined atpB/rbcL analysis, whereas with atpB alone, Amborellaceae, Nymphaeaceae, and Illiciaceae/Schisandraceae form a grade at the base of the angiosperms. The phylogenetic information at each codon position and the different types of substitutions (observed transitions and transversions in the trees vs. pairwise comparisons) were examined; taking into account their respective consistency and retention indices, we demonstrate that third-codon positions and transitions are the most useful characters in these phylogenetic reconstructions. This study further demonstrates that phylogenetic analysis of large matrices is feasible.     

Synthesis and in vitro/in vivo evaluation of novel oral N-alkyl- and N,N-dialkyl-carbamate esters of entacapone

Entacapone has a relatively low oral bioavailability which may, in part, be due to its low aqueous solubility at low pH and/or its hydrophilic character at neutral pH. Various novel N-alkyl and N,N-dialkyl carbamate esters of entacapone were synthesized as possible prodrugs of entacapone in order to increase its aqueous solubility at an acidic pH and to increase its lipophilicity at neutral pH. Oral bioavailability of entacapone and selected carbamate esters were investigated in rats. Both N-alkyl and N,N-dialkyl carbamate esters were relatively stable against chemical hydrolysis at pH 7.4 (t1/2 = 14.9-20.7 h), but hydrolyzed rapidly (t1/2 = 0.8-2.7 h) in human serum. However, in contrast to N-alkyl carbamates, N,N-dialkyl carbamates did not release entacapone in in vitro enzymatic hydrolysis (human serum) studies. N-Alkyl carbamates, 2a-c, showed increased aqueous solubility at pH 7.4, of which 2a and 2c also show increased aqueous solubility at pH 5.0, compared to entacapone. In addition to increased aqueous solubility, 2c showed increased lipophilicity at pH 7.4. However, two N-alkyl carbamates of entacapone did not increase the oral bioavailability of the parent drug in rats. Thus, it can be concluded that the relatively low lipophilicity of entacapone is not the cause of its low bioavailability.     

Ecological speciation in sympatric palms: 4. Demographic analyses support speciation of Howea in the face of high gene flow

The idea that populations must be geographically isolated (allopatric) to evolve into separate species has persisted for a long time. It is now clear that new species can also diverge despite ongoing genetic exchange, but few accepted cases of speciation in sympatry have held up when scrutinized using modern approaches. Here, we examined evidence for speciation of the Howea palms of Lord Howe Island, Australia, in light of new genomic data. We used coalescence‐based demographic models combined with double digest restriction site associated DNA sequencing of multiple individuals and provide support for previous claims by Savolainen et al. that speciation in Howea did occur in the face of gene flow.