Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.     

Hyperspectral Remote Sensing of Canopy Biodiversity in Hawaiian Lowland Rainforests

Mapping biological diversity is a high priority for conservation research, management and policy development, but few studies have provided diversity data at high spatial resolution from remote sensing. We used airborne imaging spectroscopy to map woody vascular plant species richness in lowland tropical forest ecosystems in Hawai’i. Hyperspectral signatures spanning the 400–2,500 nm wavelength range acquired by the NASA Airborne Visible and Infrared Imaging Spectrometer (AVIRIS) were analyzed at 17 forest sites with species richness values ranging from 1 to 17 species per 0.1–0.3 ha. Spatial variation (range) in the shape of the AVIRIS spectra (derivative reflectance) in wavelength regions associated with upper-canopy pigments, water, and nitrogen content were well correlated with species richness across field sites. An analysis of leaf chlorophyll, water, and nitrogen content within and across species suggested that increasing spectral diversity was linked to increasing species richness by way of increasing biochemical diversity. A linear regression analysis showed that species richness was predicted by a combination of four biochemically-distinct wavelength observations centered at 530, 720, 1,201, and 1,523 nm (r ² = 0.85, p < 0.01). This relationship was used to map species richness at approximately 0.1 ha resolution in lowland forest reserves throughout the study region. Future remote sensing studies of biodiversity will benefit from explicitly connecting chemical and physical properties of the organisms to remotely sensed data.     

Activators of phosphorylase kinase alter the cross-linking of its catalytic subunit to the C-terminal one-sixth of its regulatory alpha subunit

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.     

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.     

Donor-acceptor tetrahydrochrysenes, inherently fluorescent, high-affinity ligands for the estrogen receptor: binding and fluorescence characteristics and fluorometric assay of receptor.

We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-11 positions, is required for the highest relative binding affinity (RBA), and the trans-5,11-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NO2) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (phi f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (approximately 10(-9) M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal-dextran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when bound to ER, or when bound to non-receptor proteins. It is also the first demonstration of ER assay by fluorescence under equilibrium conditions.     

Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

How many conservation units are there for the endangered grassland earless dragons?

Species are the most commonly recognised unit for conservation management, yet significant variation can exist below the level of taxonomic recognition and there is a lack of consensus around how a species might be defined. This definition has particular relevance when species designations are used to apportion conservation effort and when definitions might be made through legislation. Here, we use microsatellite DNA analyses to test the proposition that the last remaining populations of the endangered grassland earless dragon (Tympanocryptis pinguicolla) harbour substantial cryptic genetic variation. Our study provides strong evidence that long historical isolation and the recent impacts of urbanization, have led to genetic differentiation in microsatellite DNA allele frequencies and high numbers of private alleles among three genetic clusters. This differentiation is partially concordant with previous mitochondrial DNA analyses, which show the two regions (Canberra and Monaro) where this species exists, to be reciprocally monophyletic, but differs through the identification of a third genetic cluster that splits a northern Canberra cluster from that of southern Canberra. Our data also identify a stark contrast in population genetic structure between clusters such that high levels of genetic structure are evident in the highly urbanised Canberra region but not in the largely rural Monaro region. We conclude that this species, like many reptiles, harbours considerable cryptic variation and currently comprises three distinct and discrete units. These units could be classified as separate species for the purpose of conservation under the relevant Australian and international Acts drawing management appropriate to that status.