The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

Effects of gasteroid fruiting body morphology on diversification rates in three independent clades of fungi estimated using binary state speciation and extinction analysis

Gasteroid fungi include puffballs, stinkhorns, and other forms that produce their spores inside the fruiting body. Gasteroid taxa comprise about 8.4% of the Agaricomycetes (mushroom-forming fungi) and have evolved numerous times from nongasteroid ancestors, such as gilled mushrooms, polypores, and coral fungi, which produce spores on the surface of the fruiting body. Nongasteroid Agaricomycetes have a complex mechanism of forcible spore discharge that is lost in gasteroid lineages, making reversals to nongasteroid forms very unlikely. Our objective was to determine whether gasteromycetation affects the rate of diversification of lineages "trapped" in the gasteroid state. We assembled four datasets (the Sclerodermatineae, Boletales, Phallomycetidae, and Lycoperdaceae), representing unique origins of gasteroid fungi from nongasteroid ancestors and generated phylogenies using BEAST. Using the program Diversitree, we analyzed these phylogenies to estimate character-state-specific rates of speciation and extinction, and rates of transitions between nongasteroid and gasteroid forms. Most optimal models suggest that the net diversification rate of gasteroid forms exceeds that of nongasteroid forms, and that gasteroid forms will eventually come to predominate over nongasteroid forms in the clades in which they have arisen. The low frequency of gasteroid forms in the Agaricomycetes as a whole may reflect the recent origins of many gasteroid lineages.     

Fossil mushrooms from Miocene and Cretaceous ambers and the evolution of Homobasidiomycetes

Two species of fossil mushrooms that are similar to extant Tricholomataceae are described from Cretaceous and Miocene ambers. Archaeomarasmius leggetti gen. et sp. nov., from mid-Cretaceous amber of New Jersey, resembles the extant genera Marasmius and Marasmiellus. Two fruiting bodies of Archaeomarasmius were found. One consists of a complete pileus with stipe, and the other consists of a fragment of a pileus. The latter was accidentally exposed, and zxsubsequently was used for molecular systematics studies (attempts to amplify ribosomal DNA were unsuccessful) and electron microscopy. The spores are smooth and broadly elliptic with a distinct hilar appendage. Protomycena electra gen. et sp. nov., which is represented by a single complete fruiting body from Miocene amber of the Dominican Republic, is similar to the extant genus Mycena. Based on comparison to extant Marasmieae and Myceneae, Archaeomarasmius and Protomycena were probably saprophytes of leaf litter or wood debris. The poor phylogenetic resolution for extant homobasidiomycetes limits the inferences about divergence times of homobasidiomycete clades that can be drawn from Archaeomarasmius and Protomycena. The ages of these fossils lend support to hypotheses that the cosmopolitan distributions of certain mushroom taxa could be due to fragmentation of ancestral ranges via continental drift. Anatomical and molecular studies have suggested that there has been extensive convergence and parallelism in the evolution of homobasidiomycete fruiting body form. Nevertheless, the striking similarity of these fossils to extant forms suggests that in certain lineages homobasidiomycete macroevolution has also involved long periods during which there has been little morphological change.     

Phylogenetic evidence for horizontal transmission of group I introns in the nuclear ribosomal DNA of mushroom-forming fungi

Group I introns were discovered inserted at the same position in the nuclear small-subunit ribosomal DNA (nuc-ssu-rDNA) in several species of homobasidiomycetes (mushroom-forming fungi). Based on conserved intron sequences, a pair of intron-specific primers was designed for PCR amplification and sequencing of intron-containing rDNA repeats. Using the intron-specific primers together with flanking rDNA primers, a PCR assay was conducted to determine presence or absence of introns in 39 species of homobasidiomycetes. Introns were confined to the genera Panellus, Clavicorona, and Lentinellus. Phylogenetic analyses of nuc-ssu-rDNA and mitochondrial ssu-rDNA sequences suggest that Clavicorona and Lentinellus are closely related, but that Panellus is not closely related to these. The simplest explanation for the distribution of the introns is that they have been twice independently gained via horizontal transmission, once on the lineage leading to Panellus, and once on the lineage leading to Lentinellus and Clavicorona. BLAST searches using the introns from Panellus and Lentinellus as query sequences retrieved 16 other similar group I introns of nuc-ssu-rDNA and nuclear large-subunit rDNA (nuc-lsu-rDNA) from fungal and green algal hosts. Phylogenetic analyses of intron sequences suggest that the mushroom introns are monophyletic, and are nested within a clade that contains four other introns that insert at the same position as the mushroom introns, two from different groups of fungi and two from green algae. The distribution of host lineages and insertion sites among the introns suggests that horizontal and vertical transmission, homing, and transposition have been factors in intron evolution. As distinctive, heritable features of nuclear rDNAs in certain lineages, group I introns have promise as phylogenetic markers. Nevertheless, the possibility of horizontal transmission and homing also suggest that their use poses certain pitfalls.      

Phylogenetic relationships of Auriculoscypha based on ultrastructural and molecular studies

The phylogeny of Auriculoscypha anacardiicola, an associate of scale insects in India, is investigated using subcellular characters and MP and Bayesian analyses of combined nuLSU-rDNA, nuSSU-rDNA and 5.8S rDNA sequence data. It has simple septa with a pulley-wheel-shaped pore plug, which is diagnostic of phytoparasitic members of the Pucciniomycetes, and hyphal wall break on branching, a phenomenon unique to some simple septate heterobasidiomycetes. The septal ultrastructure of A. anacardiicola is similar to that of the genus Septobasidium. The close relationship to Septobasidium is also confirmed by rDNA sequence analyses. The polyphyletic nature of the order Platygloeales, noted in earlier studies, is evident from the present molecular analysis as well. The placement of Auriculoscypha in the Platygloeales can no longer be justified and both ultrastructural and molecular evidence strongly support the placement of Auriculoscypha in the Septobasidiales.     

Rainfall-driven sex-ratio genes in African buffalo suggested by correlations between Y-chromosomal haplotype frequencies and foetal sex ratio

Background  

The Y-chromosomal diversity in the African buffalo (Syncerus caffer) population of Kruger National Park (KNP) is characterized by rainfall-driven haplotype frequency shifts between year cohorts. Stable Y-chromosomal polymorphism is difficult to reconcile with haplotype frequency variations without assuming frequency-dependent selection or specific interactions in the population dynamics of X- and Y-chromosomal genes, since otherwise the fittest haplotype would inevitably sweep to fixation. Stable Y-chromosomal polymorphism due one of these factors only seems possible when there are Y-chromosomal distorters of an equal sex ratio, which act by negatively affecting X-gametes, or Y-chromosomal suppressors of a female-biased sex ratio. These sex-ratio (SR) genes modify (suppress) gamete transmission in their own favour at a fitness cost, allowing for stable polymorphism.