Multiple Sex Pheromones and Receptors of a Mushroom-producing Fungus Elicit Mating in Yeast

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone–receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone–receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone–receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

Localization of Expression of Three Cold-Induced Genes, blt101, blt4.9, and blt14, in Different Tissues of the Crown and Developing Leaves of Cold-Acclimated Cultivated Barley1

Tissues expressing mRNAs of three cold-induced genes, blt101, blt14, and blt4.9, and a control gene, elongation factor 1α, were identified in the crown and immature leaves of cultivated barley (Hordeum vulgare L. cv Igri). Hardiness and tissue damage were assessed. blt101 and blt4.9 mRNAs were not detected in control plants; blt14 was expressed in control plants but only in the inner layers of the crown cortex. blt101 was expressed in many tissues of cold-acclimated plants but most strongly in the vascular-transition zone of the crown; blt14 was expressed only in the inner layers of the cortex and in cell layers partly surrounding vascular bundles in the vascular-transition zone; expression of blt4.9, which codes for a nonspecific lipid-transfer protein, was confined to the epidermis of the leaf and to the epidermis of the older parts of the crown. None of the cold-induced genes was expressed in the tunica, although the control gene was most strongly expressed there. Thus, the molecular aspects of acclimation differed markedly between tissues. Damage in the vascular-transition zone of the crown correlated closely with plant survival. Therefore, the strong expression of blt101 and blt14 in this zone may indicate a direct role in freezing tolerance of the crown.

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Molecular and cell biology aspects of plague

A 70-kb virulence plasmid (sometimes called pYV) enables Yersinia spp. to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. The Ysc apparatus forms a channel composed of 29 proteins. Of these, 10 have counterparts in almost every type III system. Secretion of some Yops requires the assistance, in the bacterial cytosol, of small individual chaperones called the Syc proteins. These chaperones act as bodyguards or secretion pilots for their partner Yop. Yop proteins fall into two categories. Some are intracellular effectors, whereas the others are “translocators” needed to deliver the effectors across the eukaryotic plasma membrane, into eukaryotic cells. The translocators (YopB, YopD, LcrV) form a pore of 16–23 Å in the eukaryotic cell plasma membrane. The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT. YopH is a powerful phosphotyrosine phosphatase playing an antiphagocytic role by dephosphorylating several focal adhesion proteins. YopE and YopT contribute to antiphagocytic effects by inactivating GTPases controlling cytoskeleton dynamics. YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription factor NF-κB. It also induces rapid apoptosis of macrophages. Less is known about the role of the phosphoserine kinase YopO/YpkA and YopM.

Molecular phylogeny analysis of fiddler crabs: test of the hypothesis of increasing behavioral complexity in evolution.

The current phylogenetic hypothesis for the evolution and biogeography of fiddler crabs relies on the assumption that complex behavioral traits are assumed to also be evolutionary derived. Indo-west Pacific fiddler crabs have simpler reproductive social behavior and are more marine and were thought to be ancestral to the more behaviorally complex and more terrestrial American species. It was also hypothesized that the evolution of more complex social and reproductive behavior was associated with the colonization of the higher intertidal zones. Our phylogenetic analysis, based upon a set of independent molecular characters, however, demonstrates how widely entrenched ideas about evolution and biogeography led to a reasonable, but apparently incorrect, conclusion about the evolutionary trends within this pantropical group of crustaceans. Species bearing the set of "derived traits" are phylogenetically ancestral, suggesting an alternative evolutionary scenario: the evolution of reproductive behavioral complexity in fiddler crabs may have arisen multiple times during their evolution. The evolution of behavioral complexity may have arisen by coopting of a series of other adaptations for high intertidal living and antipredator escape. A calibration of rates of molecular evolution from populations on either side of the Isthmus of Panama suggest a sequence divergence rate for 16S rRNA of 0.9% per million years. The divergence between the ancestral clade and derived forms is estimated to be approximately 22 million years ago, whereas the divergence between the American and Indo-west Pacific is estimated to be approximately 17 million years ago.

Molecular phylogeny of the extinct ground sloth Mylodon darwinii.

DNA was extracted from the remains of 35 ground sloths from various parts of North and South America. Two specimens of Mylodon darwinii, a species that went extinct at the end of the last glaciation, yielded amplifiable DNA. However, of the total DNA extracted, only approximately 1/1000 originated from the sloth, whereas a substantial part of the remainder was of bacterial and fungal origin. In spite of this, > 1100 bp of sloth mitochondrial rDNA sequences could be reconstructed from short amplification products. Phylogenetic analyses using homologous sequences from all extant edentate groups suggest that Mylodon darwinii was more closely related to the two-toed than the three-toed sloths and, thus, that an arboreal life-style has evolved at least twice among sloths. The divergence of Mylodon and the two-toed sloth furthermore allows a date for the radiation of armadillos, anteaters, and sloths to be estimated. This result shows that the edentates differ from other mammalian orders in that they contain lineages that diverged before the end of the Cretaceous Period.

Molecular evidence for multiple origins of woodiness and a New World biogeographic connection of the Macaronesian Island endemic Pericallis (Asteraceae: Senecioneae)

The prevalence of woody species in oceanic islands has attracted the attention of evolutionary biologists for more than a century. We used a phylogeny based on sequences of the internal-transcribed spacer region of nuclear ribosomal DNA to trace the evolution of woodiness in Pericallis (Asteraceae: Senecioneae), a genus endemic to the Macaronesian archipelagos of the Azores, Madeira, and Canaries. Our results show that woodiness in Pericallis originated independently at least twice in these islands, further weakening some previous hypotheses concerning the value of this character for tracing the continental ancestry of island endemics. The same data suggest that the origin of woodiness is correlated with ecological shifts from open to species-rich habitats and that the ancestor of Pericallis was an herbaceous species adapted to marginal habitats of the laurel forest. Our results also support Pericallis as closely related to New World genera of the tribe Senecioneae.

The phylogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus

Molecular differentiation between races or closely related species is often incongruent with the reproductive divergence of the taxa of interest. Shared ancient polymorphism and/or introgression during secondary contact may be responsible for the incongruence. At loci contributing to speciation, these two complications should be minimized (1, 2); hence, their variation may more faithfully reflect the history of the species' reproductive differentiation. In this study, we analyzed DNA polymorphism at the Odysseus (OdsH) locus of hybrid sterility between Drosophila mauritiana and Drosophila simulans and were able to verify such a prediction. Interestingly, DNA variation only a short distance away (1.8 kb) appears not to be influenced by the forces that shape the recent evolution of the OdsH coding region. This locus thus may represent a test case of inferring phylogeny of very closely related species.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Does behavior reflect phylogeny in swiftlets (Aves: Apodidae)? A test using cytochrome b mitochondrial DNA sequences.

Swiftlets are small insectivorous birds, many of which nest in caves and are known to echolocate. Due to a lack of distinguishing morphological characters, the taxonomy of swiftlets is primarily based on the presence or absence of echolocating ability, together with nest characters. To test the reliability of these behavioral characters, we constructed an independent phylogeny using cytochrome b mitochondrial DNA sequences from swiftlets and their relatives. This phylogeny is broadly consistent with the higher classification of swifts but does not support the monophyly of swiftlets. Echolocating swiftlets (Aerodramus) and the nonecholocating "giant swiftlet" (Hydrochous gigas) group together, but the remaining nonecholocating swiftlets belonging to Collocalia are not sister taxa to these swiftlets. While echolocation may be a synapomorphy of Aerodramus (perhaps secondarily lost in Hydrochous), no character of Aerodramus nests showed a statistically significant fit to the molecular phylogeny, indicating that nest characters are not phylogenetically reliable in this group.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Population dynamics of flaviviruses revealed by molecular phylogenies.

The phylogeny of 123 complete envelope gene sequences was reconstructed in order to understand the evolution of tick- and mosquito-borne flaviviruses. An analysis of phylogenetic tree structure reveals a continual and asymmetric branching process in the tick-borne flaviviruses, compared with an explosive radiation in the last 200 years in viruses transmitted by mosquitoes. The distinction between these two viral groups probably reflects differences in modes of dispersal, propagation, and changes in the size of host populations. The most serious implication of this work is that growing human populations are being exposed to an expanding range of increasingly diverse viral strains.

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.