Global phylogeography of the scalloped hammerhead shark (Sphyrna lewini).

Large marine fishes typically have little population genetic structure. The exceptions are associated with sedentary behaviour, disjunct distributions, or reproductive philopatry. Scalloped hammerhead sharks (Sphyrna lewini) incorporate the contrasting traits of oceanic habitat (usually associated with high dispersal) and possible fidelity to nursery grounds (for reproductive females). To evaluate the expectations of these contrasting behaviours, we examined the global genetic structure of S. lewini based on collections (n = 271 individuals) from 20 nursery areas. A 548-bp fragment of mitochondrial DNA control region revealed 22 polymorphic sites, 24 haplotypes, and three lineages distinguished by 2.56-3.77% sequence divergence. Coalescence analyses based on a provisional molecular clock indicate an origin in the Indo-West Pacific with late Pleistocene radiations into the central Pacific (Hawaii) and eastern Pacific (Central America), as well as recent interchange between oceans via southern Africa. Population subdivisions are strong (overall Phi(ST) = 0.749, P < 0.0001 and among oceans Phi(ST) = 0.598, P < 0.0098). Genetic discontinuity within oceans (Phi(ST) = 0.519, P < 0.0001) is primarily associated with oceanic barriers (migration across oceans M approximately 0), with much less structure along continental margins (M > 10). We conclude that nursery populations linked by continuous coastline have high connectivity, but that oceanic dispersal by females is rare. Although we cannot rule out philopatry to natal nurseries, oceanic barriers appear to have a much stronger influence on the genetic architecture of this species and may indicate a mechanism for recent evolutionary radiations in the genus Sphyrna.

Pax6 in the sepiolid squid Euprymna scolopes: evidence for a role in eye, sensory organ and brain development.

The cloning of a Pax6 orthologue from the sepiolid squid Euprymna scolopes and its developmental expression pattern are described. The data are consistent with the presence of a single gene encoding a protein with highly conserved DNA-binding paired and homeodomains. A detailed expression analysis by in situ hybridization and immunodetection revealed Pax6 mRNA and protein with predominantly nuclear localization in the developing eye, olfactory organ, brain lobes (optic lobe, olfactory lobe, peduncle lobe, superior frontal lobe and dorsal basal lobe), arms and mantle, suggestive of a role in eye, brain, and sensory organ development.

rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila.

The polynucleate myotubes of vertebrates and invertebrates form by fusion of myoblasts. We report the involvement of the Drosophila melanogaster Roughest (Rst) protein as a new membrane-spanning component in this process. Rst is strongly expressed in mesodermal tissues during embryogenesis, but rst null mutants display only subtle embryonic phenotypes. Evidence is presented that this is due to functional redundancy between Rst and its paralogue Kirre. Both are highly related single-pass transmembrane proteins with five extracellular immunoglobulin domains and three conserved motifs in the intracellular domain. The expression patterns of kirre and rst overlap during embryonic development in muscle founder cells. Simultaneous deletion of both genes causes an almost complete failure of fusion between muscle founder cells and fusion-competent myoblasts. This defect can be rescued by one copy of either gene. Moreover, Rst, like Kirre is a myoblast attractant.

Novel proteins interacting with the leucine-rich repeat domain of human flightless-I identified by the yeast two-hybrid system.

The flightless-I gene encodes a member of the gelsolin-like family of actin-binding proteins linked to a leucine-rich repeat (LRR) domain. It is required for cellularization during early embryogenesis and normal development of the indirect flight muscles in Drosophila melanogaster. Although the association between actin and the gelsolin-like domain of the human Flightless-I homologue (FLI) has been established, its biological role is unknown. The human FLI gene is mapped within the Smith-Magenis microdeletion region of chromosome 17. We report the identification of two related genes, LRRFIP1 and LRRFIP2, encoding proteins that interact with the LRR domain of human FLI using the yeast two-hybrid system. LRRFIP1 exhibits sequence identity with the TRIP RNA-binding protein and GCF-2 transcriptional repressor, which are also related to the murine FLAP-1 gene. LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1. LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue. A coiled-coil domain, conserved within each encoded protein, serves as a potential interaction motif for FLI LRR. The occurrence of multiple proteins able to interact with FLI within the same tissue suggests that they may compete for the same binding site. Sequencing and PCR-directed genomic analysis indicate that LRRFIP1 and LRRFIP2 are related genes that arose from gene duplication.

Data transferability from model organisms to human beings: insights from the functional genomics of the flightless region of Drosophila.

At what biological levels are data from single-celled organisms akin to a Rosetta stone for multicellular ones? To examine this question, we characterized a saturation-mutagenized 67-kb region of the Drosophila genome by gene deletions, transgenic rescues, phenotypic dissections, genomic and cDNA sequencing, bio-informatic analysis, reverse transcription-PCR studies, and evolutionary comparisons. Data analysis using cDNA/genomic DNA alignments and bio-informatic algorithms revealed 12 different predicted proteins, most of which are absent from bacterial databases, half of which are absent from Saccharomyces cerevisiae, and nearly all of which have relatives in Caenorhabditis elegans and Homo sapiens. Gene order is not evolutionarily conserved; the closest relatives of these genes are scattered throughout the yeast, nematode, and human genomes. Most gene expression is pleiotropic, and deletion studies reveal that a morphological phenotype is seldom observed when these genes are removed from the genome. These data pinpoint some general bottlenecks in functional genomics, and they reveal the acute emerging difficulties with data transferability above the levels of genes and proteins, especially with complex human phenotypes. At these higher levels the Rosetta stone analogy has almost no applicability. However, newer transgenic technologies in Drosophila and Mus, combined with coherency pattern analyses of gene networks, and synthetic neural modeling, offer insights into organismal function. We conclude that industrially scaled robogenomics in model organisms will have great impact if it can be realistically linked to epigenetic analyses of human variation and to phenotypic analyses of human diseases in different genetic backgrounds.

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron-exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans.

Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans.

Position-specific expression of the annulin protein during grasshopper embryogenesis.

Annulin, named for its annular expression in developing limb buds, is a approximately 100 kDa membrane-associated protein that is expressed in a complex and changing pattern during grasshopper embryogenesis. Its expression is dynamic along the developing midline and in the mesoderm, transient in neuroepithelial sheath cells around mitotic neuroblasts, and position-specific in circumferential stripes in each limb bud segment. Annulin expression begins along the midline of the embryo at the onset of gastrulation. Mesoderm cells express the protein as they migrate away from the midline as do new cells that come to lie at the midline. During neurogenesis, annulin expression disappears from many midline cells until only a specific subset of midline glial cells expresses high levels of the protein. Starting at the beginning of neurogenesis, sheath cells express annulin in correlation with the mitotic activity of the neuroblasts they surround.

Extraretinal photoreceptors in the brain of the crayfish Cherax destructor.

Two clusters of red-brown pigmented cell somata lie among other cell somata along the anterior margin of the cerebral ganglion in the crayfish Cherax destructor. Electron micrographs show these cells to contain round electron dense pigment granules and that the cell membranes of two or more adjacent cells fold together to form rhabdom-like structures. The pigmented cells specifically bind a monoclonal antibody against the major species of opsin in R1-7 retinula cells of the compound eye of Cherax. When stimulated with light, the pigmented cells respond with a receptor potential-like depolarization. The axons of the pigmented cells terminate in the neuropil of the protocerebral bridge, together with neuronal elements that label with antibodies against serotonin and substance P. We suggest that the brain photoreceptors of the crayfish are important in the entrainment of circadian rhythms.

The mutations previously designated as flightless-I3, flightless-O2 and standby are members of the W-2 lethal complementation group at the base of the X-chromosome of Drosophila melanogaster.

By using a well defined panel of chromosomal deficiencies, duplications and lethals, we have mapped three mutations causing flightlessness, flightless-I3, flightless-O2 and standby, to a single lethal complementation group (termed W-2) at the base of the X-chromosome of D. melanogaster. We also show that a fourth flightless mutation, termed grounded, previously mapped near to the base of the X-chromosome, is distal to the cytogenetic interval 18F to 20F. Mutants homozygous for the flightless-I3, flightless-O2 and standby mutations exhibit abnormalities of myofibrillar arrangements in the indirect flight muscles. They have distorted Z-bands and the myofibrils are often displaced from their normal parallel arrangement. These viable flightless mutations are all hypomorphs since the homozygous deficiency of the W-2 X-chromosomal region is lethal to the organism.

Haemocytes secrete basement membrane components in embryonic locusts.

Several monoclonal antibodies raised against a glycoprotein-enriched fraction of adult muscle membranes of Locusta migratoria selectively stain particles within haemocytes and basement membrane in developing locust embryos. Haemocytes containing immunoreactive particles are found associated with areas where basement membrane is being laid down. The underlying ectoderm does not show immunoreactivity. We conclude that haemocytes contribute to basement membrane formation in embryonic locusts.

The partial characterization of alcohol dehydrogenase null alleles from natural populations of Drosophila melanogaster.

Twenty-three alcohol dehydrogenase (ADH) putative null alleles extracted from four Tasmanian (Australia) populations of Drosophila melanogaster produce no ADH activity and are unable to form active heterodimers with either AdhF or AdhS. Twelve of these nulls were tested by enzyme-linked immunosorbent assay (ELISA) and did not produce any ADH cross-reacting material (CRM). The null homozygotes had similar, but slightly lower, mortalities on ethanol-supplemented media compared to an artificially induced null allele. Heterozygotes between the null alleles and standard AdhF and AdhS alleles had intermediate ADH activity and CRM levels.

Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity.

Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.

Complex glycoproteins associated with the detergent-resistant membrane matrix of the rhabdomeral microvilli of crayfish photoreceptors.

The lectin-binding properties of crayfish photoreceptor membrane were examined using isolated rhabdoms and frozen sections of whole retinas after permeabilization. Fluorescently-labelled concanavalin A (Con A) and wheat germ agglutinin (WGA) bind specifically to the rhabdoms, the accessory pigment cells, and the basement membrane of the retina in permeabilized frozen sections. Con A also binds to the clear zone in sectioned material. Peanut agglutinin (PA) binds strongly to domains associated with the periphery of rhabdoms. Intact isolated rhabdoms also bind Con A and WGA with high affinity. The major lectin-binding proteins of the photoreceptor membrane were characterized on nitrocellulose blots after SDS electrophoresis of purified photoreceptor membrane. Two major glycoproteins were found to bind a number of lectins. A 130 000 molecular weight peptide (GPII) specifically bound Con A, WGA, Dolichos biflorus agglutinin, (DBA) Ulex europaeus agglutinin (UEA) and PA, whereas the major 142 000 d protein (GPI) of the photoreceptor membrane bound only Con A and WGA implying a rather simple hexose chain consisting mainly of mannose and N-acetylglucosamine. Photoreceptor membrane vesicles, extracted with a high concentration of non-ionic detergent and centrifuged, show a specific enrichment of both proteins as well as actin and a 40 000 d component. Significant amounts of both glycoproteins are removed from the membrane residue by 0.6 M KI, even under conditions known to stabilize actin-based cytoskeletons. Both the stoichiometric amounts of these peptides in relation to the actin complement and their physicochemical properties are consonant with the hypothesis that one or both may be involved in linking a submembranous, actin-based cytoskeleton to the lipid bilayer. Rhodopsin, the major component of photoreceptor membrane, appears to be a glycoprotein by periodic acid-Schiff (PAS) staining methods but fails to bind significant amounts of lectins on Western blots. These findings are discussed in the light of post-translational processing and intracellular pathways for photoreceptor membrane components in the compound eye.

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Infiltration of compound eyes of crayfish, Cherax destructor, with the thiol protease inhibitor Ep-475 or with trifluoperazine prior to fixation for electron microscopy was found to stabilize an axial filament of 6-12 nm diam within each rhabdomeral microvillus of the photoreceptors. Rhabdoms isolated from retinal homogenates by sucrose gradient centrifugation under conditions that stabilize cytoskeletal material contained large amounts of a 42-kd polypeptide that co-migrated with insect flight muscle actin in one- and two-dimensional PAGE, inhibited pancreatic DNase l, and bound to vertebrate myosin. Vertebrate skeletal muscle actin added to retinal homogenates did not co-purify with rhabdoms, implying that actin was not a contaminant from nonmembranous structures. DNase l inhibition assays of detergent-lysed rhabdoms indicated the presence of large amounts of filamentous actin provided ATP was present. Monomeric actin in such preparations was completely polymerizable only after 90 min incubation with equimolar phalloidin. More than half of the actin present could be liberated from the membrane by sonication, indicating a loose association with the membrane. However, a large proportion of the actin was tightly bound to the rhabdomeral membrane, and washing sonicated membrane fractions with solutions of a range of ionic strengths and nonionic detergents failed to remove it. Antibodies to scallop actin only bound to frozen sections of rhabdoms after gentle permeabilization and very long incubation periods, probably because of steric hindrance and the hydrophobicity of the structure. The F-actin probe nitrobenzoxadiazol phallacidin bound to rhabdoms and labeled F-actin aggregates in other retinal components, but rhabdom fluorescence was not abolished by preincubation with phalloidin. The biochemical data indicate the existence of two distinct actin-based cytoskeletal systems, one being closely membrane associated. The other may possibly constitute the axial filament, although the evidence for this is equivocal.

Studies on the antigenic sites of actin: a comparative study of the immunogenic crossreactivity of invertebrate actins.

The structural homologies of invertebrate actins with cytoplasmic vertebrate actins have recently been substantiated by comparative sequence analyses. This suggests that cytoplasmic actin is the ancestral precursor of smooth and striated muscle actin in vertebrates. We have raised antibodies in rabbits against a number of invertebrate muscle actins and have characterized the antisera by means of the highly sensitive ELISA method, which allows quantitation of nanomolar amounts of actin. Despite the fact that the invertebrate actins examined are very similar in primary structure, our results indicate that antibodies raised against them clearly distinguish between only a few amino-acid substitutions, and that the immunoreactivities quantitatively reflect the genetic divergence of this ubiquitous conservative protein. Examination of several proteolytic fragments of scallop actin for immunoreactivity with the homologous antiserum suggests that the major antigenic sites of actin are located within the amino terminal region of the molecule, while a carboxy terminal fragment comprising residues 69-372 exhibits very weak crossreactivity. Immunoadsorption experiments further indicate that species-specific antibodies are directed to antigenic determinants in the N-terminal region. This finding is supported by an examination of the effects of chemical modifications to Tyr, His, Arg, and Cys residues on the immunoreactivity of actin. Interaction with DNAase I markedly decreases the immunoreactivity of actin. This is consonant with the finding that the amino terminal peptide comprising residues 1-207 inhibits DNAase I, whilst a tryptic fragment fails to bind to the enzyme. The interaction is abolished by EDTA and the removal of the tightly bound cation is accompanied by a conformational change, shown by shifts in circular dichroic spectra. The possible involvement of the amino terminal peptide of actin in cation binding is discussed.

Actin in cellular components of the basement membrane of the compound eye of a blowfly.

The so-called 'basement membrane' of arthropod compound eyes is known to be of heterogeneous origin (Odselius and Eloffson 1981). A major contribution in Diptera with open rhabdoms is provided by a pigmented component which lies at the basal end of the extracellular space of each ommatidium and fills it, the glial plug. Ancillary components consist of the expanded tips of cone cell processes. Each glial plug exhibits two distinct regions: ramifying processes extend into the extracellular space and contain numerous pigment granules, while proximally the cytoplasm is devoid of granules but packed with bundles of cross-linked microfilaments that bind the fluorescent F-actin probe NBD-phallacidin strongly and antibodies to scallop actin weakly. Cone cell expansions also contain microfilaments and exhibit the same binding properties. The proximal faces of the cells of the glial plugs and of the cone cell expansions are covered with a coarsely fibrillar extracellular matrix. Some actin bundles appear to be attached to the plasma membranes at their ends, although the reality of this arrangement is still in question. Cellular components of the basement membrane are bonded together by their extracellular matrices, so that collectively they provide a reinforced network that retains the retina. Bundles of axons from the photoreceptors and tracheae that supply the retina with tracheoles pass through the spaces in this network.