PubMed Health⌕ Search

Biomedical subjects

Hugh M Robertson

Publications and source records attributed to Hugh M Robertson.

17 recordsLinked to original sources
PubMed2006

Functional CpG methylation system in a social insect.

DNA methylation systems are well characterized in vertebrates, but methylation in Drosophila melanogaster and other invertebrates remains controversial. Using the recently sequenced honey bee genome, we present a bioinformatic, molecular, and biochemical characterization of a functional DNA methylation system in an insect. We report on catalytically active orthologs of the vertebrate DNA methyltransferases Dnmt1 and Dnmt3a and b, two isoforms that contain a methyl-DNA binding domain, genomic 5-methyl-deoxycytosine, and CpG-methylated genes. The honey bee provides an opportunity to study the roles of methylation in social contexts.

Amino Acid Sequence↗
PubMed2006

The chemoreceptor superfamily in the honey bee, Apis mellifera: expansion of the odorant, but not gustatory, receptor family.

The honey bee genome sequence reveals a remarkable expansion of the insect odorant receptor (Or) family relative to the repertoires of the flies Drosophila melanogaster and Anopheles gambiae, which have 62 and 79 Ors respectively. A total of 170 Or genes were annotated in the bee, of which seven are pseudogenes. These constitute five bee-specific subfamilies in an insect Or family tree, one of which has expanded to a total of 157 genes encoding proteins with 15%-99% amino acid identity. Most of the Or genes are in tandem arrays, including one with 60 genes. This bee-specific expansion of the Or repertoire presumably underlies their remarkable olfactory abilities, including perception of several pheromone blends, kin recognition signals, and diverse floral odors. The number of Apis mellifera Ors is approximately equal to the number of glomeruli in the bee antennal lobe (160-170), consistent with a general one-receptor/one-neuron/one-glomerulus relationship. The bee genome encodes just 10 gustatory receptors (Grs) compared with the D. melanogaster and A. gambiae repertoires of 68 and 76 Grs, respectively. A lack of Gr gene family expansion primarily accounts for this difference. A nurturing hive environment and a mutualistic relationship with plants may explain the lack of Gr family expansion. The Or family is the most dramatic example of gene family expansion in the bee genome, and characterizing their caste- and sex-specific gene expression may provide clues to their specific roles in detection of pheromone, kin, and floral odors.

Animals↗
PubMed2006

Canonical TTAGG-repeat telomeres and telomerase in the honey bee, Apis mellifera.

The draft assembly of the honey bee Apis mellifera genome sequence reveals that the 17 centromeric-distal telomeres are of a simple, shared, and canonical structure, with 3-4 kb of a unique subtelomeric sequence, followed by several kilobases of TTAGG or variant telomeric repeats. This simple subtelomeric structure differs from the centromeric-proximal telomeres on the short arms of the 15 acrocentric chromosomes, which are apparently composed primarily of the 176-bp AluI tandem repeat. This dichotomy between the distal and proximal telomeres may involve differential participation of the telomeres of the 15 acrocentric chromosomes in the Rabl configuration after mitosis and the chromosome bouquet in meiotic prophase I. As expected from the presence of canonical TTAGG telomeric repeats, we identified a candidate telomerase gene in the bee, as well as the silkmoth Bombyx mori and the flour beetle Tribolium castaneum.

Animals↗
PubMed2006

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock.

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

Amino Acid Motifs↗
PubMed2005

Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

Animals↗
PubMed2005

Adaptive evolution in the SRZ chemoreceptor families of Caenorhabditis elegans and Caenorhabditis briggsae.

We investigated the possibility of positive selection acting on members of the putative seven-pass chemoreceptor superfamily in Caenorhabditis elegans, which comprises approximately 1,300 genes encoding seven-pass G protein-coupled receptors (GPCRs). Using a maximum-likelihood approach, we conducted statistical tests for evidence of codon sites where the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (d(N)/d(S)) was >1. Evidence for positive selection was found only for the srz family, about which virtually nothing specific is known. We extended the annotation of the srz gene family, establishing gene models for 60 srz genes in C. elegans and 28 srz genes in Caenorhabditis briggsae. d(N)/d(S) ratios varied dramatically in different regions of the SRZ proteins, peaking in predicted extracellular regions. These regions included 23 sites where evidence of positive selection was highly significant, corresponding remarkably well with regions implicated in ligand binding in other GPCR family members. We interpret these results as indicating that the srz family is under positive selection, probably driven by ligand binding.

Adaptation, Physiological↗
PubMed2005

Drosophila N-cadherin functions in the first stage of the two-stage layer-selection process of R7 photoreceptor afferents.

Visual information received from the three types of photoreceptor neurons (R1-R6, R7 and R8) in the fly compound eyes converges to the external part of the medulla neuropil (M1-M6 layers) in a layer-specific fashion: R7 and R8 axons terminate at the M6 and M3 layers, respectively, whereas lamina neurons (L1-L5) relay R1-R6 to multiple medulla layers (M1-M5). Here, we show that during development, R7 and R8 neurons establish layer-specific projections in two separate stages: during the first stage, R7 and R8 axons sequentially target to the R7- and R8-temporary layers, respectively; and at the second stage, R7 and R8 growth cones progress synchronously to their destined layers. Using a set of mutations that delete different afferent subsets or alter R7 connectivity, we defined the mechanism of layer selection. We observed that R8, R7 and L1-L5 afferents target to their temporary layers independently, suggesting that afferent-target, but not afferent-afferent, interactions dictate the targeting specificity. N-cadherin is required in the first stage for R7 growth cones to reach and remain in the R7-temporary layer. The Ncad gene contains three pairs of alternatively spliced exons and encodes 12 isoforms. However, expressing a single Ncad isoform in Ncad mutant R7s is sufficient to rescue mistargeting phenotypes. Furthermore, Ncad isoforms mediate promiscuous heterophilic interactions in an in vitro cell-aggregation assay. We propose that Ncad isoforms do not form an adhesion code; rather, they provide permissive adhesion between R7 growth cones and their temporary targets.

Alternative Splicing↗
PubMed2003

A new member of the GM130 golgin subfamily is expressed in the optic lobe anlagen of the metamorphosing brain of Manduca sexta.

During metamorphosis of the insect brain, the optic lobe anlagen generate the proliferation centers for the visual cortices. We show here that, in the moth Manduca sexta, an 80 kDa Golgi complex protein (Ms-golgin80) is abundantly expressed in the cytoplasm of neuroblasts and ganglion mother cells in the optic lobe anlagen and proliferation centers. The predicted amino acid sequence for Ms-golgin80 is similar to that of several members of the GM130 subfamily of Golgi-associated proteins, including rat GM130 and human golgin-95. Homologs of Ms-golgin80 from Drosophila melanogaster, Caenorhabditis elegans, andBrugia malayi were identified through homology sequence search. Sequence similarities are present in three regions: the N-terminus, an internal domain of 89 amino acids, and another domain of 89 amino acids near the C-terminus. Structural similarities further suggest that these molecules play the same cellular role as GM130. GM130 is involved in the docking and fusion of coatomer (COP I) coated vesicles to the Golgi membranes; it also regulates the fragmentation and subsequent reassembly of the Golgi complex during mitosis. Abundant expression of Ms-golgin80 in neuroblasts and ganglion mother cells and its reduced expression in the neuronal progeny of these cells suggest that this protein may be involved in the maintenance of the proliferative state.

Amino Acid Sequence↗
PubMed2003

Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster.

The insect chemoreceptor superfamily in Drosophila melanogaster is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes through alternative splicing. We include two previously undescribed Or genes and two previously undescribed Gr genes; two previously predicted Or genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the Gr family, and the Or family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the Drosophila genome, with some relatively recently diverged genes still clustered in the genome. The Gr5a gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has Gr61a, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods.

Alternative Splicing↗
PubMed2003

Expression patterns of odorant-binding proteins in antennae of the moth Manduca sexta.

Monoclonal antibodies (MAbs) were generated to six recombinant proteins (odorant-binding proteins; OBPs) of Manduca sexta. The specificity of each MAb was demonstrated by labeling six immunoblots, each of which contained samples of all six recombinant OBPs. The expression patterns of the six OBPs could be grouped into three classes: (1) one (GOBP1) was expressed in sensilla located throughout each annulus; (2) two (ABPX and ABP2) were expressed in the long sensilla trichoidea bordering a zone that was arranged as an arch on the periphery of each annulus; (3) three (PBP2, PBP3, and GOBP2) were expressed in shorter sensilla occupying a wedge-shaped mid-annular zone of each annulus. In female antennae, sensilla expressing these OBPs were intermixed, and the distinct zonation observed in the male antenna was absent. In males, PBP2 was co-expressed in exactly the same cells of the mid-annular zone as those expressing PBP3 and most of the same cells expressing GOBP2, although its expression overlapped with no or only a few sensilla expressing OBPs of class 1 (GOBP1) or class 2 (ABPX, ABP2). This overlap of expression or lack of overlap between PBP2 and the other OBPs for male antennae was mirrored in female antennae. In view of the restricted spatial expression of OBPs within an annulus and the diversity of possible dimeric combinations of OBPs that arises from the co-expression of multiple OBPs in a given sensillum, OBPs could contribute to the specificity of the olfactory responses of insects.

Animals↗
PubMed2003

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer.

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.

Amino Acid Sequence↗
PubMed2003

Neutral evolution of ten types of mariner transposons in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae.

Ten types of mariner transposable elements (232 individual sequences) are present in the completed genomic DNA sequence of Caenorhabditis elegans and the partial sequence of Caenorhabditis briggsae. We analyze these replicated instances of mariner evolution and find that elements of a type have evolved within their genomes under no selection on their transposase genes. Seven of the ten reconstructed ancestral mariners carry defective transposase genes. Selection has acted during the divergence of some ancestral elements. The neutrally-evolving mariners are used to analyze the pattern of molecular evolution in Caenorhabditis. There is a significant mutational bias against transversions and significant variation in rates of change across sites. Deletions accumulate at a rate of 0.034 events/bp per substitution/site, with an average size of 166 bp (173 gaps observed). Deletions appear to obliterate preexisting deletions over time, creating larger gaps. Insertions accumulate at a rate of 0.019 events/bp per substitution/site, with an average size of 151 bp (61 events). Although the rate of deletion is lower than most estimates in other species, the large size of deletions causes rapid elimination of neutral DNA: a mariner's "half-life" (the time by which half an element's sequence should have been deleted) is approximately 0.1 subsitutions/site. This high rate of DNA deletion may explain the compact nature of the nematode genome.

Amino Acid Sequence↗
PubMed2003

PCR-RFLP identification of Diptera (Calliphoridae, Muscidae and Sarcophagidae)--a generally applicable method.

A simple, rapid method using restriction fragment length polymorphisms (RFLPs) within the internal transcribed spacer (ITS) regions of the ribosomal DNA gene repeat allows identification of insects and other organisms. We used the method to identify the morphologically similar Diptera larvae that are important in forensic entomology for estimating the time and location of death. Polymerase chain reaction (PCR) was used to amplify a region from the 18S to the 28S rRNA genes. The ITS1 and ITS2 regions provided variation between species and homogeneity within species, with the exception of Cochliomya macellaria. Combinations of the restriction enzymes DdeI, HinfI and Sau3AI provided diagnostic bands for identification of the ten species from three families of Diptera (Calliphoridae, Muscidae and Sarcophagidae).

Animals↗
PubMed2002

G protein-coupled receptors in Anopheles gambiae.

We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

Alternative Splicing↗
PubMed2002

Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.]

Animals↗
PubMed2002

Assessment of parasitism of house fly and stable fly (Diptera: Muscidae) pupae by pteromalid (Hymenoptera: Pteromalidae) parasitoids using a polymerase chain reaction assay.

The internal transcribed spacer (ITS) regions of the ribosomal DNA of house flies, Musca domestica L., the stable flies, Stomoxys calcitrans (L.), and four parasitoid species in the genus Muscidifurax (Hymenoptera: Pteromalidae) were characterized to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid parasitism of pupae of the house fly and stable fly. Two parasitoid-specific primers were designed to anneal to the 5' end of the 5.8S rRNA gene in the parasitoid species. When paired with a universal primer at the 3' end of the 18S rRNA, the primers amplified the target ITS1 region in 10 pteromalid species. PCR allowed detection of parasitoid DNA within 24 h after females of Spalangia endius Walker oviposited into house fly puparia. PCR failed to amplify parasitoid DNA or detect parasitism in puparia that were exposed to parasitoid oviposition, allowed to develop 7 d, then killed by freezing and held at 20-24 degrees C for 4 d to allow DNA degradation. Digestion of the PCR products with restriction enzymes produced restriction fragment length polymorphisms that allowed identification of individual parasitoid species. Significantly greater levels of parasitism (P < 0.05) were detected by PCR for two of the five field collection dates in 1997. On the dates when PCR detected higher levels of parasitism than estimates provided by emergence of adult insects from samples taken at Feedlot M in 1997, more than 65% of all puparia in the emergence samples failed to produce an adult insect. Three puparia collected in 1997 produced double PCR bands that corresponded to PCR band sizes of Muscidifurax spp. and Spalangia sp., possibly indicating multiple parasitism or hyperparasitism.

Animals↗