Biomedical subjects

David L Baillie

Publications and source records attributed to David L Baillie.

At least 19 recordsLinked to original sources

PubMed2006

Comparative genomics and adaptive selection of the ATP-binding-cassette gene family in caenorhabditis species.

ABC transporters constitute one of the largest gene families in all species. They are mostly involved in transport of substrates across membranes. We have previously demonstrated that the Caenorhabditis elegans ABC family shows poor one-to-one gene orthology with other distant model organisms. To address the evolution dynamics of this gene family among closely related species, we carried out a comparative analysis of the ABC family among the three nematode species C. elegans, C. briggsae, and C. remanei. In contrast to the previous observations, the majority of ABC genes in the three species were found in orthologous trios, including many tandemly duplicated ABC genes, indicating that the gene duplication took place before speciation. Species-specific expansions of ABC members are rare and mostly observed in subfamilies A and B. C. briggsae and C. remanei orthologous ABC genes tend to cluster on trees, with those of C. elegans as an outgroup, consistent with their proposed species phylogeny. Comparison of intron/exon structures of the highly conserved ABCE subfamily members also indicates a closer relationship between C. briggsae and C. remanei than between either of these species and C. elegans. A comparison between insect and mammalian species indicates lineage-specific duplications or deletions of ABC genes, while the family size remains relatively constant. Sites undergoing positive selection within subfamily D, which are implicated in very-long-chain fatty acid transport, were identified. The evolution of these sites might be driven by the changes in food source with time.

ATP-Binding Cassette Transporters↗

PubMed2006

The ELT-2 GATA-factor and the global regulation of transcription in the C. elegans intestine.

A SAGE library was prepared from hand-dissected intestines from adult Caenorhabditis elegans, allowing the identification of >4000 intestinally-expressed genes; this gene inventory provides fundamental information for understanding intestine function, structure and development. Intestinally-expressed genes fall into two broad classes: widely-expressed "housekeeping" genes and genes that are either intestine-specific or significantly intestine-enriched. Within this latter class of genes, we identified a subset of highly-expressed highly-validated genes that are expressed either exclusively or primarily in the intestine. Over half of the encoded proteins are candidates for secretion into the intestinal lumen to hydrolyze the bacterial food (e.g. lysozymes, amoebapores, lipases and especially proteases). The promoters of this subset of intestine-specific/intestine-enriched genes were analyzed computationally, using both a word-counting method (RSAT oligo-analysis) and a method based on Gibbs sampling (MotifSampler). Both methods returned the same over-represented site, namely an extended GATA-related sequence of the general form AHTGATAARR, which agrees with experimentally determined cis-acting control sequences found in intestine genes over the past 20 years. All promoters in the subset contain such a site, compared to <5% for control promoters; moreover, our analysis suggests that the majority (perhaps all) of genes expressed exclusively or primarily in the worm intestine are likely to contain such a site in their promoters. There are three zinc-finger GATA-type factors that are candidates to bind this extended GATA site in the differentiating C. elegans intestine: ELT-2, ELT-4 and ELT-7. All evidence points to ELT-2 being the most important of the three. We show that worms in which both the elt-4 and the elt-7 genes have been deleted from the genome are essentially wildtype, demonstrating that ELT-2 provides all essential GATA-factor functions in the intestine. The SAGE analysis also identifies more than a hundred other transcription factors in the adult intestine but few show an RNAi-induced loss-of-function phenotype and none (other than ELT-2) show a phenotype primarily in the intestine. We thus propose a simple model in which the ELT-2 GATA factor directly participates in the transcription of all intestine-specific/intestine-enriched genes, from the early embryo through to the dying adult. Other intestinal transcription factors would thus modulate the action of ELT-2, depending on the worm's nutritional and physiological needs.

Animals↗

PubMed2006

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport.

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.

Animals↗

PubMed2006

Caenorhabditis elegans dpy-14: an essential collagen gene with unique expression profile and physiological roles in early development.

We describe the molecular characterisation of Caenorhabditis elegans dpy-14, a gene encoding an essential cuticular collagen annotated as col-59. Expression of dpy-14 starts at the 16 E cell stage, making it the earliest-expressing collagen reported to date. SAGE data and dpy-14 promoter::GFP reporter constructs indicate that the gene is transcribed mainly during embryogenesis, specifically in ciliated neurons and hypoderm. Water permeability assays and lectin staining showed that a mutation in the DPY-14 collagen results in defects in the channels of the amphids, which are a class of ciliated neuron, while the amphids appear morphologically normal by dye filling methods. Behavioural assays showed that the ciliated neurons expressing the gene are functional in dpy-14 mutants. All together, our data suggest that ciliated neurons and their hypodermal support cells collaborate in the transcription and synthesis of DPY-14, which then becomes a component of the amphid channels but not of the amphids proper. Interestingly, seam cells of dpy-14 mutants do not properly fuse to form a syncytium. This novel phenotype due to collagen mutations further stresses that dpy-14 plays a fundamental role in C. elegans physiology, since it is required for the proper development of the hypoderm.

Amino Acid Sequence↗

PubMed2006

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics.

BACKGROUND: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. RESULTS: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. CONCLUSION: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Animals↗

PubMed2005

Distinct regulatory elements mediate similar expression patterns in the excretory cell of Caenorhabditis elegans.

Identification of cis-regulatory elements and their binding proteins constitutes an important part of understanding gene function and regulation. It is well accepted that co-expressed genes tend to share transcriptional elements. However, recent findings indicate that co-expression data show poor correlation with co-regulation data even in unicellular yeast. This motivates us to experimentally explore whether it is possible that co-expressed genes are subject to differential regulatory control using the excretory cell of Caenorhabditis elegans as an example. Excretory cell is a functional equivalent of human kidney. Transcriptional regulation of gene expression in the cell is largely unknown. We isolated a 10-bp excretory cell-specific cis-element, Ex-1, from a pgp-12 promoter. The significance of the element has been demonstrated by its capacity of converting an intestine-specific promoter into an excretory cell-specific one. We also isolated a cDNA encoding an Ex-1 binding transcription factor, DCP-66, using a yeast one-hybrid screen. Role of the factor in regulation of pgp-12 expression has been demonstrated both in vitro and in vivo. Search for occurrence of Ex-1 reveals that only a small portion of excretory cell-specific promoters contain Ex-1. Two other distinct cis-elements isolated from two different promoters can also dictate the excretory cell-specific expression but are independent of regulation by DCP-66. The results indicate that distinct regulatory elements are able to mediate the similar expression patterns.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Functional genomics of the cilium, a sensory organelle.

Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Biedl syndrome (BBS). To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the ciliogenic transcription factor, DAF-19. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

Animals↗

PubMed2005

Autosomal genes of autosomal/X-linked duplicated gene pairs and germ-line proliferation in Caenorhabditis elegans.

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-alpha homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena.

Animals↗

PubMed2005

Comparison of the yeast proteome to other fungal genomes to find core fungal genes.

The purpose of this research was to search for evolutionarily conserved fungal sequences to test the hypothesis that fungi have a set of core genes that are not found in other organisms, as these genes may indicate what makes fungi different from other organisms. By comparing 6355 predicted or known yeast (Saccharomyces cerevisiae) genes to the genomes of 13 other fungi using Standalone TBLASTN at an e-value <1E-5, a list of 3340 yeast genes was obtained with homologs present in at least 12 of 14 fungal genomes. By comparing these common fungal genes to complete genomes of animals (Fugu rubripes, Caenorhabditis elegans), plants (Arabidopsis thaliana, Oryza sativa), and bacteria (Agrobacterium tumefaciens, Xylella fastidiosa), a list of common fungal genes with homologs in these plants, animals, and bacteria was produced (938 genes), as well as a list of exclusively fungal genes without homologs in these other genomes (60 genes). To ensure that the 60 genes were exclusively fungal, these were compared using TBLASTN to the major sequence databases at GenBank: NR (nonredundant), EST (expressed sequence tags), GSS (genome survey sequences), and HTGS (unfinished high-throughput genome sequences). This resulted in 17 yeast genes with homologs in other fungal genomes, but without known homologs in other organisms. These 17 core, fungal genes were not found to differ from other yeast genes in GC content or codon usage patterns. More intensive study is required of these 17 genes and other common fungal genes to discover unique features of fungi compared to other organisms.

Computational Biology↗

PubMed2004

Identification of a nematode chemosensory gene family.

Taking advantage of the recent availability of the whole genome sequence of Caenorhabditis briggsae, a closely related nematode to Caenorhabditis elegans, we have examined the chemosensory gene superfamily by using comparative genomic methods. We have identified a chemosensory gene family, serpentine receptor class ab (srab), which exists in both species with 25 members in C. elegans and 14 members in C. briggsae. More than 20% of these gene models are reannotated. The srab family is similar to, but distinct from, the previously described serpentine receptor class a (sra) family and shows a differential expansion in C. elegans similar to that previously described for sra. The cellular expression patterns for multiple members of the srab family in both phasmid neurons in the tail and amphid neurons in the head supports the conclusion that they are chemosensory genes and suggests that they may play a role in integrating chemosensory inputs from both ends of the organism. The expansion of both the srab and sra gene families in C. elegans relative to C. briggsae is due to multiple rounds of tandem duplication and translocation of individual genes.

Animals↗

PubMed2004

Expression analysis of ABC transporters reveals differential functions of tandemly duplicated genes in Caenorhabditis elegans.

We have previously identified 60 predicted ABC transporter genes in the Caenorhabditis elegans genome and classified them into eight groups. As an initial step towards understanding how these putative ABC genes work in worms, we generated promoter-fluorescent protein fusions for the entire family to address when and where these genes are turned on in vivo. Both Aequoria green fluorescent protein (GFP) and Discosoma red fluorescent protein (RFP) were used as reporters in our transgenic assay. Observable expression is more frequently seen from fusions to genes in subfamilies B, C, D and E than those in subfamilies A and G. Sixteen worm ABC genes are found in tandem duplications, forming two four-gene clusters and four two-gene clusters. Fifteen out of the 16 duplicated gene promoters drove different or partially overlapping expression patterns, suggesting active functions for these duplicated genes. Furthermore, our results suggest that an internal promoter can cause differential expression of genes within an operon. Finally, our observations suggest that it is possible for coding sequences to function as a regulatory region for a neighbouring gene.

ATP-Binding Cassette Transporters↗

PubMed2004

The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans.

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3'-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.

Actin Depolymerizing Factors↗

PubMed2004

Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs.

BACKGROUND: The complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions. RESULTS: The experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes. CONCLUSIONS: By combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.

Animals↗

PubMed2004

Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome.

RAB, ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins belong to the Ras superfamily of small GTP-binding proteins and are essential for various membrane-associated intracellular trafficking processes. None of the approximately 50 known members of this family are linked to human disease. Using a bioinformatic screen for ciliary genes in combination with mutational analyses, we identified ARL6 as the gene underlying Bardet-Biedl syndrome type 3, a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities and cognitive impairment. We uncovered four different homozygous substitutions in ARL6 in four unrelated families affected with Bardet-Biedl syndrome, two of which disrupt a threonine residue important for GTP binding and function of several related small GTP-binding proteins. Analysis of the Caenorhabditis elegans ARL6 homolog indicates that it is specifically expressed in ciliated cells, and that, in addition to the postulated cytoplasmic functions of ARL proteins, it undergoes intraflagellar transport. These findings implicate a small GTP-binding protein in ciliary transport and the pathogenesis of a pleiotropic disorder.

ADP-Ribosylation Factors↗

PubMed2004

Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility of the IFT proteins OSM-5/Polaris and CHE-11, and to a notably lesser extent, CHE-2. We propose that BBS proteins play important, selective roles in the assembly and/or function of IFT particle components. Our findings also suggest that some of the cardinal and secondary symptoms of BBS, such as obesity, diabetes, cardiomyopathy, and learning defects may result from cilia dysfunction.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes.

BACKGROUND: Many drugs of natural origin are hydrophobic and can pass through cell membranes. Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment. Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function. All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest. The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective. RESULTS: We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank. Classification according to a unified nomenclature is presented. Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times. CONCLUSIONS: ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence'. This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life. Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication.

ATP-Binding Cassette Transporters↗

PubMed2003

The Sac1 lipid phosphatase regulates cell shape change and the JNK cascade during dorsal closure in Drosophila.

The Sac1 lipid phosphatase dephosphorylates several phosphatidylinositol (PtdIns) phosphates and, in yeast, regulates a diverse range of cellular processes including organization of the actin cytoskeleton and secretion. We have identified mutations in the gene encoding Drosophila Sac1. sac1 mutants die as embryos with defects in dorsal closure (DC). DC involves the migration of the epidermis to close a hole in the dorsal surface of the embryo occupied by the amnioserosa. It requires cell shape change in both the epidermis and amnioserosa and activation of a Jun N-terminal kinase (JNK) MAPK cascade in the leading edge cells of the epidermis [2]. Loss of Sac1 leads to the improper activation of two key events in DC: cell shape change in the amnioserosa and JNK signaling. sac1 interacts genetically with other participants in these two events, and our data suggest that loss of Sac1 leads to upregulation of one or more signals controlling DC. This study is the first report of a role for Sac1 in the development of a multicellular organism.

Animals↗

PubMed2002

A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans.

BACKGROUND: F-actin is enriched at the cortex of embryonic cells in the nematode Caenorhabditis elegans and is required for multiple processes that include the establishment of an anterior-posterior (A-P) axis and cytokinesis. However, the mechanisms that regulate cortical microfilament (MF) assembly remain poorly understood. RESULTS: We show here that a profilin called PFN-1 accumulates at the cortex independent of the actin cytoskeleton and is required for the assembly or maintenance of cortical MFs and myosin. Reducing PFN-1 levels by RNAi results in cytokinesis and A-P polarity defects. PFN-1 binds to the Formin Homology (FH) protein CYK-1, which also is required for cortical MFs. In contrast to PFN-1 and CYK-1, the Arp2/3 complex appears to be dispensable for the assembly of cortical MFs, for A-P polarity, and for cytokinesis. Instead, the Arp2/3 complex is required for cell migrations that occur during gastrulation and may also be involved in cellular rearrangements required for epidermal enclosure prior to elongation of ovoid embryos into vermiform larvae. CONCLUSIONS: We conclude that the FH protein CYK-1 and the profilin PFN-1 mediate the Arp2/3-independent assembly of MFs and are required for cytokinesis in the early embryo. These data suggest that CYK-1 and PFN-1 may nucleate MFs, as has recently been shown for an FH protein and a profilin in yeast.

Actin Cytoskeleton↗