Biomedical subjects

Howard A Baylis

Publications and source records attributed to Howard A Baylis.

9 recordsLinked to original sources

PubMed2006

The frataxin-encoding operon of Caenorhabditis elegans shows complex structure and regulation.

The genome of the nematode Caenorhabditis elegans is unusual among eukaryotes, in that it contains operons. Approximately 15% of genes in the worm are clustered into groups of between two and eight genes, which are under the control of shared regulatory sequences. Polycistronic transcripts from such operons are trans-spliced, during transcription, to produce mature monocistronic messengers. The C. elegans frataxin gene, frh-1, is encoded in the operon CEOP2232. This is one of the largest operons identified thus far in the C. elegans genome. Here we describe in detail the structure of all of the coding units within this operon. The operon is composed of eight genes of a diverse nature, organized in a complex structure. We have produced transgenic strains carrying fusions between gfp and a number of genes from the operon. These constructs show complex differential expression patterns that suggest the presence of internal promoters and regulatory sequences in the operon. This organization would permit both coordinated expression and differential expression of the components of the CEOP2232 operon. The heterogeneity of the genes, and their complex expression patterns, suggests that the clustering of CEOP2232 is not due to a need for synchronized expression of genes involved in the same physiological pathway.

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PubMed2005

Reduction of Caenorhabditis elegans frataxin increases sensitivity to oxidative stress, reduces lifespan, and causes lethality in a mitochondrial complex II mutant.

Friedreich ataxia is an autosomal recessive neurological disorder caused by deficiency of the mitochondrial protein frataxin. Studies in patient cells, mouse knockout animals, and Saccharomyces cerevisiae models have suggested several hypotheses on the frataxin function, but the full physiology of frataxin in mitochondria has not been well established yet. We have characterized the genomic structure of frh-1, the Caenorhabditis elegans frataxin gene, and we have developed a transient knockdown model of C. elegans frataxin deficiency by RNA interference. frh-1(RNAi) worms show a consistent pleiotropic phenotype that includes slow growth, lethargic behavior, egg laying defects, reduced brood size, abnormal pharyngeal pumping, and altered defecation. Lifespan is significantly reduced, and worms have increased sensitivity to oxidative stress that, in turn, might explain the reduction of longevity of the worms. We also demonstrate synthetic genetic interaction between frh-1 and mev-1, the gene encoding the succinate dehydrogenase cytochrome b subunit of complex II in mitochondria, suggesting a possible role of the C. elegans frataxin in the electron transport chain; thus, the respiratory chain might be involved in the pathogenesis of the disease. We propose that this C. elegans model may be a useful biological tool for drug screening in Friedreich ataxia.

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PubMed2005

VAV's got rhythm.

Biological rhythms with periods of less than a day are physiologically important but poorly understood. In this issue of Cell, Norman, Maricq, and colleagues (Norman et al., 2005) show that VAV-1, a guanine nucleotide exchange factor for Rho-family GTPases, is necessary for three rhythmic behaviors in the nematode Caenorhabditis elegans: feeding, defecation, and ovulation.

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PubMed2005

Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males.

Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP(3)) is required at several points during mating. Disruption of IP(3) receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

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PubMed2004

The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit.

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.

Alleles↗

PubMed2004

Inositol 1,4,5-trisphosphate signaling regulates rhythmic contractile activity of myoepithelial sheath cells in Caenorhabditis elegans.

Intercellular communication between germ cells and neighboring somatic cells is essential for reproduction. Caenorhabditis elegans oocytes are surrounded by and coupled via gap junctions to smooth muscle-like myoepithelial sheath cells. Rhythmic sheath cell contraction drives ovulation and is triggered by a factor secreted from oocytes undergoing meiotic maturation. We demonstrate for the first time that signaling through the epidermal growth factor-like ligand LIN-3 and the LET-23 tyrosine kinase receptor induces ovulatory contractions of sheath cells. Reduction-of-function mutations in the inositol 1,4,5-trisphosphate (IP(3)) receptor gene itr-1 and knockdown of itr-1 expression by RNA interference inhibit sheath contractile activity. itr-1 gain-of-function mutations increase the rate and force of basal contractions and induce tonic sheath contraction during ovulation. Sheath contractile activity is disrupted by RNAi of plc-3, one of six phospholipase C-encoding genes in the C. elegans genome. PLC-3 is a PLC-gamma homolog and is expressed in contractile sheath cells of the proximal gonad. Maintenance of sheath contractile activity requires plasma membrane Ca(2+) entry. We conclude that IP(3) generated by LET-23 mediated activation of PLC-gamma induces repetitive intracellular Ca(2+) release that drives rhythmic sheath cell contraction. Calcium entry may function to trigger Ca(2+) release via IP(3) receptors and/or refill intracellular Ca(2+) stores.

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PubMed2004

IRI-1, a LIN-15B homologue, interacts with inositol-1,4,5-triphosphate receptors and regulates gonadogenesis, defecation, and pharyngeal pumping in Caenorhabditis elegans.

Inositol-1,4,5-triphosphate receptors (IP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores. They are central to a wide range of cellular responses. IP(3)Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP(3) and IP(3)Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP(3)R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.

Amino Acid Sequence↗

PubMed2002

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans.

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.

Amino Acid Sequence↗

PubMed2002

Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.

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