Calx, a Na-Ca exchanger gene of Drosophila melanogaster

We have cloned Calx, a gene that encodes a Na-Ca exchanger of Drosophila melanogaster. Calx encodes two repeated motifs, Calx-α and Calx-β, that overlap domains required for exchanger activity and regulation. Calx has multiple transcripts in adults, including at least one expressed in the retina. The Calx genomic locus comprises ≥35 kb between the Atpα and rudimentary-like genes in chromosomal region 93B. In Xenopus oocytes, microinjected Calx cRNA induces calcium uptake like that of its homolog, the 3Na^+-1Ca^(2+) exchanger of mammalian heart. Implications of Calx-α motifs for the mechanism of Na-Ca exchange are discussed

Calx, A Sodium-Calcium Exchanger of Drosophila melanogaster

Calcium extrusion is necessary for cellular survival and suspected to modulate cellular activity. Drosophila phototransduction is a promising system in which to study calcium export, since it is dominated by calcium activity yet, unlike most calcium-dependent signalling pathways, genetically pliable. The multiple roles of calcium flux in Drosophila phototransduction are reviewed in Chapter One. Calx, a Drosophila ortholog of mammalian 3Na+/1Ca2+ exchangers, was isolated and characterized (Chapter Two). Calx's gene product has ~50% identity to its direct mammalian homologs, with more distant similarities to an exchanger-related superfamily. There exist at least seven alternately spliced adult Calx transcripts, with an alternatively spliced miniexon in Calx's protein-coding region. A full-length Calx cDNA of 5408 bp has lengthy, elaborate 5' and 3' UTRs. Calx transcripts are ubiquitously expressed in embryos and adult heads, with one 5.7 kb transcript expressed in photoreceptors; Calx protein is also ubiquitous in adult heads, with a notable presence in photoreceptors and neuropil. Heterologous expression of Calx in Xenopus oocytes shows that it encodes a bona fide sodium-calcium exchanger; unlike mammalian retinal exchangers, it does not depend on potassium for activity. Calx encodes two novel protein motifs, Calx-α and Calx-β. Both are intragenically duplicated, but they probably have different functions: Calx-α is likely to encode residues central to calcium export, while Calx-β may mediate intracellular signalling or cytoskeletal anchoring.</p

Identification of DVA Interneuron Regulatory Sequences in Caenorhabditis elegans

Background: The identity of each neuron is determined by the expression of a distinct group of genes comprising its terminal gene battery. The regulatory sequences that control the expression of such terminal gene batteries in individual neurons is largely unknown. The existence of a complete genome sequence for C. elegans and draft genomes of other nematodes let us use comparative genomics to identify regulatory sequences directing expression in the DVA interneuron. Methodology/Principal Findings: Using phylogenetic comparisons of multiple Caenorhabditis species, we identified conserved non-coding sequences in 3 of 10 genes (fax-1, nmr-1, and twk-16) that direct expression of reporter transgenes in DVA and other neurons. The conserved region and flanking sequences in an 85-bp intronic region of the twk-16 gene directs highly restricted expression in DVA. Mutagenesis of this 85 bp region shows that it has at least four regions. The central 53 bp region contains a 29 bp region that represses expression and a 24 bp region that drives broad neuronal expression. Two short flanking regions restrict expression of the twk-16 gene to DVA. A shared GA-rich motif was identified in three of these genes but had opposite effects on expression when mutated in the nmr-1 and twk-16 DVA regulatory elements. Conclusions/Significance: We identified by multi-species conservation regulatory regions within three genes that direct expression in the DVA neuron. We identified four contiguous regions of sequence of the twk-16 gene enhancer with positive and negative effects on expression, which combined to restrict expression to the DVA neuron. For this neuron a single binding site may thus not achieve sufficient specificity for cell specific expression. One of the positive elements, an 8-bp sequence required for expression was identified in silico by sequence comparisons of seven nematode species, demonstrating the potential resolution of expanded multi-species phylogenetic comparisons

Multigenome DNA sequence conservation identifies Hox cis-regulatory elements

To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced ∼0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide

Transducing touch in Caenorhabditis elegans

Mechanosensation has been studied for decades, but understanding of its molecular mechanism is only now emerging from studies in Caenorhabditis elegans and Drosophila melanogaster. In both cases, the entry point proved to be genetic screens that allowed molecules needed for mechanosensation to be identified without any prior understanding of the likely components. In C. elegans, genetic screens revealed molecules needed for touch sensation along the body wall and other regions of force sensitivity. Members of two extensive membrane protein families have emerged as candidate sensory mechanotransduction channels: mec-4 and mec-10, which encode amiloride-sensitive channels (ASCs or DEG/ENaCs), and osm-9, which encodes a TRP ion channel. There are roughly 50 other members of these families whose functions in C. elegans are unknown. This article classifies these channels in C. elegans, with an emphasis on insights into their function derived from mutation. We also review the neuronal cell types in which these channels might be expressed and mediate mechanotransduction