Molecular characterization of the Notch homologue from the Australian sheep blowfly, Lucilia cuprina

Abstract

The Drosophila melanogaster Notch gene product functions as a receptor of intercellular signals and is central to cell fate specification. The Scalloped wings (Scl) gene is the homologue of Notch in the Australian sheep blowfly, Lucilia cuprina. An allele of Scl is thought to be involved in the modification of Darwinian fitness and bristle asymmetry in flies resistant to organophosphorous chemicals (OPs). As a first step towards the testing of this hypothesis we cloned and sequenced Scl. A full-length cDNA segment representing the mRNA of Scl is 8503 bp and encodes a protein of 2653 amino acids, which shares 73.6% identity with Notch. All functional motifs including EGF-like repeats, LNR repeats, cdc10/ankyrin repeats, opa and PEST elements are present in the same order as in Notch and the sequence identities peak in these motifs. With respect to genomic structure, intron/exon boundaries are conserved but, in most cases, the Scl introns are larger. Sequence analysis of the upstream genomic region reveals that the gene has a TATA-less promoter. Consistent with a central role in embryogenesis and imaginal development, high levels of Scl expression were detected in the early embryonic and pupal stages.

Introduction

The Notch gene in Drosophila melanogaster encodes a large transmembrane protein that functions as a receptor of intercellular signals and is central to cell fate specification in many developmental processes (Fortini and Artavanis-Tsakonas, 1993), including the formation of the nervous system (Artavanis-Tsakonas et al., 1991, Campos-Ortega and Jan, 1991). Notch is regulated by several elements in the signalling pathway. Its extracellular domain contains 36 EGF-like repeats, and the ligands Delta and Serrate bind to specific EGF repeats (Rebay et al., 1991). The intracellular domain of Notch has six cdc10/ankyrin repeats that interact with the cytoplasmic proteins Deltex and Suppressor of Hairless (Diederich et al., 1994, Fortini and Artavanis-Tsakonas, 1994, Matsuno et al., 1995, Axelrod et al., 1996).

Notch homologues have been studied in the nematode, Caenorhabditis elegans (Tax et al., 1994), the amphibian, Xenopus laevis (Coffman et al., 1990), zebrafish (Bierkamp and Campos-Ortega, 1993) and various mammals (Ellisen et al., 1991, Weinmaster et al., 1991, Reaume et al., 1992, Kopan and Weintraub, 1993). Beyond exhibiting significant sequence identity, these Notch homologues are involved in mediating cell-fate decisions by multipotent precursor cells in a number of different tissues and stages of development (Ahmad et al., 1995, Artavanis-Tsakonas et al., 1995).

In the Australian sheep blowfly, Lucilia cuprina, the Scalloped wings (Scl) gene has been identified as the homologue of Notch (Davies et al., 1996). Notch and Scl mutants have a recessive lethal embryonic phenotype associated with hypertrophy of the central and peripheral nervous systems due to a mis-routing of cells to the neural fate (Skeath and Carroll, 1992, Davies et al., 1996). Loss-of-function mutations in each gene result in similar dominant wing notching and bristle phenotypes in adults. At the molecular level, a 2.7 kb genomic Cla I fragment (C4) from L. cuprina with homology to a part of exon E of Notch has been cloned and sequenced (Davies et al., 1996). The C4 clone, which contains the last two EGF repeats and a portion of the intracellular domain including the cdc10/ankyrin repeats, hybridizes in situ to the cytological locus of Scl (Davies et al., 1996).

We are interested in the Scl gene as its function could be associated with resistance to the organophosphorous insecticides (OPs) in L. cuprina. Diazinon resistance in L. cuprina is mediated by allelic substitution at the Rop-1 locus, which encodes the carboxylesterase E3 (Hughes and Raftos, 1985, Newcomb et al., 1997a, Newcomb et al., 1997b). The Rop-1 mutation is pleiotropic, reducing Darwinian fitness and increasing bristle asymmetry in addition to conferring OP resistance (Clarke and McKenzie, 1987, McKenzie and Clarke, 1988, Batterham et al., 1996). A mutation at a second locus, Modifier (M), increases the fitness and decreases the asymmetry associated with the Rop-1 mutation (McKenzie and Game, 1987, McKenzie and Clarke, 1988). M has been mapped to a region tightly linked to the Scl locus and many lines of genetic evidence suggest the allelism of M to Scl gene (Batterham et al., 1996, Davies et al., 1996, McKenzie et al., 1990). Models for direct or indirect interactions between the products of Scl and Rop-1 have been proposed (Batterham et al., 1996, Davies et al., 1996). The molecular characterization of the Scl gene is a prerequisite for testing the hypothesis that M is a Scl allele. This paper reports the cloning and sequencing of the Scl cDNA and the description of the genomic structure and expression of the gene. The cloning of Scl provides the first complete sequence for a Notch homologue from a non-drosophilid insect and the most detailed molecular characterization of any L. cuprina gene studied to date.