Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene.

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.

Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing.

Genome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in alpha-type nicotinic acetylcholine receptor subunits. The subunits are designated Dalpha5, Dalpha6, and Dalpha7. Cloning of the Dalpha5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dalpha6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dalpha6 involves exons encoding nAChR functional domains. The Dalpha6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dalpha6, four of which are also edited in the Dalpha6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels.

A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.

A growing number of medical research teams have begun to explore the experimental advantages of using a genetic animal model, the nematode worm Caenorhabditis elegans, with a view to enhancing our understanding of genes underlying human congenital disorders. In this study, we have compared sequences of positionally cloned human disease genes with the C.elegans database of predicted genes. Drawing on examples from spinal muscular atrophy, polycystic kidney disease, muscular dystrophy and Alzheimer's disease, we illustrate how data from C.elegans can yield new insights into the function and interactions of human disease genes.

The Caenorhabditis elegans orthologue of the human gene responsible for spinal muscular atrophy is a maternal product critical for germline maturation and embryonic viability.

Spinal muscular atrophy (SMA) is a common disorder characterized by loss of lower motor neurones of the spinal cord. The disease is caused by mutations in the survival motor neurone ( SMN ) gene. SMN is ubiquitously expressed and evolutionarily conserved, and its role in RNA processing has been well established. However, these properties do not explain the observed specificity of motor neurone death. To gain further insight into the function of SMN, we have isolated and characterized the Caenorhabditis elegans orthologue of the SMN gene ( CeSMN ). Here we show that CeSMN is transmitted maternally as a predominantly nuclear factor, which remains present in all the blastomeres throughout embryonic development and onwards into adulthood. In adult nematodes, a CeSMN-green fluorescent protein fusion protein is expressed in a number of cell types including the germline. Both disruption of the endogenous CeSMN function and overexpression of the gene result in a severe decrease in the number of progeny and in locomotive defects. In addition, its transient knockdown leads to sterility caused by a defect in germ cell maturation. The expression pattern and functional properties so far observed for CeSMN, together with its unusual behaviour in the germline, indicate that SMN may be involved in specific gene expression events at these very early developmental stages. We have also identified a deletion in the CeSMN promoter region in egl-32. This mutant may become a useful genetic tool with which to explore regulation of CeSMN and hence provide possible clues for novel therapeutic strategies for SMA.

Structure and promoter activity of the 5' flanking region of ace-1, the gene encoding acetylcholinesterase of class A in Caenorhabditis elegans.

We report the structure and the functional activity of the promoter region of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. We found that ace-1 was trans -spliced to the SL1 spliced leader and that transcription was initiated at a cluster of multiple starts. There was neither a TATA nor a CAAT box at consensus distances from these starts. Interspecies sequence comparison of the 5' regions of ace-1 in C. elegans and in the related nematode Caenorhabditis briggsae identified four blocks of conserved sequences located within a sequence of 2.4 kilobases upstream from the initiator ATG. In vitro expression of CAT reporter genes in mammalian cells allowed the determination of a minimal promoter in the first 288 nucleotides. In phenotype rescue experiments in vivo, the ace-1 gene containing 2.4 kilobases of 5' flanking region of either C. elegans or C. briggsae was found to restore a coordinated mobility to the uncoordinated double mutants ace-1(-);ace-2(-)of C. elegans. This showed that the ace-1 promoter was contained in 2.4 kilobases of the 5' region, and indicated that cis -regulatory elements as well as coding sequences of ace-1 were functionally conserved between the two nematode species. The pattern of ace-1 expression was established through microinjection of Green Fluorescent Protein reporter gene constructs and showed a major mesodermal expression. Deletion analysis showed that two of the four blocks of conserved sequences act as tissue-specific activators. The distal block is a mesodermal enhancer responsible for the expression in body wall muscle cells, anal sphincter and vulval muscle cells. Another block of conserved sequence directs expression in pharyngeal muscle cells pm5 and three pairs of cephalic sensory neurons.

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans.

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

Three genes, ace-1, ace-2 and ace-3, respectively located on chromosomes X, I and II, were reported to encode acetylcholinesterases (AChEs) of classes A, B and C in the nematode Caenorhabditis elegans. We have previously cloned and sequenced ace-1 in the two related species C. elegans and C. briggsae. We report here partial sequences of ace-2 (encoding class B) and of two other ace sequences located in close proximity on chromosome II in C. elegans and C. briggsae. These two sequences are provisionally named ace-x and ace-y, because it is not possible at the moment to establish which of these two genes corresponds to ace-3. Ace-x and ace-y are transcribed in vivo as shown by RT-PCR and they are likely to be included in a single operon.

Sequence comparison of ACE-1, the gene encoding acetylcholinesterase of class A, in the two nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

The ace-1 gene, which encodes acetylcholinesterase of class A, has been cloned and sequenced in C. briggsae and compared to its homologue in C. elegans. Both genes present an open reading frame of 1860 nucleotides. The percentages of identity are 80% and 95% at the nucleotide and aminoacid levels respectively. All residues characteristic of an acetylcholinesterase are found in conserved positions in C. briggsae ACE-1. The deduced C-terminus is hydrophilic, thus resembling the catalytic peptide T of vertebrate cholinesterases. Codon usage in both ace-1 genes appears to be lowly biased. This may indicate that these genes are lowly expressed. The splicing sites of the eight introns of ace-1 in C. elegans are conserved in C. briggsae, but introns are shorter in C. briggsae. No homology was found between intronic sequences in both species, except for the consensus border sequences.

Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans.

Two genes (ace-1 and ace-2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace-1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TGG (W99)-->TGA (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace-1 transcripts to only 10% of that in wild-type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace-2 takes place with unchanged ace-1 mRNA level and enzymatic activity similar to class A AChE.