Worm Breeder's Gazette 13(5): 43 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | Differenciation cellulaire et croissance. Physiologie animale INRA. Montpellier. France. |
| 2 | Biologie des invertebres. Zoologie INRA. Antibes. France. |
| 3 | Department of Experimental Medicine. University of Perugia, Perugia. Italy. |
| 4 | e-mail: arpagaus@montpellier.inra.fr |
Three genes ace-1, ace-2, ace-3, encode three pharmacological classes of acetylcholinesterase (AChE) in C. elegans. The coding sequence of ace-1 has been elucidated (Arpagaus et al., J. Biol. Chem. 1994, 269:9957-9965). It presents 42percent of identity at the amino acid level with human AChE. In order to establish the mechanism of expression of ace-1 and its regulation we have cloned and sequenced 1.2 kb of the 5'-flanking region. We have started to define the promoter region by mapping the transcription start site using 5' RACE experirnents. We found that a portion of ace-1 mRNA is trans-spliced by SL1. We identified, on the untranspliced mRNA, at least two start sites at -168 bp and -196 bp upstream of the ATG initiatior of translation. There is neither a TATA box nor a CAAT box in close proximity of these two sites. In addition one of the transcription start site matches the consensus sequence of an initiator. To test whether this 5' flanking region carried a functional promoter we have used transient transfection of this upstream region linked to a reporter gene (CAT assay) in mouse fibroblasts 10Tl/2. The preliminary results show that the 1.2 kb as well as a 5' deleted 795 bp fragment exhibit promoter activity. Thus the ace-1 promoter is recognized by the transcription apparatus of the mouse cells. The 5' flanking region was searched for eukaryotic DNA binding consensus sequences using a data base of transcription factor binding sites. This analysis revealed potential binding sequences for transcription factors such as: NF-D, myo-D, mef-2, AP-1, CREB. We want now to clone the 5' flanking region of ace-1 gene from C. briggsae for selection of conserved regions with the C. elegans ace-1 promoter. The C. briggsae genomic DNA library (kindly provided by Baillie's lab) was screened with the ace-1 cDNA from C. elegans. Six clones have been isolated. So far, only one of these clones has been further characterised. In particular, we have sequenced a 500 bp fragment of the transcribed region upstream of the active site serine. This C. briggsae fragment presents 95percent and 78percent identity with the corresponding sequence in C. elegans, at the amino acid and nucleotide levels respectively. We expect that the comparaison between the ace-1 5'flanking region in the two species of Caenorhabditis will permit the identification of conserved sequences that will be analyzed by mutagenesis and / or deletions.