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.

Analysis of the 5' Transcriptional Regulatory Region of the ace-1 Gene in C. elegans.

Emmanuel Culetto1,2, Yann Fedon1, Marta Grauso1,3, Jean-Baptiste Berge2, Jean-Pierre Toutant1, Martine Arpagaus1,2,4

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.