ABSTRACT
We have sequenced genomic fragments which encode the N-terminus of the TIMELESS (TIM) clock protein in
Drosophila simulans
and
D.yakuba
. We observe that in these two species, the initiating methionine appears to lie
downstream of the one proposed to encode the translational start in
D
.
melanogaster
, thereby truncating the N-terminus by 23 amino acids. We then sequenced the corresponding 5
'
fragment in a number of
D.melanogaster
individuals from different strains. We observed a polymorphism which strongly
suggests that the originally proposed start site cannot be utilised in some
individuals, and that these flies will initiate translation of TIM at the
downstream ATG. Given the current interest in TIM regulation in
D.melanogaster
, it is important to correctly define the N-terminus in this species.
Circadian rhythmicity is a widespread and ancient adaptation of living organisms
to the rotation of the earth. Mutations affecting circadian rhythms have been
identified in several organisms ranging from prokaryotes (
1
) to plants (
2
) and mammals (
3
). In
Drosophila melanogaster
two clock genes which encode an integral component of the pacemaker have been
cloned and characterized,
period
(
per
) (
4
-
7
) and
timeless
(
tim
) (
8
,
9
). It has been demonstrated that PER and TIM physically interact (
9
-
11
) and both proteins are involved in a mutual negative feedback regulation of
their own expression (
12
). Moreover,
tim
+
activity is necessary for the nuclear localization of a PER reporter (
13
).
The
D.melanogaster
tim
cDNA sequence was obtained by sequencing several clones from independently
derived head specific cDNA libraries as well as genomic DNA (
8
). The data reveal that the sequence contains two open reading frames (ORFs)
coding for two conceptual proteins of 1122 or 1389 amino acids (
8
). Consequently, there may be two forms of the TIM protein. The two ORFs share
the residues 1-1104 but have different C-termini depending on the removal or not of a particular intron.
As part of our continuing comparative analyses of clock genes in diptera (
14
-
16
), we designed a series of oligoprimers in order to amplify fragments of the
tim
gene from other species. Primers which focused on the region encoding the N-terminus, amplified a fragment in the
D.melanogaster
sibling species,
D.simulans
, and a more distantly related member of the
melanogaster
subgroup of species,
D.yakuba
. We observe in both species a different translational start from the one
identified by Myers
et al
. (
8
) in
D
.
melanogaster
. Furthermore, the same alternative translational start is also observed in some
D.melanogaster
individuals. Given the current interest in PER and TIM (
17
,
18
) and the ongoing biochemical analysis of these two proteins in
D.melanogaster
(
10
,
11
), particularly with regard to TIM degradation, the N-terminus of TIM may play an important role in the regulation of the two
proteins.
Single
D.melanogaster
individuals were obtained from the following laboratory strains: Canton-S,
per
+
, per
S
and
per
01
mutant lines. In addition, isofemale lines were collected from a natural
population from Cognac in France (see ref.
19
for details). Individual male descendants were crossed to attached-
X
females to generate self perpetuating iso-
X
chromosome lines. Two of these lines,
Co-TG14
and
Co-TG20
, were used. Consequently the second chromosomes in these lines will carry
either the natural Cognac-derived or the attached-
X
strain-derived
tim
allele.
A
D.yakuba
strain was obtained from J. David (University of Paris) and the
D.simulans
strain was obtained from the Bowling Green
Drosophila
Species Stock Center, USA.
For the amplification of the
tim
fragment from
D.melanogaster
the 5' primer used was 5'- CACAATCACATCTGGAATAA-3' corresponding to nucleotide positions 264-283 in the sequence from Myers
et al
. (
8
). The 3' primer corresponded to positions 384-403 (5'-GCATTCGGGTTGACCACATA-3'). For the amplification from
D.simulans
and
D.yakuba
the following degenerate primers were used: 5' primer was 5'- CAYAAYCAYATHTGGAAYAA-3' (264-283); 3' primer was 5'-GCRTTSGGRTTNACNACR-3' (384-403).
Single fly DNA was prepared using the method of Gloor and Engels (
20
). DNA amplification by PCR was carried out according to Jeffreys
et al
. (
21
) in a MJ Research thermocycler for 30 cycles: 95oC for 1 min, 65oC for 1 min, 72oC for 1 min for
D.melanogaster
and 95oC for 1 min, 48oC for 1 min 10 s, 72oC for 1 min 20 s, for
D.simulans
and
D.yakuba.
BioTaq polymerase from Bioline was used.
The PCR amplified fragments to be sequenced were cloned into the pMOSBlueT-vector (Amersham). The recombinant DNAs were used to transform MOSBlue
competent cells (Amersham). A minimum of three clones was sequenced from two
independent PCR amplifications for each fly's DNA. The DNA sequencing was carried out either using the ThermoSequenase cycle sequencing kit US78500 (Amersham) or by an automatic system using the Perkin-Elmer Turbo-Catalyst robotic station and ABI 377 sequencing apparatus. The M13R
and -40 oligos were used to sequence the cloned fragment.
Figure
1
illustrates the genomic sequences obtained from the 139 or 140 bp
tim
fragment. The 5' stretch (from nucleotide 1 to 403) of the
tim
cDNA sequence of
D.melanogaster
obtained by Myers
et al
. (
8
) is also reported for comparison (GenBank accession number U37018). Note that
there are a number of ATG motifs. The first two (represented in bold italics in
Fig.
1
, positions 208-210 and 227-229) give rise to premature translational stop codons, and the
third (positions 243-245, underlined in Fig.
1
), has been suggested by Myers
et al
. (
8
) to represent the initiating motif.
The intraspecific and interspecific pattern of sequences described above,
strongly suggest that the first methionine of the TIM protein is coded by the
ATG motif (positions 312-314, represented in bold in Fig.
1
). This is located downstream from position 294 where the polymorphism for the
deletion of the corresponding base (G) has been observed. This scenario would
maintain the same reading frame initiated by the upstream ATG motif (position
243-245) suggested by Myers
et al
. (
8
), but would truncate the N-terminus by 23 amino acids (Fig.
2
).
The sequences surrounding the downstream ATG (positions 306-315, boxed in Fig.
1
) are in agreement with the most stringent requirements dictated by Kozak's
consensus translation initiation sequence (GCC
R
CC
The regulation of PER and TIM is currently of great interest (
10
,
11
,
17
,
18
) and the stability and degradation of these two proteins has major implications
for the negative feedback model of circadian timing (
24
). A precise definition of the N-terminus of TIM may therefore be of considerable importance.
We acknowledge a NERC grant to CPK, a CEC grant from the Biotechnology programme
(ERBBIO4CT960096), and a MURST-British Council grant to CPK and RC for Anglo-Italian cooperation, and a MURST studentship to FS.
REFERENCES
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