Copyright  2000 by the Genetics Society of America
The Clock Gene period of the Housefly, Musca domestica,
Rescues Behavioral Rhythmicity in Drosophila melanogaster:
Evidence for Intermolecular Coevolution?
Alberto Piccin,*,1 Martin Couchman,*,2 Jonathan D. Clayton,* David Chalmers,*,3
Rodolfo Costa† and Charalambos P. Kyriacou*
*Department of Genetics, University of Leicester, Leicester LE1 7RH, United Kingdom and †Dipartimento di Biologia,
Università di Padova, 35131 Padova, Italy
Manuscript received April 25, 1999
Accepted for publication October 22, 1999
ABSTRACT
In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via
a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have
revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per
expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial
step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in
the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence
similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate
with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple
hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER
PAS domain can explain the behavioral data gathered from transformants.
T
HE molecular basis of the circadian clock has been
studied in such diverse model systems as the fly
Drosophila melanogaster, the bread mold Neurospora crassa,
and the cyanobacterium Synechococcus (for review see
Rosato et al. 1997; Dunlap 1999). In these organisms,
a general mechanism appears to have evolved in which
a negative autoregulatory feedback loop plays a prominent role (e.g., Hardin et al. 1990). In Drosophila, for
example, there is a succession of temporally regulated
events, involving delays between peak levels of period
(per) and timeless (tim) mRNA and protein, subsequent
post-translational modification, and PER-TIM dimerization. This is followed by nuclear entry of the PER-TIM
partners and repression of per and tim transcription. As
the PER-TIM heterodimer degrades, the block is lifted
and the cycle of per and tim transcription/translation/
autoregulation begins again (reviewed in Rosato et al.
1997; Dunlap 1999).
More recently, new clock genes have been discovered
and incorporated into the Drosophila feedback model.
Two members of the bHLH PAS family, Clock (Vita-
Corresponding author: C. P. Kyriacou, Department of Genetics, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom. E-mail: [email protected]
1
Present address: Dipartimento di Biologia, Università di Padova,
35131 Padova, Italy.
2
Present address: Department of Biology, Imperial College, Silwood
Park, Ascot, Berks SL5 7PY, England.
3
Present address: ETS/FC 1Bd A. Fleming, 25020 Besançon, France.
Genetics 154: 747–758 (February 2000)
terna et al. 1994; King et al. 1997; also known as jerk,
Allada et al. 1998) and bmal1 (Darlington et al. 1998;
also known as cyc, Rutila et al. 1998), provide the transcription factors for per and tim that are negatively regulated by PER-TIM nuclear entry. Furthermore, the identification of doubletime (dbt), which encodes casein kinase
1ε, has illuminated our understanding of how the delay
between peak per mRNA and protein levels can be generated (Kloss et al. 1998; Price et al. 1998). Finally, dcry
encodes a fly cryptochrome that may be relevant for
the clock’s photoentrainment (Emery et al. 1998; Stanewsky et al. 1998; Ceriani et al. 1999; Lucas and Foster 1999).
Studies of clock genes in mammals have also suggested
a negative feedback role for mper homologues (reviewed
in Dunlap 1999), but the data for mtim are less clear
(Sangoram et al. 1998; Zylka et al. 1998). Furthermore,
although Drosophila Clock (dClock) mRNA cycles with a
circadian profile (Lee et al. 1998), mClock does not (Sun
et al. 1997; Tei et al. 1997). More intriguingly, in the
silkmoth Antheraea pernyi, the temporal expression of
per and tim mRNA and the spatial expression of PER
and TIM proteins in the brain do not easily fit with the
negative feedback loop model (Reppert et al. 1994;
Sauman and Reppert 1996). Thus, it appears that a
general clock mechanism has undergone several variations on a theme, with perhaps the same molecules
carrying on slightly modified tasks, even in rather close
evolutionary lineages. To study this further, we have
developed the housefly, Musca domestica, as a compara-
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A. Piccin et al.
tive clock model, and here we describe the initial cloning of the per homologue from the housefly, the unusual
phylogenetic position of the PAS dimerization domain,
and how the behavioral study of various interspecific
per transformants, including those carrying the Musca
per orthologue, indicates a possible case of intermolecular coevolution between PER and one of its partners,
the clock protein TIM.
MATERIALS AND METHODS
Fly strains: D. melanogaster and M. domestica strains were
reared in a light-dark (LD) 12:12 cycle at 258. Drosophila
adults and larvae were fed on sugar medium (6.5% sucrose,
11.5% baker’s yeast, 1% agar, 0.2% nipagin). Adult houseflies’
diet consisted of sucrose and dry milk. Eggs were laid in larval
medium (prepared with 50 g bran, 0.1 g dried yeast, 80 ml
milk, 30 ml H2O, and 1.5 ml of 20% nipagin), where they
developed into adults.
Isolation and sequencing of the housefly per gene: The per
homologue from Musca was cloned using a PCR-DOP strategy
based on the sequences from Colot et al. (1988). The housefly
probe was amplified from Musca genomic DNA using the
degenerate primers deg5 59-CCCGAATTCATGGARACNYTNA
TGGAYGA-39 and deg3 59-CCCGAATTCRTCRTARTARTCRT
GRTG-39 (binding at position corresponding to amino acids
560–566 and 598–603 of the D. melanogaster protein sequence
depicted in Figure 2, respectively), both carrying EcoRI-cleavable extensions. The amplified 132-bp fragment, which encompasses the perS region, was then used to screen an EMBL3
Musca genomic library. A 16-kb positive clone was isolated
and subcloned into pUC19. Various different subclones were
subsequently obtained and sequenced. The resulting 16-kb
genomic clone was characterized, partially sequenced, and
compared with cDNA sequence obtained from reverse transcriptase (RT)-PCR and 59 RACE fragments performed on
mRNA isolated from Musca heads.
Construction of the M. domestica and D. yakuba transgenes:
The M. domestica per construct pMM1 for P-element transformation was prepared using the D. melanogaster per promoter and
59 UTR, fused to the coding sequences and 39 UTR of the
Musca gene. To reduce the size of the transgene, the large
(z5 kb) Musca intron 2 was removed from the construct; with
this exception, the construct was assembled using genomic
DNA. A PCR strategy was adopted to fuse the untranslated D.
melanogaster portion of exon 2 to the Musca gene at the initial
methionine codon. A 422-bp D. melanogaster DNA fragment,
containing per sequence from 2422 to 21 with respect to the
translation start, was amplified from a D. melanogaster per clone,
using primers 55dro (59-CGAAGCAACATTCGGAATTTG-39)
and 53dro (59-ATTCACCTTCCATGGTGCTTAGGTTCTCCA
GCTTG-39). 53dro carries a tail (underlined) complementary
to part of the Musca coding sequence. A 160-bp Musca cDNA
fragment (from 0 to 1160 with respect to the starting methionine, corresponding to the genomic region encompassing the
large intron 2) was amplified using 35mus (59-AACCTAAGCAC
CATGGAAGGTGAATCTACGGAAT-39) in conjunction with
amp6 (59-GCGGGATCCGATGGTTTGCCGCCATAACC-39).
The underlined region of 35mus does not bind to the Musca
sequence but represents a region of complementarity to the
59 UTR D. melanogaster sequence. Comparable amounts of the
amplified fragments were then pooled together and again
subjected to PCR so that the two complementary tracts would
allow the fusion of the two fragments to give the chimeric
DNA, and the two primers (55dro and amp6) would only allow
amplification of the chimeric fragment. A proof-reading DNA
polymerase (Vent polymerase; New England Biolabs, Beverly,
MA) was used to minimize the risk of mutagenesis and the
amplified product was then sequenced to ensure no mutations
were incorporated. The PCR product was then cut with the
enzymes XbaI and BglI to give the resulting 0.5-kb chimeric
exon 2 fragment, which was then joined to a 5-kb BglI-SalI
genomic fragment (the remaining Musca sequences) and to
a 6-kb BamHI-XbaI fragment (the D. melanogaster 59 regulatory
region), and inserted into the transformation vector pW8
(Klemenz et al. 1987) linearized with BamHI and XhoI.
The D. yakuba per orthologue (Thackeray and Kyriacou
1990) was cloned into the Carnegie 20 transformation vector
(Spradling 1986), after replacing D. yakuba’s upstream sequences with those of D. melanogaster, by swapping a 4-kb
XhoI-SalI fragment between the two species. The resultant
transgene, pMY1, retains the D. melanogaster 59 sequence up
to the SalI site in the first untranslated exon (Citri et al.
1987). All coding sequences are therefore from D. yakuba per,
whereas nearly all the regulatory material is from D. melanogaster.
The published results from the transformants carrying the
following per transgenes are also cited in this study: mps1,
which carries the D. pseudoobscura per coding sequences and
39 UTR, fused to the D. melanogaster 59 regulatory region at a
point close to the 39 end of the large first intron (Petersen
et al. 1988); mps3, a chimeric D. melanogaster/D. pseudoobscura
per, in which D. melanogaster provides the 59 regulatory region
and N-terminal coding sequences up to just before the ThrGly encoding repeat, and D. pseudoobscura contributes the
C-terminal half of the coding sequence and 39 UTR (Peixoto
et al. 1998); per1 transgenes from D. melanogaster, carrying the
13.2-kb per transcription unit and either rosy1 (ry1) or white1
(w1) eye markers (Citri et al. 1987; Sawyer et al. 1997; Peixoto et al. 1998); Ap, a transgene carrying the Antheraea pernyi
per cDNA and 39 UTR fused to the D. melanogaster 59 regulatory
regions (Levine et al. 1995)
P-element transformation: Transformation of Drosophila
embryos was carried out according to Spradling (1986). The
strain used for the microinjections of pMM1 was w; 1/1; Sb,
e, D2-3/TM6, which contains a stable P element (D2-3) on the
third chromosome as a source of transposase (Robertson et
al. 1988). The transformation vector used was pW8, which
carries the mini-white gene. Plasmid for injections was purified
with the QIAGEN tip-500 (QIAGEN, Chatsworth, CA) columns, using manufacturer’s instructions. PCR on the transformants’ genomic DNA showed that the D2-3 element was
successively crossed out of the transformed lines.
Microinjection of the pMY1 (D. yakuba) transgene, carrying
a ry1-selectable marker, was performed using per01; ry506 hosts
and the helper plasmid pp25.7wc (Karess and Rubin 1984).
The chromosomal location of pMM1 and pMY1 inserts was
determined using appropriate balancer stocks, while the number of inserts within each line was checked by means of Southern blotting.
Behavioral analyses: Fly locomotor activity was monitored
with the use of an activity event recorder (e.g., see Hamblen
et al. 1986) produced by Biodata Ltd. (Manchester, United
Kingdom), consisting of many individual activity units, each
sandwiched between two infrared photocells. Single flies were
loaded into glass tubes and each glass tube was clamped between the diodes of the photocells. The flies were entrained
in a 12:12 LD photoperiod for 2 days prior to the start of
the activity recording in constant darkness (DD). Data were
collected over 7 days in a 30-min-bin format. The periodicity
was calculated by spectral analyses, performed with the CLEAN
algorithm of Roberts et al. (1987), which was run on a Silicon
Graphics platform. Significance levels were determined by
Monte Carlo simulation as described in Peixoto et al. (1998).
Musca domestica per Gene
In addition, all activity data were analyzed by autocorrelation,
from which significant periods, at least at the 5% level, were
extracted. Only flies with significant periods from both the
spectral and autocorrelation procedures were judged as
“rhythmic” (see Sawyer et al. 1997; Peixoto et al. 1998).
Computer analyses: DNA and protein data analyses were
performed using various programs of the GCG package for
molecular biology (version 8; University of Wisconsin Genetics
Computer Group, Madison, WI; Devereux et al. 1984). Multiple sequence alignment was performed with the program ClustalW (Higgins and Sharp 1988) and corrected by eye. The
phylogenetic analyses were computed with the PHYLIP (Phylogeny Inference Package, version 3.57c) package provided
by J. Felsenstein (University of Washington, Seattle, Washington). DNA phylogeny was performed applying Kimura’s twoparameter model (Kimura 1980), while protein distance matrices were calculated using either the PAM (Dayhoff 1979)
or Kimura (1983) methods, and the phylogenetic trees were
generated with the UPGMA algorithm (Sneath and Sokal
1973). PEST sequence analyses were performed with the PESTFIND program (Rogers et al. 1986).
RESULTS
Cloning of the M. domestica per homologue: M. domestica per spans 9 kb from the starting codon to the putative polyadenylation signal (GenBank accession nos.
AF142662, AF142663, and AF142664). This dramatic
increase in size compared to its Drosophila orthologues
(e.g., Citri et al. 1987; Colot et al. 1988; Thackeray
and Kyriacou 1990) is deceptive, in that the encoded
1048-residue protein is shorter than that of D. melanogaster. The size of the gene is increased by a modification
in its intron-exon structure, with four additional introns
(Figure 1). Apart from the large intron 2, which has
expanded to 5 kb compared to its positional homologues in D. melanogaster and D. virilis, which are 60 and
70 bp, respectively (Colot et al. 1988), the other introns
falling within the coding sequence are small, ranging
in size between 50 and 72 bp. Intron 2 is located at
exactly the same position in all dipteran per genes and
the intron/exon boundaries are well conserved. The
available 1.6-kb sequence from Musca intron 2 shows
traces of at least one duplication event involving 84 bp,
indicating how this intron may have expanded to its
final size from a shorter ancestor. All the remaining
introns of the Musca per gene are short, resembling
those in D. melanogaster, including introns 3, 4, 5, and
9, which do not have a Drosophila counterpart. Interestingly, one RT-PCR product carried the sequence corresponding to amino acids RLKLKSPFPYYSETNCNFFSIN
TQ, which is found in the Musca genomic sequence,
inserted just N terminal to the putative Musca NLS.
However, subsequent RT-PCRs failed to confirm this
cDNA, and so this sequence corresponds either to a
very rare head transcript or is an RT-PCR artefact. In
any case, it represents the novel intron number 3 (see
Figure 1). In the conserved region known as “c2”
(Colot et al. 1988) are introns 5, 6, and 7, the latter two
having a Drosophila positional homologue occurring in
749
the same codon and in the same phase. Introns 8, 10,
and 11 of Musca have a Drosophila equivalent even
though the divergence between the two per genes does
not allow an alignment of the neighbouring exon sequence. All the introns in the Musca gene are rich in
A 1 T (66%) as in D. pseudoobscura and D. virilis (62
and 60%, respectively) but in contrast with D. melanogaster and D. yakuba (52–53%).
A rather different situation is found for the coding
sequence. Here the Drosophila species show a relatively
high C 1 G content (62, 64, 60, and 56%, respectively,
in melanogaster, yakuba, pseudoobscura, and virilis), while
Musca displays a 44% C 1 G content. This observation
reflects a different codon usage in per between the different groups: among Drosophilids, C- and G-ending codons are strongly preferred while in Musca codons that
terminate in either A or T are favored. Published codon
usage tables for D. melanogaster (Sharp et al. 1992) display similar per-like values for highly expressed genes.
The Musca PER protein: The Musca per transcript
contains an ORF encoding for the 1048-amino-acid long
polypeptide depicted in Figure 2, with a putative molecular weight of 116 kD. The division of per into conserved
(c) and nonconserved (nc) regions was introduced
upon comparison of the gene in three different species,
D. melanogaster, D. pseudoobscura, and D. virilis (Colot
et al. 1988). A fourth Drosophila per gene, cloned from
D. yakuba (Thackeray and Kyriacou 1990), given the
short evolutionary distance of this species from melanogaster (6–15 million years; Lachaise et al. 1988; Russo
et al. 1995), does not show much variation, even in the
so-called nonconserved regions, when compared to its
closely related homologue. This overall pattern of variation is largely preserved in the housefly gene; the six
conserved blocks are clearly apparent upon comparison
of Musca per with any of the Drosophila homologues.
As in Drosophila, c1 and c2 constitute most of the
N-terminal half of the protein, while c3, c4, c5, and c6
are localized in the C-terminal half and are generally
less well conserved (Figure 2). In Musca the similarity
of c1, c2, and c3 to the Drosophila proteins is very high,
between 80 and 94%, slightly lower in c6 (75–82%),
and considerably lower in c4 and c5 (57–71%), as scored
by the GCG program Bestfit.
The N-terminal block c1 contains the NLS (Vosshall
et al. 1994; Saez and Young 1996). Interestingly, a second conserved putative NLS is found in c3, but a functional analysis of this signal has not been reported. The
longest conserved block is c2, representing almost half
the length of the entire Musca PER protein and containing the PAS dimerization region (Huang et al. 1993)
and the cytoplasmic localization domain (CLD; Saez
and Young 1996). Our definition of PAS includes residues 238–496 (in the D. melanogaster sequence; e.g., see
Pellequer et al. 1998), and begins a few amino acids
upstream of the first 51-residue PASA degenerate repeat, and ends downstream of the PASB repeat after
750
A. Piccin et al.
Figure 1.—Intron/exon comparison of D. melanogaster (top) and M. domestica per (bottom). Open boxes represent the 59 UTR.
The regions of high homology (conserved regions) are depicted in black, while the nonconserved regions are in gray. The NLS,
PAS (including PAC/CLD), and Thr-Gly domains are indicated. Introns are numbered. Musca intron 1 is depicted by a broken
line to indicate that its length and complete sequence have not been determined.
the PAC domain (Ponting and Aravind 1997). The
PAC domain includes the CLD as defined by the deletion studies of Saez and Young (1996), except for a
few C-terminal residues that cannot be unaligned between the species. This broad definition of PAS encompasses all the N-terminal regions that physically interact
with TIM (Saez and Young 1996). The sites to which
the perL, perS, and per01 mutations have been mapped
(Baylies et al. 1987; Yu et al. 1987) are included within
this region and are perfectly conserved in all PER proteins (Figure 2).
Secondary structure analyses of the predicted Musca
protein sequence with the PHDsec program (EMBL)
identified an HLH domain located in c2 [amino acids
(aa) 450–512], at the end of the CLD (Figure 2). The
same structural motif was also found in the D. melanogaster sequence (aa 525–571). The Musca candidate HLH
lies in a different region of the protein from the one
suggested in the mammalian mper1 homologue (Sun et
al. 1997). Musca c5 also contains an opa repeat (CAG),
which generates a cluster of glutamines at positions
907–917, a feature associated with transcriptional activators (Courey and Tjian 1988; Emili et al. 1994); despite
this poly-Q stretch being localized in a conserved region,
none of the other Drosophila orthologues display a similar motif. A poly-Q stretch is also found in nc1 of D.
virilis. In nc2 lies the Thr-Gly repeat, and as reported
in other non-Drosophilid dipterans (Nielsen et al.
1994), the Musca repeat of two Thr-Gly pairs has not
undergone the dramatic expansion in size observed in
the Drosophila genus (Costa et al. 1991; Peixoto et al.
1992, 1993). Various PEST sequences (Rogers et al.
1986) and phosphorylation sites are also found within
the PER proteins (Figure 2). One putative site for casein
kinase II phosphorylation is found in all the Dipteran
sequences within the C-terminal conserved PEST motif
(see Figure 2).
Molecular phylogeny of the PER proteins: The phylogeny of the six species D. melanogaster, D. yakuba, D.
pseudoobscura, D. virilis, M. domestica, and A. pernyi is well
known from traditional taxonomic approaches. A. pernyi
belongs to the order Lepidoptera, which was already
well differentiated at the end of the Triassic era 200
mya (Boudreaux 1978). The group Calyptratae (to
which M. domestica belongs) diverged from the group
Acalyptratae (which includes the Drosophilidae) 100
mya (Hennig 1981); the time of divergence of D. melanogaster and D. virilis is estimated to be z40 mya (Schlotterer et al. 1994), the obscura group (to which D. pseudoobscura belongs) separated from the melanogaster group
between 25 mya (Russo et al. 1995) and 30 mya
(Schlotterer et al. 1994), and the phylogenetic dis-
Musca domestica per Gene
Figure 2.—Alignment of the conserved regions of the Per proteins from D. melanogaster, D. yakuba, D. pseudoobscura, D. virilis, M. domestica, and A. pernyi (see materials
and methods). An alignment is also attempted in nonconserved region 1 (nc1), in nc4, and in nc5. The ends of the sequences are indicated by asterisks. Dots represent
equal amino acids and dashes represent deletions. The positions of introns are indicated by arrows on the melanogaster and Musca sequences. The nuclear translocation
signals (NLS) are boxed in gray, with the label NLS below. The putative HLH domain is indicated by a double underline and the CLD is boxed on the melanogaster
sequence. Shown in boldface type are also the polyQ stretches. PEST regions are underlined; the casein kinase II site within the C-terminal PEST is boxed in gray. The
PAS region (from residue 238 to 496 of the D. melanogaster sequence and that includes PAC/CLD) is shown with a thick underline, with the two degenerate PAS repeats
underlined in gray.
751
752
A. Piccin et al.
Figure 2.—Continued.
753
Figure 2.—Continued.
Musca domestica per Gene
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A. Piccin et al.
Figure 3.—Phylogeny of different fragments of the per coding sequence or PER protein. Tree A represents the molecular
phylogeny of the PAS-encoding DNA, and was obtained by
applying the Kimura two-parameter method (Kimura 1980)
to the UPGMA tree-drawing method (Sneath et al. 1973).
The following two trees, based on amino acid sequence, were
obtained with the PAM distance matrix (Dayhoff 1979) and
the UPGMA method. B represents the phylogeny of the c1 1
c3 regions and C that of the PAS region (residues 238–496
of the D. melanogaster sequence). The numbers represent the
confidence limits of the branching on their right. The length
of each branch is proportional to the value of the distance
calculated by the program.
tance between D. melanogaster and D. yakuba is 6–15 mya
(Lachaise et al. 1988; Russo et al. 1995). Although there
are uncertainties about the exact time of divergence,
there are no ambiguities in the branching order of these
species (see below). We used the phylogenetic approach
to examine whether there is any significant difference
between the species tree and the PER protein tree,
which could be taken as an indicator of unusual events
in the evolution of PER protein sequences. We were
particularly interested in analyzing the PAS domain,
which has been implicated in the protein-protein interactions between PER and TIM, and comparing it with
the evolution of non-PAS sequences (Huang et al. 1993;
Gekakis et al. 1995; Saez and Young 1996).
First, a molecular phylogeny was computed on the
DNA sequence coding for the PAS domain (Figure 3A).
We used Kimura’s two-parameter method (Kimura
1980) to estimate the evolutionary distance. As can be
seen, there is no ambiguity in that the species tree is
faithfully reproduced. We then generated a phylogeny
based on the alignable amino acid sequence from nonPAS regions c1 1 c3 (Figure 3B), employing the PAM
distance matrix (Dayhoff 1979), which takes into account the fact that some amino acid replacements occur
at higher frequencies than others, irrespective of the
necessary number of nucleotide substitutions. A tree
similar to the DNA tree from Figure 3A was obtained,
except that D. virilis and D. pseudoobscura had swapped
positions.
The third fragment of PER used in this analysis was
the PAS region from c2 (including PAC/CLD, residues
238–496 of the D. melanogaster sequence), which represents a functional domain of PER. The PAS tree places
Musca PAS closer to D. melanogaster than D. pseudoobscura
and D. virilis (see Figure 3C), contradicting the species
tree drawn from per DNA (Figure 3A). A similar switching of positions of the Musca and D. pseudoobscura/virilis
groups, with similarly high bootstrap values, was also
observed using Kimura’s (1983) protein distance matrix (data not shown), so the tree is reasonably robust.
This unusual PAS phylogeny is reflected in the smaller
number of differences between the D. melanogaster/M.
domestica pairwise comparison (29 aa changes 1 1 aa
deletion) compared to that of D. melanogaster/D. pseudoobscura (33 aa changes) or D. melanogaster/D. virilis (44
replacements).
Rescue of circadian rhythmicity in transgenic D. melanogaster carrying the housefly per: To study any possible
functional implications of the unusual phylogenies observed for the PAS domain, we transformed the Musca
per gene into arrhythmic Drosophila per01 mutants. The
transgene, pMM1, carries the 59 D. melanogaster regulatory sequences until the first coding methionine, and
the coding sequence and 39 UTR of Musca per. Two
transgenic lines, each carrying one autosomal copy of
pMM1, were studied for rescue of free-running circadian
locomotor activity. In addition, we also studied one
transgenic line, pMY1-M7, which carried an autosomal
copy of the D. yakuba per coding sequences (Thackeray
and Kyriacou 1990) fused to the D. melanogaster 59
region. Both the pMM1 and pMY1 transgenes were studied in per01 males. Table 1 reveals the patterns of rescue
observed in these various Musca and D. yakuba transgenic lines. Included in Table 1 are the results obtained
in our laboratory for transgenic lines carrying a single
autosomal copy of the D. pseudoobscura per coding sequences fused to the 59 regions of D. melanogaster per
(the mps1 transgene; see materials and methods). In
addition, the data are also illustrated from transformants carrying a single copy of the chimeric transgene,
mps3, also from our laboratory, in which the 59 regulatory and coding regions of D. melanogaster were fused
to the 39 sequences of D. pseudoobscura at a position
Musca domestica per Gene
755
TABLE 1
Rescue of rhythmic behavior by various per transgenes
Genotype
per1a
per1a
per1a
per1a
pMM1
pMM1
pMY1
mps1a
mps1a
mps3a
mps3a
Apb
Line
No. flies tested
% Arrhythmic
2a
34a
17a
116a
34a
34c
M7
120
126
65
67
M4-15
25
47
24
33
35
36
44
31
37
44
32
48
8.0
17.02
16.67
9.09
0
19.44
13.7
42.0
54.1
4.5
0
77.1
Mean period 6 SEM (hr)
25.0
25.0
24.3
25.5
22.4
22.2
23.5
28.3
28.9
25.7
24.4
20.9
6
6
6
6
6
6
6
6
6
6
6
6
0.1
0.1
0.7
0.1
0.1
0.2
0.1
1.2
0.8
0.1
0.1
0.5
Summary of locomotor activity data at 258 of transgenic D. melanogaster per01 mutants carrying the following
transgenes: per1 (wild-type D. melanogaster per), pMM1 (M. domestica per), pMY1 (D. yakuba per), mps1 (D.
pseudoobscura per), mps3 (a chimeric D. melanogaster/D. pseudoobscura, see text), and Ap (A. pernyi per). All
transgenes had their respective coding sequences fused to the D. melanogaster per regulatory region.
a
Data from Peixoto et al. (1998).
b
Data from Levine et al. (1995).
corresponding to D. melanogaster residue 639, z180 bp
upsteam of the Thr-Gly-encoding repeat region (see
materials and methods). The data from mps1 and
mps3 are taken directly from Peixoto et al. (1998). Also
from this study, the data are included from control
transformant lines carrying a single autosomal copy of
the D. melanogaster per transgene, with either ry1 or w1
markers. Finally, and also in Table 1, are the results
obtained by Levine et al. (1995) for a per01 transformant
line, M4-15, which gave the best reported rescue of
rhythmicity when carrying a transgene encoding the
corresponding A. pernyi per cDNA (Ap) fused to the D.
melanogaster 59 regulatory region. All these transgenes
therefore carried D. melanogaster regulatory sequences
ligated to various species-coding sequences, and all the
Drosophila and Musca constructs were derived from
genomic DNA, except that the large intron 2 of Musca
per was removed.
The results reveal a striking correlation between the
level of rescue of rhythmicity and the phylogeny of the
PAS region (Figure 3C). The D. pseudoobscura per
transgene, mps1, rescues rhythmicity in a per01 background relatively poorly, with significant rhythmicity observed in z50% of individuals, but with longer-thannormal periods. The rescue obtained in our study with
the mps1 transgene is better than that reported by
Petersen et al. (1988) for the same transgenic strains,
in which rhythmicity was z10%. The difference in our
results is due to our use of a more sensitive statistical
measure of rhythmicity (see Sawyer et al. 1997; Peixoto
et al. 1998).
In contrast, the Musca per transgene, pMM1, rescues
behavior remarkably robustly, with 80–100% of individuals showing statistically significant rhythms, although
the periods are z2 hr shorter than the corresponding
D. melanogaster per transformants. Furthermore, our
spectral analyses (see Sawyer et al. 1997; Peixoto et al.
1998), mean that the strength of individual rhythms will
broadly correlate with the proportion of flies that are
rhythmic within each genotype class, and not surprisingly, pMM1 transformants have much stronger individual rhythms than mps1 (data not shown). The D. yakuba
per transgene, pMY1, also yields very robust rhythms with
.80% of individuals giving statistically significant cycles
with a free-running period of z23.5 hr. The line carrying the A. pernyi per transgene, which best rescues
rhythmicity, generated only z20% rhythmic individuals
with very short periods (Levine et al. 1995). Finally,
the mps3 chimeric transgene, in which the N-terminal
coding sequences of D. pseudoobscura have been replaced
with those of D. melanogaster, generates essentially wildtype rhythms (Peixoto et al. 1998). The D. virilis per
gene has yet to be transformed into D. melanogaster hosts.
Thus the M. domestica per transgene provides a robust
rescue of per01 rhythms, which belies its evolutionary
position relative to D. pseudoobscura.
DISCUSSION
Conservation of the Musca per homologue: The cloning of the M. domestica per orthologue has revealed
stretches of similarity in all the conserved regions first
identified by Colot et al. (1988) in Drosophilid per
genes. However, the structure of the Musca gene is
different from its Drosophila orthologues; even though
the full length of the primary transcript is not known, it
must be considerably longer than that of D. melanogaster
given the increase in the size of intron 2. Also, the
756
A. Piccin et al.
number of introns is increased in the housefly gene,
reflecting the size of the genome of M. domestica, which
is about five times larger than that of D. melanogaster
(John and Miklos 1988). It is tempting to say that the
increase in genomic complexity must correlate with an
increase in both number and size of introns. Unfortunately, most of the available Musca gene sequences
come from cDNA libraries, so there are not sufficient
genomic data to test this hypothesis.
In D. melanogaster, per is sex-linked, being located at
the 3B1-2 region, close to the tip of the X chromosome
(Young and Judd 1978). In D. pseudoobscura, per has also
been mapped to the X chromosome (Petersen et al.
1988) while the chromosome location of per in D. virilis
is not known. In situ mapping of the Musca per gene to
polytene chromosomes was unsuccessful, but as the X
chromosome of M. domestica is entirely heterochromatic
(Malacrida et al. 1985) it may contain very few, if any,
genes. Indications that per might be located on chromosome III of Musca come from Malacrida et al. (1985),
who described the correspondence between various
linkage groups of D. melanogaster and M. domestica. In
particular, the Musca genes corresponding to Drosophila yellow and white, which lie close to per, and are called
brown body and w, respectively, are found on the right
arm of the housefly chromosome III.
The A 1 T content is relatively high in intron sequences from Musca, D. pseudoobscura and D. virilis, but
in contrast to Drosophila, this high A 1 T profile is
maintained in the Musca coding sequence. This reflects
a bias in Drosophila per for the usage of codons that
either terminate in C or G and that is seen particularly
in highly expressed genes (Sharp et al. 1992). These
authors used the term “optimal codons” to describe
these frequently used triplets, which may reflect translational selection among synonymous codons, mutational
trends (Moriyama and Gojobori 1992), or selection
for particular structures in DNA. The few Musca genes
analyzed cannot provide much insight for elucidating
the codon usage in this organism, but if Musca per is as
extensively expressed as Drosophila per, at least in the
adult (e.g. Plautz et al. 1997), then unlike Drosophila,
any bias in codon usage toward a higher frequency of
optimal codons may be A- and T-ending.
At the protein level, c1 and c2 regions show high
similarity between all species (Figure 2), underscoring
the importance of PAS in the biochemical function of
PER, and suggesting that an equally important role may
be played by c1, in which is found the NLS. In c2,
two sites are found within the PAS domain that, when
mutated, decrease dimerization efficiency: the per L site
and a cluster of amino acids at position 413–419 (in
the second PAS repeat) of the D. melanogaster protein
(Huang et al. 1993). The effect on protein-protein interactions of this amino acid cluster was assayed because
the residues contained in the fragment are highly conserved in the PAS regions of AHR, ARNT, SIM, and
PER (Huang et al. 1993). Both of these areas of PAS
are highly conserved in Musca. Another area in c2 has
been identified as a short period domain in which mutations consistently shorten the circadian period (Baylies
et al. 1987; Rutila et al. 1992). It extends from 3 amino
acids upstream to 16 downstream of the perS site. The
high degree of similarity suggests that in Musca this area
retains its functional importance. An intriguing feature
of the C-terminal region of both Musca and Drosophila
PAS domains was the suggestion of an HLH motif, which
was generated with the use of the PHDsec structural
algorithm. The report of a similar motif in mammalian
PER, albeit in another region (Sun et al. 1997), seems
more than coincidence, so whether these regions can
act as an HLH domain should be tested.
Phosphorylation of PER by the DBT casein kinase 1ε
has been shown to play an important role in contributing to the delay observed in peak levels of per transcript
and protein (Kloss et al. 1998; Price et al. 1998). In
the yeast fructose-1,6-biphosphatase, phosphorylation
transforms a weak PEST region into a strong proteolytic
signal (Rechsteiner 1988). Similarly, the degradation
of PER appears to occur mainly at the level of the phosphorylated forms, whose appearance triggers the negative feedback on per transcription (Edery et al. 1994).
By analogy to the yeast protein, a conditional PEST
region(s) in PER could be activated by phosphorylation.
Although many consensus phosphorylation sites are
found in the different PER proteins, it is interesting
that the conserved C-terminal PEST region also carries
a conserved potential phosphorylation site for casein
kinase II.
Circadian rhythmicity of locomotor activity is restored
in D. melanogaster per01 mutants expressing one copy of
Musca per. More than 80% of the transformants display
rhythmic behavior. This is in stark contrast with what
has been reported by Petersen et al. (1988) and later
by Peixoto et al. (1998), where a comparable transgene,
mps1, containing the D. pseudoobscura per coding sequence fused to the D. melanogaster promoter, generates
a significantly weaker rescue. As the processing of the
59 introns of mps1 has been assayed by Petersen et al.
(1988) and found to be normal, and the mps3 construct,
which encodes the C-terminal half of the D. pseudoobscura protein, gives wild-type rescue of behavior (Peixoto et al. 1998), suggesting that the 39 introns are also
processed normally, it is highly unlikely that the poor
rescue of mps1 is due to problems in producing the
transcript. In addition, mps3 enhances rhythmic behavior back to wild-type levels, thus mapping the poorer
rescue of mps1 to the D. pseudoobscura coding sequences
in the N-terminal half of PER (Peixoto et al. 1998).
Thus, attention is drawn to the N terminus and the PAS
domain, implicated in protein-protein interactions with
TIM, for explaining the different levels of transgenic
rescue (Gekakis et al. 1995; Saez and Young 1996).
Given the unusual phylogeny of PAS (Figure 3C),
Musca domestica per Gene
the ability of Musca PER to direct efficient rescue of
rhythmicity in D. melanogaster aperiodic mutants, in contrast to D. pseudoobscura PER, could mean that PASmediated PER-TIM interactions can take place in an
almost normal fashion between Musca PER and the
host D. melanogaster TIM, as opposed to the melanogasterpseudoobscura pairing. This idea could also be extended
to any PAS-mediated interactions between Musca PER
with the hosts CLOCK and BMAL1. This simple but
compelling explanation for the functional data may represent an example of intermolecular coevolution between PER and its various partners. Testing this hypothesis experimentally for the TIM interaction would require
the simultaneous transformation into D. melanogaster
double mutant per01; tim0 hosts, of both D. pseudoobscura
tim and per, with the expectation that the levels of rescue
should be significantly improved over those seen with
the D. pseudoobscura mps1 transgene (Petersen et al.
1988; Peixoto et al. 1998). In addition, coevolution
between PER and TIM (or CLOCK and BMAL1) might
also be seen at the sequence level in phylogenetic trees,
with a switching of the relative positions of the relevant
D. pseudoobscura and M. domestica TIM sequences as observed for PAS (Figure 3C).
In conclusion, the identification and isolation of
Musca per has provided some initial surprises. Without
the phylogenetic perspective, the results obtained from
the transformation experiments would have been difficult to interpret. We suggest an initial, simple, testable
hypothesis based on PER-TIM coevolution to explain
the differential success of interspecific clock gene transformations to rescue clock function in D. melanogaster,
and to this end we are attempting to identify PER partners in both Musca and D. pseudoobscura.
M.C. thanks the Biotechnology and Biological Research Council
(BBSRC) for a studentship. C.P.K. and R.C. were supported by a
European Community grant under the Human Capital and Mobility
programme. In addition, C.P.K. acknowledges grants from BBSRC
and Human Frontiers Science Programme and R.C. acknowledges
grants from Ministero Universitá e Ricerca Scientifica e Technologica
and Ministero delle Risorse Agricole, Alimentari e Forestali.
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Communicating editor: J. J. Loros