A Mutation in Drosophila simulans that Lengthens the Circadian Period of Locomotor Activity

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly)24 repeat, but the flanking region of a (Thr-Gly)23 allele. One of the five isofemale lines that carried this 'mutant' Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Molecular Analysis of Circadian Clocks in Drosophila simulans

The Drosophila simulans per gene is polymorphic for the length of a repeat that encodes a series of Thr-Gly pairs. We have examined the circadian behaviour of flies derived from isofemale lines that carry the major variants, and find some significant differences in the way that the clock responds to temperature challenge, that might relate to the observed frequencies of these alleles in nature. We also observe that circadian thermal behaviour is also predictably influenced by subtle differences in the temperature of the locality from which these flies have been originally collected. There appear to be species-specific differences in the circadian locomotor patterns of D. melanogaster and D. simulans and in the way they may respond to temperature. Using chimeric per transgenes which carry the different species Thr-Gly fragments, we have been able to identify components of the behaviour that are modulated by this region of the PER protein.     

In search of clinal variation in the period and clock timing genes in Australian Drosophila melanogaster populations

Clinal variation for repeat number in the Thr-Gly region of the period circadian timing gene in Drosophila melanogaster was described in Europe and has subsequently been used as evidence of thermal selection on period alleles. To test for clinal variation in this gene along the east coast of Australia, the period polymorphism was scored on flies from multiple samples collected repeatedly over a 5-year interval, along with variation at another circadian rhythm locus, clock. For period, there was no consistent evidence of clinal variation in the 17 and/or 20 repeat alleles, although when average allele length was examined a weak consistent clinal pattern was detected. For clock there was no evidence of clinal variation in the two most common alleles or in average repeat size. These data are inconsistent with the reported patterns in Europe and suggest that clinal variation in timing genes needs to be re-examined in this region.     

Mutational mechanisms,phylogeny, and evolution of a repetitive region within a clock gene ofDrosophila melanogaster

The D. melanogaster clock gene period (per) is an internally repetitive gene encoding a tandem array of Thr-Gly codons that are highly polymorphic in length in European natural populations. The two major length variants, (Thr-Gly)₂₀ and (Thr-Gly)₁₇, show a highly significant latitudinal cline. In this study we present the complete sequence of the Thr-Gly region of 91 individuals from 6 natural populations of D. melanogaster, 5 from Europe and 1 from North Africa. We further characterized these 91 individuals for polymorphic sites in two other regions, one upstream and one downstream of the Thr-Gly repeat. We used the haplotypic combinations of Thr-Gly allele with flanking markers in an attempt to identify the mechanisms involved in the evolution of the D. melanogaster Thr-Gly region and to infer the phylogenetic relationship existing among the Thr-Gly alleles. We observe evidence for both intra- and interallelic mutational mechanisms, including replication slippage, unequal crossing-over, and gene conversion. Received: 22 August 1995 / Accepted: 17 October 1995     

Linkage disequilibrium, mutational analysis and natural selection in the repetitive region of the clock gene, period, in Drosophila melanogaster

We have used the method of disequilibrium pattern analysis to examine associations between the threonine-glycine (Thr-Gly) encoding repeat region of the clock gene period (per) of Drosophila melanogaster, and polymorphic sites both upstream and downstream of the repeat, in a number of European fly populations. The results are consistent with the view that selection may be operating on various haplotypes which share the Thr-Gly length alleles encoding 17, 20 and 23 dipeptide pairs, and that the repeat itself may be the focus for selection. These conclusions lend support to a number of other population and behavioural investigations which have provided evidence that selection is acting on the Thr-Gly region. The linkage analysis was also used to infer an approximate mutation rate (mu) for the repeat, of 10(-5) < mu < 4 x 10(-5) per gamete per generation. Direct measurements of the mutation rate using the polymerase chain reaction in a pedigree analysis of tens of thousands of individuals do not contradict this value. Consequently, the Thr-Gly repeat does not have a mutation rate that is as high as some of the non-coding minisatellites, but it is several orders of magnitude higher than the nucleotide substitution rate. The implications of this elevated mutation rate for linkage disequilibria and selection are discussed.     

Molecular Polymorphism in the Period Gene of Drosophila Simulans

The threonine-glycine (Thr-Gly) repeat region of the period (per) gene of eight natural populations of Drosophila simulans from Europe and North Africa was analyzed by polymerase chain reaction, DNA sequencing and heteroduplex formation. Five different length alleles encoding 21, 23, 25 and two different kinds of 24 Thr-Gly pairs in the uninterrupted repeat were found. In the 3' region flanking the repeat 6 nucleotide substitutions (3 synonymous, 3 replacement) were observed in three different combinations that we called haplotypes I, II and III. The complete linkage disequilibrium observed between the haplotypes and these length variants allowed us to infer from the repeat length, the DNA sequence at the 3' polymorphic sites. The haplotypes were homogeneously distributed across Europe and North Africa. The data show statistically significant departures from neutral expectations according to the Tajima test. The results suggest that balancing selection might have played a role in determining the observed levels and patterns of genetic diversity at the per gene in D. simulans.     

The period gene Thr-Gly polymorphism in Australian and African Drosophila melanogaster populations: implications for selection

The period gene is a key regulator of biological rhythmicity in Drosophila melanogaster. The central part of the gene encodes a dipeptide Thr-Gly repeat that has been implicated in the evolution of both circadian and ultradian rhythms. We have previously observed that length variation in the repeat follows a latitudinal cline in Europe and North Africa, so we have sought to extend this observation to the southern hemisphere. We observe a parallel cline in Australia for one of the two major length variants and find higher levels of some Thr-Gly length variants, particularly at the tropical latitudes, that are extremely rare in Europe. In addition we examined >40 haplotypes from sub-Saharan Africa and find a very different and far more variable profile of Thr-Gly sequences. Statistical analysis of the periodicity and codon content of the repeat from all three continents reveals a possible mechanism that may explain how the repeat initially arose in the ancestors of the D. melanogaster subgroup of species. Our results further reinforce the view that thermal selection may have contributed to shaping the continental patterns of Thr-Gly variability.     

Conformational analysis of peptide T and of its C-pentapeptide fragment

The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.     

Length polymorphism in the threonine-glycine-encoding repeat region of theperiod gene inDrosophila

Summary Single-fly polymerase chain reaction amplification and direct DNA sequencing revealed high levels of length polymorphism in the threonine-glycine encoding repeat region of theperiod (per) gene in natural populations ofDrosophila melanogaster. DNA comparison of two alleles of identical lengths gave a high number of synonymous substitutions suggesting an ancient time of separation. However detailed examination of the sequences of different Thr-Gly length variants indicated that this divergence could be understood in terms of four deletion/insertion events. InDrosophila pseudoobscura a length polymorphism is observed in a five-amino acid degenerate repeat, which corresponds tomelanogaster's Thr-Gly domain. In spite of the differences betweenD. melanogaster andD. pseudoobscura in the amino acid sequence of the repeats, the predicted secondary structures suggest evolutionary and mechanistic constraints on theper protein of these two species.     

Evolutionary analysis of PHLPP1 gene in humans and non-human primates

The chromosome 18q22-23 region has been shown to be implicated in bipolar disorder (BPAD) by several studies. PHLPP1 gene, in the locus (chromosome 18q22-23), is involved in circadian pathways and bears modules like ‘PH domain and leucine rich repeat protein phosphatase’. This gene also contains a polyglutamine (CAG or PolyQ) repeat motif at the carboxyl terminal end. A comparative analysis of the PolyQ repeats of the PHLPP1 gene in humans, non-human primates and other species has been attempted in order to investigate the possible significance of repeat length as seen in other triplet-repeat associated diseases. Sequencing of the CAG repeat in humans and in non-human primates revealed that the CAG repeat is not polymorphic in humans; whereas, in other species it shows an area of high variability, both in length and sequence composition. Despite the conservation of circadian clock components in different species, there is remarkable diversity in the protein structure, regulation and biochemical functions of the circadian orthologs. These can be due to specific adaptations in accordance with the physiology of the particular species providing a species-specific biological advantage.     

Insights into the structural variation between pentapeptide repeat proteins--crystal structure of Rfr23 from Cyanothece 51142

Cyanothece sp. PCC 51142 contains 35 pentapeptide repeat proteins (PRPs), proteins that contain a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. Published crystal structures of PRPs show that the tandem pentapeptide repeats adopt a type of right-handed quadrilateral beta-helix called an Rfr-fold. To characterize how structural features of Rfr-folds might vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.4A resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue alpha-helix (M26(*)-E34). The Rfr-fold of Rfr23 contains four structural features previously unobserved in Rfr-folds. First, Rfr23 is composed entirely of type II beta-turns. Second, the pentapeptide repeats are not consecutive in the primary amino acid sequence. Instead, Rfr23 contains 24-residues protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56-P79) (24-residue insertion). Third, a disulfide bond between C39 and C42 bridges the beta-turn between the first and second pentapeptide repeats in the first coil (disulfide bracket). NMR spectroscopy indicates that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue perturbs the Rfr-fold slightly in the last coil between the C-terminal two pentapeptide repeats (single-residue bulge).     

The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant strain 543 of the cyanobacterium Anabaena sp. strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis. Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall. Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography. The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids. The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX. The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region. A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543. We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.     

Interspecific comparison of the period gene of Drosophila reveals large blocks of non-conserved coding DNA.

We have cloned and sequenced the coding region of the period (per) gene from Drosophila pseudoobscura and D. virilis. A comparison with that of D. melanogaster reveals that the conceptual translation products consist of interspersed blocks of conserved and non-conserved amino acid sequence. The non-conserved portion, comprising approximately 33% of the protein sequence, includes the perfect Thr-Gly repeat of D. melanogaster, which is absent from the D. pseudoobscura and D. virilis proteins. Based on these observations and cross-species transformation experiments, we suggest that the interspecific variability in the per primary amino acid sequence contributes to the control of species-specific behaviors.     

Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles.

Synaptophysin is a major integral membrane protein of synaptic vesicles. Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy-terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure. The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin. By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure. Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.     

Timekeeping in bacteria: the cyanobacterial circadian clock

The prokaryotes known as cyanobacteria possess an endogenous 24h biological (circadian) clock that provides temporal coordination for physiological processes. Although the cyanobacterial clock has the same fundamental properties as circadian clocks in eukaryotes, its components are non-homologous to those of animals, plants or fungi. Moreover, its mechanism is likely to be very different from that depicted in eukaryotic clock models. The picture that is emerging for the timing mechanism in cyanobacteria is of a multiprotein, multimeric, molecular machine composed of proteins whose domains exhibit twists on common themes. Signal transduction into and out of the clock core appears to occur via histidine protein kinase-based phosphorylation relays.