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.     

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.     

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.     

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.     

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.     

Genetic variability in the complete mitochondrial control region of the Eurasian otter (Lutra lutra) in the Iberian Peninsula

Sequencing of the complete mitochondrial DNA control region from 31 samples of the Eurasian otter, Lutra lutra , enabled us to establish the length and structure of this fragment, as well as to describe, for the first time, the RS3 repetitive region located at the 3' end. In addition, genetic variability of the 5' end was examined in 63 individuals, 57 of which were wild otters from the Iberian Peninsula and six captive reared otters. This analysis resulted in extremely low variability. All the samples from the Iberian Peninsula share a single haplotype, Lut 1, the most common haplotype in Europe. Captive otters showed two haplotypes: Lut 3, which has been described in wild otters from eastern Germany, and Lut 6, an haplotype not described to date. Higher variability was observed in the repetitive RS3 region. The tandem repeat was composed of an array of ten repeat units of 22 bp with differences in the repetitive motifs that differed in the arrays of different specimens. In total, 20 different haplotypes from 31 individuals were found. However, the geographical distribution of these haplotypes did not generate a phylogeographical signal. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 397–403.     

Polyploidy,phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL-trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian-Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian-Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long-term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.     

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.     

Geographical segregation in Dunlin Calidris alpina populations wintering along the East Atlantic migratory flyway – evidence from mitochondrial DNA analysis

Dunlin Calidris alpina is one of the most abundant shorebirds using coastal habitats in the East Atlantic migratory flyway, that links arctic breeding locations (Greenland to Siberia) with wintering grounds (West Europe to West Africa). Differential migration and winter segregation between populations have been indicated by morphometrics and ringing recoveries. Here, we analyse the potential of genetic markers (mitochondrial DNA – mtDNA) to validate and enhance such findings. We compared mtDNA haplotypes frequencies at different wintering sites (from north-west Europe to West Africa). All birds from West Africa had western (European) haplotypes, while the eastern (Siberian) haplotypes were only present in European winter samples, reaching higher frequencies further north in Europe. Compilation of published results from migrating birds also confirmed these differences, with the sole presence of European haplotypes in Iberia and West Africa and increasingly higher frequencies of Siberian haplotypes from south-west to north-west Europe. Comparison with published haplotype frequencies of breeding populations shows that birds from Greenland, Iceland, and North Europe were predominant in wintering grounds in West Africa, while populations wintering in West Europe originated from more eastern breeding grounds (e.g. North Russia). These results show that genetic markers can be used to enhance the integrative monitoring of wintering and breeding populations, by providing biogeographical evidence that validate the winter segregation of breeding populations.     

A latitudinal cline in a Drosophila clock gene.

The clock gene period determines biological rhythmicity in Drosophila melanogaster and encodes a protein characterized by an alternating series of threonine-glycine pairs. The minisatellite region encoding the threonine-glycine repeat is polymorphic in length in natural Drosophila melanogaster populations. In this paper we report the geographical analysis of this polymorphism within Europe and North Africa. A robust clinal pattern is observed along a north-south axis. We suggest the possibility that the length polymorphism could be maintained by thermal selection because the threonine-glycine region has been shown to provide thermostability to the circadian phenotype.     

Different period gene repeats take 'turns' at fine-tuning the circadian clock

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.     

The Rosy Region of Drosophila Melanogaster and Drosophila Simulans. I. Contrasting Levels of Naturally Occurring DNA Restriction Map Variation and Divergence

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.     

Time to the most recent common ancestor and divergence times of populations of common chaffinches (Fringilla coelebs) in Europe and North Africa: insights into Pleistocene refugia and current levels of migration

We analyzed sequences from a 275-bp hypervariable region in the 5' end of the mitochondrial DNA control region in 190 common chaffinches (Fringilla coelebs) from 19 populations in Europe and North Africa, including new samples from Greece and Morocco. Coalescent techniques were applied to estimate the time to the most recent common ancestor (TMRCA) and divergence times of these populations. The first objective of this study was to infer the locations of refugia where chaffinches survived the last glacial episode, and this was achieved by estimating the TMRCA of populations in regions surrounding the Mediterranean that were unglaciated in the late Pleistocene. Although extant populations in Iberia, Corsica, Greece, and North Africa harbor haplotypes that are basal in a phylogenetic tree, this information alone cannot be used to infer that these localities served as refugia, because it is impossible to infer the ages of populations and their divergence times without also considering the population genetic processes of mutation, migration, and drift. Provided we assume the TMRCAs of populations are a reasonable estimate of a population's age, coalescent-based methods place resident populations in Iberia, Corsica, Greece, and North Africa during the time of the last glacial maximum, suggesting these regions served as refugia for the common chaffinch. The second objective was to determine when populations began diverging from each other and to use this as a baseline to estimate current levels of gene flow. Divergence time estimates suggest that European populations began diverging about 60,000 years before present. The relatively recent divergence of populations in North Africa, Italy, and Iberia may explain why classic migration estimates based on equilibrium assumptions are high for these populations. We compare these estimates with nonequilibrium-based estimates and show that the nonequilibrium estimates are consistently lower than the equilibrium estimates.     

Speciation in Pararge (Satyrinae: Nymphalidae) butterflies – North Africa is the source of ancestral populations of all Pararge species

Abstract.  The genus Pararge comprises three species: P. aegeria , distributed in Europe and North Africa; P. xiphia , endemic to Madeira; and P. xiphioides , endemic to the Canary Islands. Two subspecies are recognized in P. aegeria , P. a. tircis and P. a. aegeria , distributed in northern and southern Europe, respectively. In the 1970s, P. aegeria appeared on Madeira. However, despite the status of P. aegeria as a model species in ecological studies, the evolutionary history of Pararge remains unknown. We studied the phylogenetic relationships of the three Pararge species, using the mitochondrial gene cytochrome oxidase subunit I and the nuclear gene wingless to infer modes and times of speciation. On the basis of our analyses, Pararge forms a strongly supported monophyletic group, with the DNA haplotypes of the three species also forming well-supported monophyletic groups. We found that P. xiphia diverged first from the common ancestor a maximum of five million years ago, with P. xiphioides and P. aegeria being sister species that diverged a maximum of three million years ago. The two subspecies, P. a. tircis and P. a. aegeria , were not distinguishable on the basis of DNA haplotypes; instead, our data clearly distinguished between European specimens and those from North Africa. Madeiran P. aegeria has North African haplotypes and thus originated from there rather than from Europe. We hypothesize that the Mediterranean Sea forms a strong barrier to dispersal for Pararge butterflies, and has done so for approximately the past one million years.     

Genome organization and polymorphism of the murine beta-glucuronidase region

Thirty-eight kilobases of mouse genomic DNA which surround and include the coding sequences for beta-glucuronidase has been mapped. Intron-exon arrangements were determined by hybridization of genomic sequences with cDNA clones, and minimum estimates of gene length (11-17 kb) and intron number were obtained. Only a single gene was observed when genomic DNA was probed with subclones containing beta-glucuronidase coding sequence; there was no evidence of duplicated or pseudogenes. However, sequences distal to the 3' end of the gene are present elsewhere in the genome in a limited number of copies. Eight haplotypes of the beta-glucuronidase region with differing regulatory genotypes were compared for restriction fragment polymorphisms. Surprisingly little was found, considering the diverse origin of the haplotypes. Two of the polymorphisms that were found may be correlated with regulatory phenotypes. A BamHI site is missing from the CS and CL haplotypes that share regulatory properties, and a 0.2-kb insertion is consistently present in haplotypes showing increased response to induction by androgens in kidney.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.