Divergence of the Yellow Gene between Drosophila Melanogaster and D. Subobscura: Recombination Rate, Codon Bias and Synonymous Substitutions

The yellow (y) gene maps near the telomere of the X chromosome in Drosophila melanogaster but not in D. subobscura. Thus the strong reduction in the recombination rate associated with telomeric regions is not expected in D. subobscura. To study the divergence of a gene whose recombination rate differs between two species, the y gene of D. subobscura was sequenced. Sequence comparison between D. melanogaster and D. subobscura revealed several elements conserved in noncoding regions that may correspond to putative cis-acting regulatory sequences. Divergence in the y gene coding region between D. subobscura and D. melanogaster was compared with that found in other genes sequenced in both species. Both, yellow and scute exhibit an unusually high number of synonymous substitutions per site (p(s)). Also for these genes, the extent of codon bias differs between both species, being much higher in D. subobscura than in D. melanogaster. This pattern of divergence is consistent with the hitchhiking and background selection models that predict an increase in the fixation rate of slightly deleterious mutations and a decrease in the rate of fixation of slightly advantageous mutations in regions with low recombination rates such as in the y-sc gene region of D. melanogaster.     

Nucleotide variation at the yellow gene region is not reduced in Drosophila subobscura: a study in relation to chromosomal polymorphism

In contrast to Drosophila melanogaster and Drosophila simulans, the yellow (y) gene region of Drosophila subobscura is not located in a region with a strong reduction in recombination. In addition, this gene maps very close to the breakpoints of different inversions that segregate as polymorphic in natural populations of D. subobscura. Therefore, levels of variation at the y gene region in this species relative to those found in D. melanogaster and D. simulans may be affected not only by the change in the recombinational environment, but also by the presence of inversion polymorphism. To further investigate these aspects, an approximately 5.4-kb region of the A (=X) chromosome including the y gene was sequenced in 25 lines of D. subobscura and in the closely related species Drosophila madeirensis and Drosophila guanche. The D. subobscura lines studied differed in their A-chromosomal arrangements, A(st), A(2), and A(1). Unlike in D. melanogaster and D. simulans, levels of variation at the y gene region of D. subobscura are not reduced relative to those found at other genomic regions in the same species (rp49, Acp70A, and Acph-1). This result supports the effect of the change in the recombinational environment of a particular gene on the level of neutral variation. In addition, nucleotide variation is affected by chromosomal polymorphism. A strong genetic differentiation is detected between the A(1) arrangement and either A(st) or A(2), but not between A(st) and A(2). This result is consistent with the location of the y gene relative to the breakpoints of inversions A(1) and A(2). In addition, the pattern of nucleotide polymorphism in A(st)+A(2) and A(1) seems to point out that variation at the y gene region within these chromosomal classes is in the phase transient to equilibrium. The estimated ages of these arrangements assuming a star genealogy indicate that their origin cannot predate the D. madeirensis split. Therefore, the present results are consistent with a chromosomal phylogeny where Am(1), which is an arrangement present in D. madeirensis but absent in current populations of D. subobscura, would be the ancestral arrangement.     

The 5S ribosomal genes in the Drosophila melanogaster species subgroup. Nucleotide sequence of a 5S unit from Drosophila simulans and Drosophila teissieri

The 5S genes of the eight species of the D. melanogaster subgroup have been mapped. The spacers, in contrast with coding regions, differ markedly between most species. One 5S gene unit has been sequenced for both D. simulans and D. teissieri. The mature 5S RNA region in these two species is identical to the corresponding region of D. melanogaster. Only 5 nucleotide variations occur between the D. melanogaster and D. simulans 5S gene spacers. The spacer in D. teissieri is very different. Only two segments, located one at each side of the coding region, are clearly homologous to corresponding sequences of D. melanogaster and D. simulans.     

Gypsy homologous sequences inDrosophila subobscura (gypsyDS)

Summary Characterization of sequences homologous to theDrosophila melanogaster gypsy transposable element was carried out inDrosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species.GypsyDS sequences are located in both euchromatic and heterochromatic regions. A completegypsyDS sequence was isolated from aD. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of theD. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between theD. subobscura-D. virilis sequences than betweenD. subobscura andD. melanogaster. Molecular divergence ofgypsy sequences betweenD. virilis andD. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest thatgypsy sequences have been horizontally transferred between these species.Offprint requests to: T.M. Alberola     

BAC library construction and BAC end sequencing of five Drosophila species: the comparative map with the D. melanogaster genome

We constructed and characterized arrayed bacterial artificial chromosome (BAC) libraries of five Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. auraria, and D. ananassae), which are genetically well characterized in the studies of meiosis, evolution, population genetics, and developmental biology. The BAC libraries comprise 8,000 to 12,500 clones for each species, estimated to cover the most of the genomes. We sequenced both ends of most of these BAC clones with a success rate of 91%. Of these, 53,701 clones consisting of non-repetitive BAC end sequences (BESs) were mapped with reference of the public D. melanogaster genome sequences. The BES mapping estimated that the BAC libraries of D. auraria and D. ananassae covered 47% and 57% of the D. melanogaster genome, respectively, and those of D. melanogaster, D. sechellia, and D. simulans covered 94-97%. The low coverage by BESs of D. auraria and D. ananassae may be due to the high sequence divergence with D. melanogaster. From the comparative BES mapping, 111 possible breakpoints of chromosomal rearrangements were identified in these four species. The breakpoints of the major chromosome rearrangement between D. simulans and D. melanogaster on the third chromosome were determined within 20 kb in 84E and 30 kb in 93E/F. Corresponding breakpoints were also identified in D. sechellia. The BAC clones described here will be an important addition to the Drosophila genomic resources.     

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.     

Molecular Organization of the X Chromosome in Different Species of the Obscura Group of Drosophila

Nine single copy regions located on the X chromosome have been mapped by in situ hybridization in six species of the obscura group of Drosophila. Three Palearctic species, D. subobscura, D. madeirensis and D. guanche, and three Nearctic species, D. pseudoobscura, D. persimilis and D. miranda, have been studied. Eight of the regions include known genes from D. melanogaster (Pgd, zeste, white, cut, vermilion, RNA polymerase II 215, forked and suppressor of forked) and the ninth region (lambda DsubF6) has not yet been characterized. In all six species, as in D. melanogaster, all probes hybridize to a single site. Established chromosomal arm homologies of Muller's element A are only partly supported by present results since two of the probes (Pgd and zeste) hybridize at the proximal end of the XR chromosomal arm in the three Nearctic species. In addition to the centric fusion of Muller's A (= XL) and D (= XR) elements, the metacentric X chromosome of the Nearctic species requires a pericentric inversion to account for this result. Previously proposed homologies of particular chromosomal regions of the A (= X) chromosome in the three species of the D. subobscura cluster and of the XL chromosomal arm in the three species of the D. pseudoobscura cluster are discussed in light of the present results. Location of the studied markers has changed drastically not only since the divergence between the melanogaster and obscura groups but also since the Palearctic and Nearctic species of the obscura group diverged.     

Synonymous rates at the RpII215 gene of Drosophila: variation among species and across the coding region

The region encompassing the RpII215 gene that encodes the largest component of the RNA polymerase II complex (1889 amino acids) has been sequenced in Drosophila subobscura, D. madeirensis, D. guanche, and D. pseudoobscura. Nonsynonymous divergence estimates (Ka) indicate that this gene has a very low rate of amino acid replacements. Given its low Ka and constitutive expression, synonymous substitution rates are, however, unexpectedly high. Sequence comparisons have allowed the molecular clock hypothesis to be tested. D. guanche is an insular species and it is therefore expected to have a reduced effective size relative to D. subobscura. The significantly higher rate of synonymous substitutions detected in the D. guanche lineage could be explained if synonymous mutations behave as nearly neutral. Significant departure from the molecular clock hypothesis for synonymous and nonsynonymous substitutions was detected when comparing the D. subobscura, D. pseudoobscura, and D. melanogaster lineages. Codon bias and synonymous divergence between D. subobscura and D. melanogaster were negatively correlated across the RpII215 coding region, which indicates that selection coefficients for synonymous mutations vary across the gene. The C-terminal domain (CTD) of the RpII215 protein is structurally and functionally differentiated from the rest of the protein. Synonymous substitution rates were significantly different in both regions, which strongly indicates that synonymous mutations in the CTD and in the non-CTD regions are under detectably different selection coefficients.     

The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.     

Wanderings of Hobo: A Transposon in Drosophila Melanogaster and its Close Relatives

The transposon hobo is present in the genomes of Drosophila melanogaster and Drosophila simulans (and D. mauritiana and probably D. sechellia, based on Southern blots) as full-size elements and internally deleted copies. The full-size melanogaster, simulans and mauritiana hobo elements are 99.9% identical at the DNA sequence level, and internally deleted copies in these species essentially differ only in having deletions. In addition to these, hobo-related sequences are present and detectable with a hobo probe in all these species. Those in D. melanogaster are 86-94% identical to the canonical hobo, but with many indels. We have sequenced one that appears to be inserted in heterochromatin (GenBank Acc. No. AF520587). It is 87.6% identical to the canonical hobo, but quite fragmented by indels, with remnants of other transposons inserted in and near it, and clearly is defunct. Numerous similar elements are found in the sequenced D. melanogaster genome. It has recently been shown that some are fixed in the euchromatic genome, but it is probable that still more reside in heterochromatic regions not included in the D. melanogaster genome database. They are probably all relics of an earlier introduction of hobo into the ancestral species. There appear to have been a minimum of two introductions of hobo into the melanogaster subgroup, and more likely three, two ancient and one quite recent. The recent introduction of hobo was probably followed by transfers between the extant species (whether 'horizontally' or by infrequent interspecific hybridization).     

Molecular evolution of a duplication: the sex-peptide (Acp70A) gene region of Drosophila subobscura and Drosophila madeirensis

In Drosophila melanogaster, the Acp70A gene, which is involved in the postmating reactions of the female, is a single-copy gene. However, in Drosophila subobscura, the gene is duplicated and both copies are transcribed. To study the molecular evolution of the duplication, a 2.1- kb fragment encompassing both copies of the duplication was sequenced for 10 lines of D. subobscura and one line of Drosophila madeirensis. Estimates of the divergence between the two copies of the duplicated region and between the two species studied, D. subobscura and D. madeirensis, revealed that both copies of the Acp70a gene had evolved independently since their duplication. The ratio of nonsynonymous to silent divergence between copies was generally higher than one. The McDonald and Kreitman test revealed an excess of nonsynonymous changes fixed since the duplication and before the split of the D. subobscura and D. madeirensis lineages. These results point to natural selection driving protein evolution after the duplication. Specifically, adaptive evolution appears to have caused the initial differentiation between copies of the N-terminal parts of the proteins, while purifying selection could be responsible for the high conservation of the C- terminal parts.      

Localization and characterization of X chromosome inversion breakpoints separating Drosophila mojavensis and Drosophila arizonae

Ectopic exchange between transposable elements or other repetitive sequences along a chromosome can produce chromosomal inversions. As a result, genome sequence studies typically find sequence similarity between corresponding inversion breakpoint regions. Here, we identify and investigate the breakpoint regions of the X chromosome inversion distinguishing Drosophila mojavensis and Drosophila arizonae. We localize one inversion breakpoint to 13.7 kb and localize the other to a 1-Mb interval. Using this localization and assuming microsynteny between Drosophila melanogaster and D. arizonae, we pinpoint likely positions of the inversion breakpoints to windows of less than 3000 bp. These breakpoints define the size of the inversion to approximately 11 Mb. However, in contrast to many other studies, we fail to find significant sequence similarity between the 2 breakpoint regions. The localization of these inversion breakpoints will facilitate future genetic and molecular evolutionary studies in this species group, an emerging model system for ecological genetics.     

Evolution of the Autosomal Chorion Locus in Drosophila. I. General Organization of the Locus and Sequence Comparisons of Genes S15 and S19 in Evolutionarily Distant Species

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.     

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.