Comparative sequence analysis and patterns of covariation in RNA secondary structures

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Positive selection drives the evolution of the Acp29AB accessory gland protein in Drosophila.

Nucleotide sequence variation at the Acp29AB gene region has been surveyed in Drosophila melanogaster from Spain (12 lines), Ivory Coast (14 lines), and Malawi (13 lines) and in one line of D. simulans. The approximately 1.7-kb region studied encompasses the Acp29AB gene that codes for a male accessory gland protein and its flanking regions. Seventy-seven nucleotide and 8 length polymorphisms were detected. Nonsynonymous polymorphism was an order of magnitude lower than synonymous polymorphism, but still high relative to other non-sex-related genes. In D. melanogaster variation at this region revealed no major genetic differentiation between East and West African populations, while differentiation was highly significant between the European and the two African populations. Comparison of polymorphism and divergence at synonymous and nonsynonymous sites showed an excess of fixed nonsynonymous changes, which indicates that the evolution of the Acp29AB protein has been driven by directional selection at least after the split of the D. melanogaster and D. simulans lineages. The pattern of variation in extant populations of D. melanogaster favors a scenario where the fixation of advantageous replacement substitutions occurred in the early stages of speciation and balancing selection is maintaining variation in this species.     

DNA variation at the rp49 gene region of Drosophila simulans: evolutionary inferences from an unusual haplotype structure.

An approximately 1.3-kb region including the rp49 gene plus its 5' and 3' flanking regions was sequenced in 24 lines of Drosophila simulans (10 from Spain and 14 from Mozambique). Fifty-four nucleotide and 8 length polymorphisms were detected. All nucleotide polymorphisms were silent: 52 in noncoding regions and 2 at synonymous sites in the coding region. Estimated silent nucleotide diversity was similar in both populations (pi = 0.016, for the total sample). Nucleotide variation revealed an unusual haplotype structure showing a subset of 11 sequences with a single polymorphism. This haplotype was present at intermediate frequencies in both the European and the African samples. The presence of such a major haplotype in a highly recombining region is incompatible with the neutral equilibrium model. This haplotype structure in both a derived and a putatively ancestral population can be most parsimoniously explained by positive selection. As the rate of recombination in the rp49 region is high, the target of selection should be close to or within the region studied.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

Nucleotide Variation and Conservation at the Dpp Locus, a Gene Controlling Early Development in Drosophila

A study of polymorphism and species divergence of the dpp gene of Drosophila has been made. Eighteen lines from a population of D. melanogaster were sequenced for 5200 bp of the Hin region of the gene, coding for the dpp polypeptide. A comparison was made with sequence from D. simulans. Ninety-six silent polymorphisms and three amino acid replacement polymorphisms were found. The overall silent polymorphism (0.0247) is low, but haplotype diversity (0.0066 for effectively silent sites and 0.0054 for all sites) is in the range found for enzyme loci. Amino acid variation is absent in the N-terminal signal peptide, the C-terminal TGF-β peptide and in the N-terminal half of the pro-protein region. At the nucleotide level there is strong conservation in the middle half of the large intron and in the 3' untranslated sequence of the last exon. The 3' untranslated conservation, which is perfect for 110 bp among all the divergent species, is unexplained. There is strong positive linkage disequilibrium among polymorphic sites, with stretches of apparent gene conversion among originally divergent sequences. The population apparently is a migration mixture of divergent clades.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.      

Divergent structure and function of the bicoid gene in Muscoidea fly species

SUMMARY We have investigated the evolution of the bicoid ( bcd ) gene in fly species of the Muscoidea Superfamily. We obtained the complete bcd sequence from the housefly Musca domestica and found polymorphism in the coding region among Musca strains. In addition to Musca , we cloned most of the bcd coding sequences from two blowfly species Calliphora vicina and Lucilia sericata . The 5' and 3' regulatory regions flanking the Musca bcd gene are widely diverged in sequence from Drosophila; however, some important sequence motifs identified in Drosophila bcd are present. The predicted RNA secondary structures of the 3' UTRs are similar, despite sequence divergence. Comparison of Bicoid (Bcd) proteins shows a serine-rich domain of unknown function is present in the Muscoidea species, but is absent in other species. The in vivo function of bcd in Musca was tested by RNAi to mimic loss of function phenotype. We obtained a head defect phenotype similar to weak bcd alleles of Drosophila . Although our comparisons initially suggest functional conservation between species, closer inspection reveals significant differences. Divergence of structural motifs, such as regulatory elements in flanking regions and conservation of protein domains in some species but not in others, points to functional divergence between species. We suggest that the larger embryonic size in Muscoidea species restricts the morphogenetic activity of a weak Bcd activator, which has evolved a more specialized role in head determination and lost some functions in thoracic development.     

Historical Selection, Amino Acid Polymorphism and Lineage-Specific Divergence at the G6pd Locus in Drosophila Melanogaster and D. Simulans

The nucleotide diversity across 1705 bp of the G6pd gene is studied in 50 Drosophila melanogaster and 12 D. simulans lines. Our earlier report contrasted intraspecific polymorphism and interspecific differences at silent and replacement sites in these species. This report expands the number of European and African lines and examines the pattern of polymorphism with respect to the common A/B allozymes. In D. melanogaster the silent nucleotide diversity varies 2.8-fold across localities. The B allele sequences are two- to fourfold more variable than the derived A allele, and differences between allozymes are twice as among B alleles. There is strong linkage disequilibrium across the G6pd region. In both species the level of silent polymorphism increases from the 5' to 3' ends, while there is no comparable pattern in level of silent site divergence or fixation. The neutral model is not rejected in either species. Using D. yakuba as an outgroup, the D. melanogaster lineage shows a twofold greater rate of silent fixation, but less than half the rate of amino acid replacement. Lineage-specific differences in mutation fixation are inconsistent with neutral expectations and suggest the interaction of species-specific population size differences with both weakly advantageous and deleterious selection.     

The evolution of an alpha-esterase pseudogene inactivated in the Drosophila melanogaster lineage

Previous analyses of the alpha-esterase cluster of Drosophila melanogaster revealed 10 active genes and the DmalphaE4a-Psi pseudogene. Here, we reconstruct the evolution of the pseudogene from the sequences of 12 alleles from widely scattered D. melanogaster populations and single alleles from Drosophila simulans and Drosophila yakuba. All of the DmalphaE4a-Psi alleles contain numerous inactivating mutations, suggesting that pseudogene alleles are fixed in natural populations. Several lines of evidence also suggest that DmalphaE4a is now evolving without selective constraint in the D. melanogaster lineage. There are three polymorphic indels which result in frameshifts; a key nucleotide of the intron splice acceptor is polymorphic; the neutral mutation parameter is the same for replacement and silent sites; one of the nonsilent polymorphisms results in a stop codon; only 1 of the 13 replacement polymorphisms is biochemically conservative; residues that are conserved among active esterases have different states in DmalphaE4a-Psi; and there are about half as many transitional polymorphisms as transversional ones. In contrast, the D. simulans and D. yakuba orthologs DsalphaE4a and DyalphaE4a do not have the inactivating mutations of DmalphaE4a-Psi and appear to be evolving under the purifying selection typical of protein- encoding genes. For instance, there have been more substitutions in the introns than in the exons, and more in silent sites than in replacement sites. Furthermore, most of the amino acid substitutions that have occurred between DyalphaE4a and DsalphaE4a are located in sites that typically vary among active alpha-esterases rather than those that are usually conserved. We argue that the original alphaE4a gene had a function which it has lost since the divergence of the D. melanogaster and D. simulans lineages.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Large number of replacement polymorphisms in rapidly evolving genes of Drosophila. Implications for genome-wide surveys of DNA polymorphism

We present a survey of nucleotide polymorphism of three novel, rapidly evolving genes in populations of Drosophila melanogaster and D. simulans. Levels of silent polymorphism are comparable to other loci, but the number of replacement polymorphisms is higher than that in most other genes surveyed in D. melanogaster and D. simulans. Tests of neutrality fail to reject neutral evolution with one exception. This concerns a gene located in a region of high recombination rate in D. simulans and in a region of low recombination rate in D. melanogaster, due to an inversion. In the latter case it shows a very low number of polymorphisms, presumably due to selective sweeps in the region. Patterns of nucleotide polymorphism suggest that most substitutions are neutral or nearly neutral and that weak (positive and purifying) selection plays a significant role in the evolution of these genes. At all three loci, purifying selection of slightly deleterious replacement mutations appears to be more efficient in D. simulans than in D. melanogaster, presumably due to different effective population sizes. Our analysis suggests that current knowledge about genome-wide patterns of nucleotide polymorphism is far from complete with respect to the types and range of nucleotide substitutions and that further analysis of differences between local populations will be required to understand the forces more completely. We note that rapidly diverging and nearly neutrally evolving genes cannot be expected only in the genome of Drosophila, but are likely to occur in large numbers also in other organisms and that their function and evolution are little understood so far.     

Recurrent positive selection at bgcn, a key determinant of germ line differentiation, does not appear to be driven by simple coevolution with its partner protein bam

Surveys of nucleotide sequence polymorphism in Drosophila melanogaster and Drosophila simulans were performed at 2 interacting loci crucial for gametogenesis: bag-of-marbles (bam) and benign gonial cell neoplasm (bgcn). At the polymorphism level, both loci appear to be evolving under the expectations of the neutral theory. However, ratios of polymorphism and divergence for synonymous and nonsynonymous mutations depart significantly from neutral expectations for both loci consistent with a previous observation of positive selection at bam. The deviations suggest either an excess of synonymous polymorphisms or an excess of nonsynonymous fixations at both loci. Synonymous evolution appears to conform to neutrality at bam. At bgcn, there is evidence of positive selection affecting preferred synonymous mutations along the D. simulans lineage. However, there is also a significantly higher rate of nonsynonymous fixations at bgcn within D. simulans. Thus, the deviation from neutrality detected by the McDonald-Kreitman test at these 2 loci is likely due to the selective acceleration of nonsynonymous fixations. Differences in the pattern of amino acid fixations between these 2 interacting proteins suggest that the detected positive selection is not due to a simple model of coevolution.