Intron length evolution in Drosophila

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.     

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.     

Selective constraints on intron evolution in Drosophila

Intron sizes show an asymmetrical distribution in a number of organisms, with a large number of "short" introns clustered around a minimal intron length and a much broader distribution of longer introns. In Drosophila melanogaster, the short intron class is centered around 61 bp. The narrow length distribution suggests that natural selection may play a role in maintaining intron size. A comparison of 15 orthologous introns among species of the D. melanogaster subgroup indicates that, in general, short introns are not under greater DNA sequence or length constraints than long introns. There is a bias toward deletions in all introns (deletion/insertion ratio is 1.66), and the vast majority of indels are of short length (<10 bp). Indels occurring on the internal branches of the phylogenetic tree are significantly longer than those occurring on the terminal branches. These results are consistent with a compensatory model of intron length evolution in which slightly deleterious short deletions are frequently fixed within species by genetic drift, and relatively rare larger insertions that restore intron length are fixed by positive selection. A comparison of paralogous introns shared among duplicated genes suggests that length constraints differ between introns within the same gene. The janusA, janusB, and ocnus genes share two short introns derived from a common ancestor. The first of these introns shows significantly fewer indels than the second intron, although the two introns show a comparable number of substitutions. This indicates that intron-specific selective constraints have been maintained following gene duplication, which preceded the divergence of the D. melanogaster species subgroup.     

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster

The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved.     

Molecular Evolution of Drosophila Metallothionein Genes

The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy metal detoxification. Several different tandem duplications of Mtn have been shown to increase cadmium and copper tolerance, as well as Mtn expression. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, we compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee and Georgia. Restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications and a subset (327) of these lines for Mto duplications. The frequency of pooled Mtn duplications found ranged from 0% to 20%, and was not significantly higher at the contaminated sites. No Mto duplications were identified. Estimates of sequence diversity at the Mtn locus among a subsample (92) of the duplication survey were obtained using four-cutter analysis. This analysis revealed a low level of polymorphism, consistent with both selection at the Mtn locus, and a fairly recent origin for the duplications. To further examine this hypothesis, we sequenced an Mtn allele of Drosophila simulans and measured the amount of nucleotide sequence divergence between D. simulans and its sibling species D. melanogaster. The levels of silent nucleotide polymorphism and divergence in the Mtn region were compared with those in the Adh region, using the neutrality test of R.R. Hudson, M. Kreitman and M. Aguadé.     

Evolution of the AMP-forming acetyl-CoA synthetase gene in the Drosophilidae family

Analysis of the AMP-forming ACS gene was performed in 12 species of the Drosophilidae family. Systematically four introns, aligned at the same positions, were detected, but none of them showed a position similar to those known for species outside the Drosophilidae family. The average length of introns varied from 63 to 75 bp but in two species Drosophila takahashii and D. kikkawai the length of the second intron was 343 and 210 bp, respectively. In coding regions, about 80% of the third codon positions were substituted while first and second positions showed, respectively, 14% and 6% substitutions. Interestingly, the divergence observed at the protein level between species was very low. The phylogenetic tree based on the DNA sequences of the exons was mainly in agreement with taxonomic classification and previous molecular phylogenies except for D. ananassae, which appeared more closely related to D. subobscura and D. funebris than to the species of the melanogaster group.     

The correlation between intron length and recombination in drosophila. Dynamic equilibrium between mutational and selective forces

Intron length is negatively correlated with recombination in both Drosophila melanogaster and humans. This correlation is not likely to be the result of mutational processes alone: evolutionary analysis of intron length polymorphism in D. melanogaster reveals equivalent ratios of deletion to insertion in regions of high and low recombination. The polymorphism data do reveal, however, an excess of deletions relative to insertions (i.e., a deletion bias), with an overall deletion-to-insertion events ratio of 1.35. We propose two types of selection favoring longer intron lengths. First, the natural mutational bias toward deletion must be opposed by strong selection in very short introns to maintain the minimum intron length needed for the intron splicing reaction. Second, selection will favor insertions in introns that increase recombination between mutations under the influence of selection in adjacent exons. Mutations that increase recombination, even slightly, will be selectively favored because they reduce interference among selected mutations. Interference selection acting on intron length mutations must be very weak, as indicated by frequency spectrum analysis of Drosophila intron length polymorphism, making the equilibrium for intron length sensitive to changes in the recombinational environment and population size. One consequence of this sensitivity is that the advantage of longer introns is expected to decrease inversely with the rate of recombination, thus leading to a negative correlation between intron length and recombination rate. Also in accord with this model, intron length differs between closely related Drosophila species, with the longest variant present more often in D. melanogaster than in D. simulans. We suggest that the study of the proposed dynamic model, taking into account interference among selected sites, might shed light on many aspects of the comparative biology of genome sizes including the C value paradox.     

Evolution of Polypyrimidines in Drosophila

We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, bu the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polpurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.--These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA . These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.     

Changes in the recombinational environment affect divergence in the yellow gene of Drosophila

The complete coding region of the yellow (y) gene was sequenced in different Drosophila species. In the species of the melanogaster subgroup (D. melanogaster, D. simulans, D. mauritiana, D. yakuba, and D. erecta), this gene is located at the tip of the X chromosome in a region with a strong reduction in recombination rate. In contrast, in D. ananassae (included in the ananassae subgroup of the melanogaster group) and in the obscura group species (D. subobscura, D. madeirensis, D. guanche, and D. pseudoobscura), the y gene is located in regions with normal recombination rates. As predicted by the hitchhiking and background selection models, this change in the recombinational environment affected synonymous divergence in the y-gene-coding region. Estimates of the number of synonymous substitutions per site were much lower between the obscura group species and D. ananassae than between the species of the obscura group and the melanogaster subgroup. In fact, a highly significant increase in the rate of synonymous substitution was detected in all lineages leading to the species of the melanogaster subgroup relative to the D. ananassae lineage. This increase can be explained by a higher fixation rate of mutations from preferred to unpreferred codons (slightly deleterious mutations). The lower codon bias detected in all species of the melanogaster subgroup relative to D. ananassae (or to the obscura group species) would be consistent with this proposal. Therefore, at least in Drosophila, changes in the recombination rate in different lineages might cause deviations of the molecular-clock hypothesis and contribute to the overdispersion of the rate of synonymous substitution. In contrast, the change in the recombinational environment of the y gene has no detectable effect on the rate of amino acid replacement in the Yellow protein.     

Inferring Weak Selection from Patterns of Polymorphism and Divergence at ``silent' Sites in Drosophila DNA

Patterns of codon usage and ``silent'''' DNA divergence suggest that natural selection discriminates among synonymous codons in Drosophila. ``Preferred'''' codons are consistently found in higher frequencies within their synonymous families in Drosophila melanogaster genes. This suggests a simple model of silent DNA evolution where natural selection favors mutations from unpreferred to preferred codons (preferred changes). Changes in the opposite direction, from preferred to unpreferred synonymous codons (unpreferred changes), are selected against. Here, selection on synonymous DNA mutations is investigated by comparing the evolutionary dynamics of these two categories of silent DNA changes. Sequences from outgroups are used to determine the direction of synonymous DNA changes within and between D. melanogaster and Drosophila simulans for five genes. Population genetics theory shows that differences in the fitness effect of mutations can be inferred from the comparison of ratios of polymorphism to divergence. Unpreferred changes show a significantly higher ratio of polymorphism to divergence than preferred changes in the D. simulans lineage, confirming the action of selection at silent sites. An excess of unpreferred fixations in 28 genes suggests a relaxation of selection on synonymous mutations in D. melanogaster. Estimates of selection coefficients for synonymous mutations (3.6 <|N(e)s| < 1.3) in D. simulans are consistent with the reduced efficacy of natural selection (|N(e)s| < 1) in the three- to sixfold smaller effective population size of D. melanogaster. Synonymous DNA changes appear to be a prevalent class of weakly selected mutations in Drosophila.     

The hemoglobin genes of Drosophila

We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the databases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds/dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system. Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae. Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved. In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0. Both genes are conserved in various drosophilid species, but only expression of glob2 could be demonstrated by western blotting and RT-PCR. Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins. glob1 is closely related to the intracellular hemoglobin of the botfly Gasterophilus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade.     

Nucleotide variation at the runt locus in Drosophila melanogaster and Drosophila simulans.

Intra- and interspecific nucleotide variation for the major developmental gene runt in Drosophila was studied in D. melanogaster and D. simulans. The 1.5-kb protein-coding region and the 0.4-kb intron of the runt gene were sequenced for 11 alleles in each species. The D. melanogaster alleles originated from east Africa. Estimated parameters of intraspecific variation in D. melanogaster (exons: theta = 0.018, pi = 0.018; intron: theta = 0.014, pi = 0.014) and D. simulans (exons: theta = 0.007, pi = 0.005; intron: theta = 0.008, pi = 0.005) were below average for other X-linked genes, while divergence between species (exons: D = 0.094; intron: D = 0.069) fell within the normal range for both silent and replacement changes. This estimate for runt, along with published values for three other genes in regions of normal recombination, show east African D. melanogaster to be roughly twice as polymorphic as D. simulans. The majority of nucleotide variation, silent and replacement, in both species was found to be selectively neutral using various statistical tests (HKA, McDonald-Kreitman, Tajima, and Fu and Li tests). Monte Carlo simulations of the coalescent process significantly rejected a Wright-Fisher model with respect to an amino acid polymorphism and the distribution of polymorphic sites among the D. simulans lines. This indicated an old lineage and may reflect ancestral population substructuring in D. simulans.     

The intron boundaries and flanking rRNA coding sequences of Calliphora erythrocephala rDNA

We have sequenced the available cloned examples of the intron-coding sequence junctions for the rDNA of the higher Dipteran, Calliphora erythrocephala. The introns interrupt the rDNA at the same position as the type 1 intron family detected in Drosophila melanogaster and D. virilis (10,11). A duplication of 14 base pairs of the 28S rRNA coding sequence surrounds a short version of the major genomic length class of introns. This same duplication is associated with boundaries of the type 1 introns in D. virilis and D. melanogaster (10, 13,14). We have detected considerable homology between the 3' intron sequences of C. erythrocephala and D. virilis. The rRNA coding sequences flanking the introns are extremely homologous in C. erythrocephala, D. melanogaster and D. virilis, with only one small region of significant divergence. This corresponds to a variable stem region previously identified in eukaryotic 28S rRNA at a site analogous to the L1 ribosomal protein binding site of prokaryotic 23S rRNA (27).     

How intron splicing affects the deletion and insertion profile in Drosophila melanogaster

Studies of "dead-on-arrival" transposable elements in Drosophila melanogaster found that deletions outnumber insertions approximately 8:1 with a median size for deletions of approximately 10 bp. These results are consistent with the deletion and insertion profiles found in most other Drosophila pseudogenes. In contrast, a recent study of D. melanogaster introns found a deletion/insertion ratio of 1.35:1, with 84% of deletions being shorter than 10 bp. This discrepancy could be explained if deletions, especially long deletions, are more frequently strongly deleterious than insertions and are eliminated disproportionately from intron sequences. To test this possibility, we use analysis and simulations to examine how deletions and insertions of different lengths affect different components of splicing and determine the distribution of deletions and insertions that preserve the original exons. We find that, consistent with our predictions, longer deletions affect splicing at a much higher rate compared to insertions and short deletions. We also explore other potential constraints in introns and show that most of these also disproportionately affect large deletions. Altogether we demonstrate that constraints in introns may explain much of the difference in the pattern of deletions and insertions observed in Drosophila introns and pseudogenes.     

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.