Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.     

The YYRR box: a conserved dipyrimidine-dipurine sequence element in Drosophila and other eukaryotes.

We have discovered a novel DNA sequence element in Drosophila which is based upon a CTGA tandem repeat. This element has been named the YYRR box to emphasize its dipyrimidine-dipurine nature which is predicted to have unusual structural features. Southern hybridization analysis of genomic DNA indicates the presence of 25-30 copies of the YYRR box in each of three Drosophila species (melanogaster, pseudoobscura, and virilis) and conservation of genomic location within species. Similar analysis of human and rat DNA indicates the presence of YYRR related sequences in mammals as well. YYRR boxes have been localized to two genetic loci in Drosophila: Gld and a gene tentative identified as ted. These two genes exhibit correlated patterns of developmental expression and an identical mutant phenotype. Sequence analysis of the Gld YYRR box in three Drosophila species revealed a high degree of conservation despite its intronic location.     

No accelerated rate of protein evolution in male-biased Drosophila pseudoobscura genes

Sexually dimorphic traits are often subject to diversifying selection. Genes with a male-biased gene expression also are probably affected by sexual selection and have a high rate of protein evolution. We used SAGE to measure sex-biased gene expression in Drosophila pseudoobscura. Consistent with previous results from D. melanogaster, a larger number of genes were male biased (402 genes) than female biased (138 genes). About 34% of the genes changed the sex-related expression pattern between D. melanogaster and D. pseudoobscura. Combining gene expression with protein divergence between both species, we observed a striking difference in the rate of evolution for genes with a male-biased gene expression in one species only. Contrary to expectations, D. pseudoobscura genes in this category showed no accelerated rate of protein evolution, while D. melanogaster genes did. If sexual selection is driving molecular evolution of male-biased genes, our data imply a radically different selection regime in D. pseudoobscura.     

Molecular evolution of sex-biased genes in Drosophila

Studies of morphology, interspecific hybridization, protein/DNA sequences, and levels of gene expression have suggested that sex-related characters (particularly those involved in male reproduction) evolve rapidly relative to non-sex-related characters. Here we report a general comparison of evolutionary rates of sex-biased genes using data from cDNA microarray experiments and comparative genomic studies of Drosophila. Comparisons of nonsynonymous/synonymous substitution rates (d(N)/d(S)) between species of the D. melanogaster subgroup revealed that genes with male-biased expression had significantly faster rates of evolution than genes with female-biased or unbiased expression. The difference was caused primarily by a higher d(N) in the male-biased genes. The same pattern was observed for comparisons among more distantly related species. In comparisons between D. melanogaster and D. pseudoobscura, genes with highly biased male expression were significantly more divergent than genes with highly biased female expression. In many cases, orthologs of D. melanogaster male-biased genes could not be identified in D. pseudoobscura through a Blast search. In contrast to the male-biased genes, there was no clear evidence for accelerated rates of evolution in female-biased genes, and most comparisons indicated a reduced rate of evolution in female-biased genes relative to unbiased genes. Male-biased genes did not show an increased ratio of nonsynonymous/synonymous polymorphism within D. melanogaster, and comparisons of polymorphism/divergence ratios suggest that the rapid evolution of male-biased genes is caused by positive selection.     

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

Organ-specific patterns of gene expression in the reproductive tract of Drosophila are regulated by the sex-determination genes

The sex-determination genes of Drosophila act to repress the developmental pathway for the internal somatic reproductive organs of the opposite sex. By misregulating this pathway during preadult development, the organ-specific expression pattern of the glucose dehydrogenase gene (Gld) in the reproductive tract of adult flies has been changed without a concomitant sexual transformation of the reproductive organs. Misregulation of the tra, tra-2, and dsx genes leads to very similar patterns of ectopic expression of Gld. The induced ectopic patterns of Gld expression at the adult stage occur in a small subset of organs which all normally express the Gld gene during their morphogenesis. These ectopic patterns are irrevocably set during late larval-early pupal development. The normal pattern of Gld expression in several other Drosophila species is quite similar to the ectopic patterns which we have generated in D. melanogaster, suggesting that the interspecific variation in Gld expression may result from variation in the expression of the sex-determination genes.     

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.     

Rates and patterns of chromosomal evolution in Drosophila pseudoobscura and D. miranda

Comparisons of gene orders between species permit estimation of the rate of chromosomal evolution since their divergence from a common ancestor. We have compared gene orders on three chromosomes of Drosophila pseudoobscura with its close relative, D. miranda, and the distant outgroup species, D. melanogaster, by using the public genome sequences of D. pseudoobscura and D. melanogaster and approximately 50 in situ hybridizations of gene probes in D. miranda. We find no evidence for extensive transfer of genes among chromosomes in D. miranda. The rates of chromosomal rearrangements between D. miranda and D. pseudoobscura are far higher than those found before in Drosophila and approach those for nematodes, the fastest rates among higher eukaryotes. In addition, we find that the D. pseudoobscura chromosome with the highest level of inversion polymorphism (Muller's element C) does not show an unusually fast rate of evolution with respect to chromosome structure, suggesting that this classic case of inversion polymorphism reflects selection rather than mutational processes. On the basis of our results, we propose possible ancestral arrangements for the D. pseudoobscura C chromosome, which are different from those in the current literature. We also describe a new method for correcting for rearrangements that are not detected with a limited set of markers.     

Duplicative and conservative transpositions of larval serum protein 1 genes in the genus Drosophila

Interspecific comparative molecular analyses of transposed genes and their flanking regions can help to elucidate the time, direction, and mechanism of gene transposition. In the Drosophila melanogaster genome, three Larval serum protein 1 (Lsp1) genes (alpha, beta and gamma) are present and each of them is located on a different chromosome, suggesting multiple transposition events. We have characterized the molecular organization of Lsp1 genes in D. buzzatii, a species of the Drosophila subgenus and in D. pseudoobscura, a species of the Sophophora subgenus. Our results show that only two Lsp1 genes (beta and gamma) exist in these two species. The same chromosomal localization and genomic organization, different from that of D. melanogaster, is found in both species for the Lsp1beta and Lsp1gamma genes. Overall, at least two duplicative and two conservative transpositions are necessary to explain the present chromosomal distribution of Lsp1 genes in the three Drosophila species. Clear evidence for implication of snRNA genes in the transposition of Lsp1beta in Drosophila has been found. We suggest that an ectopic exchange between highly similar snRNA sequences was responsible for the transposition of this gene. We have also identified the putative cis-acting regulatory regions of these genes, which seemingly transposed along with the coding sequences.     

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.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.     

Evolution acts on enhancer organization to fine-tune gradient threshold readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.