Identification of One Intron Loss and Phylogenetic Evolution of Dfak Gene in the Drosophila melanogaster Species Group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Convergent intron loss of MRP1 in Drosophila and mosquito species

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.     

Molecular Evolution of the Metallothionein Gene Mtn in the Melanogaster Species Group: Results from Drosophila Ananassae

Three distinctly different alleles of the metallothionein gene Mtn have been identified in natural Drosophila melanogaster populations: Mtn(.3), Mtn(1), and Dp(Mtn(1)), where the latter designates a tandem duplication of Mtn(1). In Drosophila simulans, only Mtn(.3)-type alleles have been found. It has been suggested that Mtn(.3) is the ancestral allele and demonstrated that a presumed two-step transition from Mtn(.3) to Mtn(1) to Dp(Mtn(1)) is accompanied by an approximate 5-fold increase in RNA levels. We analyzed the evolutionary genetics of the Mtn locus of Drosophila ananassae, a distant relative of D. melanogaster and D. simulans within the melanogaster species group. The Mtn gene of D. ananassae is most similar to Mtn(.3). (i) it is identical with Mtn(.3) at the amino acid level, but differs from Mtn(1) in its terminal codon; (ii) its 3'' UTR contains a characteristic extra DNA segment of about 50 bp which is present in Mtn(.3), but lacking in Mtn(1); (iii) duplications of Mtn were not found in a worldwide sample of 110 wild D. ananassae chromosomes. However, the intron of the Mtn gene in D. ananassae is only 69 bp long, whereas the length of the Mtn(.3) and Mtn(1) introns is 265 bp; and it lacks a polypyrimidine stretch upstream of the 3'' splice site in contrast to the much greater pyrimidine-richness found in the Mtn(.3) and Mtn(1) introns. A short intron (67 bp) was also identified in a D. pseudoobscura Mtn allele, suggesting that the short intron is the ancestral form and that the transition from the short to the long intron occurred within the melanogaster species group. We discuss the significance of this observation with regard to the recently proposed classification of D. melanogaster introns into two groups: short introns (<90 bp) which tend to lack polypyrimidine stretches, and longer ones which have strong 3'' splice signals similar to mammalian introns. A database search revealed that this length dimorphism is an evolutionarily conserved feature of Drosophila introns; transitions from one size class to the other appear to be rare between closely related species (e.g., within the melanogaster subgroup).     

Molecular evolution of Drosophila odorant receptor genes

A total of 752 odorant receptor (Or) genes, including pseudogenes, were identified in 11 Drosophila species and named after their orthologs in Drosophila melanogaster. The 813 Or genes, including 61 from D. melanogaster, were classified into 59 orthologous groups that are well supported by gene phylogeny. By reconciling with the gene family phylogeny, we estimated the number of gene duplication/loss events and intron gain/loss events in the species phylogeny. We found that these events are particularly frequent in Drosophila grimshawi, Drosophila willistoni, and obscura group. More than half of the duplicated genes stay as tandem arrays, whose size range from 2 to 8. These genes vary in sequence and some likely underwent positive selection, indicating that the gene duplication was important for flies to acquire new olfactory functions. We hypothesize that Or genes conferred the basic olfactory repertoire to ancestral flies before the speciation of the Drosophila and Sophophora subgenera about 40 Mya. This repertoire has been largely maintained in the current species, whereas lineage-specific gene duplication seems to have led to additional specialization in some species in response to specific ecological conditions.     

Phylogenetic and genomewide analyses suggest a functional relationship between kayak, the Drosophila fos homolog, and fig, a predicted protein phosphatase 2c nested within a kayak intron

A gene located within the intron of a larger gene is an uncommon arrangement in any species. Few of these nested gene arrangements have been explored from an evolutionary perspective. Here we report a phylogenetic analysis of kayak (kay) and fos intron gene (fig), a divergently transcribed gene located in a kay intron, utilizing 12 Drosophila species. The evolutionary relationship between these genes is of interest because kay is the homolog of the proto-oncogene c-fos whose function is modulated by serine/threonine phosphorylation and fig is a predicted PP2C phosphatase specific for serine/threonine residues. We found that, despite an extraordinary level of diversification in the intron-exon structure of kay (11 inversions and six independent exon losses), the nested arrangement of kay and fig is conserved in all species. A genomewide analysis of protein-coding nested gene pairs revealed that approximately 20% of nested pairs in D. melanogaster are also nested in D. pseudoobscura and D. virilis. A phylogenetic examination of fig revealed that there are three subfamilies of PP2C phosphatases in all 12 species of Drosophila. Overall, our phylogenetic and genomewide analyses suggest that the nested arrangement of kay and fig may be due to a functional relationship between them.     

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.     

Conserved (CT) n·(GA) n Repeats in the Non-coding Regions at the Gpdh Locus are Binding Sites for the GAGA Factor in Drosophila melanogaster and its Sibling Species

The (CT)n.(GA)n rich sequences in the upstream and 5' intron enhancer regions of the sn-glycerol-3-phosphate dehydrogenase (Gpdh) gene in Drosophila melanogaster, its sibling and distantly related species are conserved in their position and in the number of repeats. Using in vitro DNA-footprint analyses we show that the GAGA factor binds to these multiple closely spaced and overlapping conserved (CT)n.(GA)n repeats in D. melanogaster and D. erecta.     

Microevolutionary divergence pattern of the segmentation gene hunchback in Drosophila

To study the microevolutionary processes shaping the evolution of the segmentation gene hunchback (hb) from Drosophila melanogaster, we cloned and sequenced the gene from 12 isofemale lines representing wild-type populations of D. melanogaster, as well as from the closely related species Drosophila sechellia, Drosophila orena, and Drosophila yakuba. We find a relatively low degree of sequence variation in D. melanogaster (theta = 0.0017), which is, however, consistent with its chromosomal location in a region of low recombination. Tests of neutrality do not reject a neutral-evolution model for the whole region. However, pairwise tests with different subregions indicate that there is a relative excess of polymorphic sites in the leader and the intron. Codon usage pattern analysis shows a particularly biased codon usage in the highly conserved regions, which is in line with the hypothesis that selection on translational accuracy is the driving force behind such a bias. A comparison of the expression pattern of hb in different sibling species of D. melanogaster reveals some regulatory changes in D. yakuba, which could be interpreted as changes in the timing of secondary expression domains.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

Rapid evolution of two odorant-binding protein genes, Obp57d and Obp57e, in the Drosophila melanogaster species group

Genes encoding odorant-binding protein (OBP) form a large family in an insect genome. Two OBP genes, Obp57d and Obp57e, were previously identified to be involved in host-plant recognition in Drosophila sechellia. Here, by comparing the genomic sequences at the Obp57d/e locus from 27 Drosophila species, we found large differences in gene number between species. Phylogenetic analysis revealed that Obp57d and Obp57e in the D. melanogaster species group arose by gene duplication of an ancestral OBP gene that remains single in the obscura species group. Further gain and loss of OBP genes were observed in several lineages in the melanogaster group. Site-specific analysis of evolutionary rate suggests that Obp57d and Obp57e have functionally diverged from each other. Thus, there are two classes of gene number differences in the Obp57d/e region: the difference of the genes that have functionally diverged from each other and the difference of the genes that appear to be functionally identical. Our analyses demonstrate that these two classes of differences can be distinguished by comparisons of many genomic sequences from closely related species.     

Evolution of the Adh locus in the Drosophila willistoni group: the loss of an intron, and shift in codon usage

We report here the DNA sequence of the alcohol dehydrogenase gene (Adh) cloned from Drosophila willistoni. The three major findings are as follows: (1) Relative to all other Adh genes known from Drosophila, D. willistoni Adh has the last intron precisely deleted; PCR directly from total genomic DNA indicates that the deletion exists in all members of the willistoni group but not in any other group, including the closely related saltans group. Otherwise the structure and predicted protein are very similar to those of other species. (2) There is a significant shift in codon usage, especially compared with that in D. melanogaster Adh. The most striking shift is from C to U in the wobble position (both third and first position). Unlike the codon-usage-bias pattern typical of highly biased genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C in the third position. (3) The phylogenetic information provided by this new sequence is in agreement with almost all other molecular and morphological data, in placing the obscura group closer to the melanogaster group, with the willistoni group farther distant but still clearly within the subgenus Sophophora.      

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

Interspecific Comparison of the Transformer Gene of Drosophila Reveals an Unusually High Degree of Evolutionary Divergence

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.