Domesticated P Elements in the Drosophila montium Species Subgroup Have a New Function Related to a DNA Binding Property

Molecular domestication of a transposable element is defined as its functional recruitment by the host genome. To date, two independent events of molecular domestication of the P transposable element have been described: in the Drosophila obscura species group and in the Drosophila montium species subgroup. These P neogenes consist of stationary, nonrepeated sequences, potentially encoding 66-kDa repressor-like (RL) proteins. Here we investigate the function of the montium P neogenes. We provide evidence for the presence of RL proteins in two montium species (D. tsacasi and D. bocqueti) specifically expressed in adult and larval brain and gonads. We tested the hypothesis that the montium P neogenes’ function is related to the repression of the transposition of distantly related mobile P elements which coexist in the genome. Our results strongly suggest that the montium P neogenes are not recruited to downregulate the P element transposition. Given that all the proteins encoded by mobile or stationary P homologous sequences show a strong conservation of the DNA binding domain, we tested the capacity of the RL proteins to bind DNA in vivo. Immunostaining of polytene chromosomes in D. melanogaster transgenic lines strongly suggests that montium P neogenes encode proteins that bind DNA in vivo. RL proteins show multiple binding to the chromosomes. We suggest that the property recruited in the case of the montium P neoproteins is their DNA binding property. The possible functions of these neogenes are discussed. [Reviewing Editor: Dr. Dmitri Petrov     

Horizontal transmission versus vertical inheritance of P elements in Drosophila and Scaptomyza: has the M-type subfamily spread from East Asia?

Polymerase chain reaction (PCR) screening for P elements was carried out in 77 species with a primer set highly specific for the M-type subfamily. In the course of this search M-type elements were detected in 29 species: In the melanogaster (subgroups montium and rhopaloa ) and obscura species groups of Drosophila (25 out of 71 species examined), and in the genus Scaptomyza (four out of six species). M-type elements are present in all species of the montium subgroup investigated so far (21), but occur only sporadically in other groups. Within the montium subgroup 20 species possess only incomplete copies, only one species ( D. lacteicornis ) harbours apparently full-sized elements. In contrast, outside the montium subgroup almost all species with M-type elements carry full-sized copies suggesting transpositional activity, at least in the recent past. The interior section of the full-sized M-type element of D. lacteicornis was partially sequenced (936 bp). In addition, the complete sequences of four internally deleted M-type elements of D. lacteicornis, D. rufa, D. quadraria , and D. triauraria were determined. Sequence comparisons (including sequence data from previous investigations) revealed striking discrepancies between P element phylogeny and the cladogenesis of their host species. Among several possible pathways for interspecific transfers of M-type elements, we favour the hypothesis assuming the invasion of Scaptomyza as well as the obscura group species of Drosophila via independent transmission routes originating from Asian species of the montium subgroup of Drosophila . The logical geographic scenario for these events would be East-Asia.     

Isolation, characterization, and localization of beta-tubulin genomic clones of three Drosophila montium subgroup species.

Genomic libraries were constructed from three Drosophila species, namely Drosophila auraria, Drosophila serrata, and Drosophila kikkawai, belonging to the Drosophila montium subgroup of the Drosophila melanogaster species group. Clones containing beta-tubulin specific sequences were isolated, characterized by restriction endonuclease digestions and Southern hybridizations, and mapped by in situ hybridization on the polytene chromosomes of the species studied. The distribution of the beta-tubulin loci was found to be similar in D. montium species and D. melanogaster.     

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.     

线粒体ND4-ND4L基因在黑腹果蝇种组中的进化特征

本实验对黑腹果蝇种组(melanogaster species group)中8个种亚组33个样品两个线粒体基因ND4和ND4L进行了测序,并分析了ND4基因的序列差异和碱基替换特点,发现近缘物种中存在很明显的转换倾向,而在远缘物种中由于重复替换导致转换数处于饱和状态,我们的实验数据证实了线粒体基因较核基因有较快的进化速度。最后根据D．melanogaster与D．yakuba的遗传距离推算了8个种亚组的分化时间,ananassae种亚组最先分化,然后依次是montium,melanogaster,ficsphila,eugracilis,elegans,suzukii和takahashii最后分化。     

Evolutionary Conservation of the Structure and Expression of Alternatively Spliced Ultrabithorax Isoforms from Drosophila

In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.     

Balbiani rings in Drosophila: the Balbiani ring 1 is a common characteristic in several montium species.

Drosophila bicornuta, D. jambulina, D. biauraria, D. triauraria and D. quadraria, belonging to the montium subgroup of the melanogaster species, have a well-formed Balbiani ring (BR) in their salivary gland chromosomes. The BR has many similarities to the BR1 of D. auraria and D. serrata, two other montium species, indicating that the BR1 is a common characteristic in several species of the montium subgroup. It is suggested that the BR1 structure and its possible function(s), in contrast to the BR2, may have been selected during evolution.     

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.     

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.     

The heat shock genes in theDrosophila montium subgroup: Chromosomal localization and evolutionary implications

The hsp70, hsp83, hsrω, and the small heat shock protein genes were mapped on the polytene chromosomes of six species, representative of the geographical distribution of the Drosophila montium subgroup of the melanogaster species group. In addition, based on hybridization conditions, the putative locus of the hsp68 gene is given. In contrast to the situation in the melanogaster subgroup species, the hsp70 locus is single in the montium species. The hsp83, hsrωand the small hsp loci are also single in the montium genomes studied here, a common feature of all Drosophila species. Among the hsp genes studied, the small hsp genes and the hsrω-homologous sequences exhibit a higher degree of divergence between the melanogaster and the montium subgroups. Our results support the idea that the montium subgroup species has a genome organization closer to that of the common ancestor compared with the melanogaster subgroup species. Received: 25 July 1995; in revised form: 31 January 1996 / Accepted: 1 April 1996     

Developmental constraints and convergent evolution in Drosophila sex comb formation

SUMMARY The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii , where the SCs remain approximately transverse, and Drosophila biarmipes , where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species ( Drosophila nikananu ) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster , forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac) , is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.     

Functional analysis of poly(ADP-ribose) polymerase in Drosophila melanogaster

Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes. To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster. The PARP gene consisted of six translatable exons and spanned more than 50 kb. The DNA binding domain is encoded by exons 1-4. Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina. There are two cDNAs species in Drosophila. One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing. The expression of these two forms of PARP in E. coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive. On the other hand PARP I is active. A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly. These results suggest that the PARP gene plays an important function during the development of Drosophila.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.