Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.     

A comparison of transplantable bicoid activity and partial bicoid homeobox sequences in several Drosophila and blowfly species (Calliphoridae)

In order to test for bicoid-like activity in insects other than Drosophila melanogaster, anterior egg cytoplasm from the following species was injected into cleavage stage embryos from mutant D. melanogaster lacking a functional bicoid (bcd) product: six other Drosophila species, the housefly, three blowfly species, the primitive cyclorrhaphic dipteran Megaselia, and the honeybee Apis mellifera; preliminary tests were made with four lower dipterans (Nematocera). Rescue effects were only observed with the drosophilids, housefly, and two of the three blowfly species. Rescue was stronger with the drosophilids than with the other flies as donors. Where checked (D. pseudoobscura), a positive correlation was found between the amount of cytoplasm injected and the number of pattern elements formed, suggesting threshold effects upon target genes as with the endogenous bcd product. By polymerase chain reaction, fragments from a bcd-orthologous homeobox were cloned from the three blowfly species. The derived sequence of 43 amino acids was identical in all blowflies and the housefly but differed at 4 positions from the orthologous D. melanogaster sequence. Localization of the mRNA recognized by the respective fragments in the blowflies Lucilia and Phormia resembled that known from D. melanogaster, while Calliphora — the blowfly species lacking rescue activity —showed remarkable differences of localization in both ovarian follicles and the deposited egg cell. This surprising divergence within a morphologically rather uniform family of cyclorrhaphic dipterans should be of interest from both functional and evolutionary points of view.     

Distribution and conservation of the foldback transposable element inDrosophila

Summary Foldback elements are a family of transposable elements described inDrosophila melanogaster. The members of this dispersed repetitive family have terminal inverted repeats that sometimes flank a central region. The inverted repeats of all the family members are homologous.The study of the distribution and conservation of the foldback elements in differentDrosophila species shows that this distribution is different from that of the hybrid dysgenesis systems (PM and IR). Sequences homologous to foldback elements were observed by Southern blots and in situ hybridization in all species of themelanogaster subgroup and in some species of themontium andtakahashii subgroups. The element was probably already present before the radiation of these subgroups. No evidence of horizontal transmission of the foldback element could be observed.     

A Ty1-copia group retrotransposon sequence in a vertebrate.

Summary We have used the polymerase chain reaction (PCR) to isolate a sequence characteristic of aTy1-copia group retrotransposon from the genome of the herring (Clupea harengus). This is the firstTy1-copia group retrotransposon sequence described in a vertebrate. Phylogenetic comparison of this sequence with other members of this group of retrotransposons shows that it resembles more closely some Tyl-copia group members fromDrosophila melanogaster than other group members in plants and fungi. These observations provide further evidence that theTy1-copia group LTR retrotransposons span many of the major eukaryote species boundaries, suggesting that horizontal transmission between different species has played a role in the evolution of this retrotransposon group.     

Eyes absent: A gene family found in several metazoan phyla

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65–67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sclera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later. Received: 23 November 1996 / Accepted: 25 February 1997     

Chromosomal localization of homeobox genes and associated markers on porcine Chromosomes 3, 5, 12, 15, 16 and 18: comparative mapping study with human and mouse

Four homeobox genes that belong to the four homeobox gene clusters known in mammals have been regionally assigned to four distinct porcine chromosomes in conserved regions between human and pig. HOXA11, HOXB6, HOXC8, and HOXD4 genes were mapped by radioactive in situ hybridization to porcine Chromosomes (Chrs) 18q21-24 (with a secondary signal in 16q14-21), 12p11-12, 5p11-12, and 15q22-23 respectively. Besides, we have also revealed the presence of a porcine homeobox (pig Hbx24) which, although showing DNA sequence homology with a mouse gene of HOXB cluster, was located on porcine Chr 3 (3p14-13) outside the Hox clusters. To support the identity of the homeobox gene clusters analyzed and in the light of the high sequence similarity among homeobox genes, we also localized markers known to be mapped near each Hox cluster in human. In this way, four genes were also mapped in pig: GAPD (5q12-21), GAD1 (15q21-22), INHBA (18q24), and IGFBP3 (18q24). Mapping of HOXA11, INHBA, and IGFBP3 on pig Chr 18 constitutes the first assignments of genes on this small chromosome. These new localizations extend the information on the conservation of four human chromosomal regions in the pig genome. Received: 7 August 1995 / Accepted: 16 October 1995     

Hmx homeobox gene function in inner ear and nervous system cell-type specification and development

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic–neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.     

The evolutionary genetics of thehobo transposable element in theDrosophila melanogaster complex

Hobo elements are a family of transposable elements found inDrosophila melanogaster and its three sibling species:D. simulans, D. mauritiana andD. sechellia. Studies inD. melanogaster have shown thathobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level somehobo-hybridizing sequences have also been found in the other members of themelanogaster subgroup and in many members of the relatedmontium subgroup. Surveys of older collected strains ofD. melanogaster suggest that completehobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history ofhobo in theD. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the groupD. melanogaster, D. simulans andD. mauritiana and the fourth speciesD. sechellia. The hypothesis of multiple transfers in the recent past into theD. melanogaster complex from a common outside source is discussed.     

Search for Drosophila genes encoding a conserved domain present in the achaete-scute complex and myc proteins

Summary Several genes of the achaete-scute complex (ASC) of Drosophila melanogaster encode a 60 amino acids long conserved domain which shares a significant homology with a region of the vertebrate myc proteins. Based on these results, the existence of a family of Drosophila genes that would share both this conserved domain and the neurogenic function of the AS-C has been postulated. To test this proposal, we have searched a D. melanogaster genomic library with a probe that encodes the conserved domain. Only under very low stringency hybridization conditions, clones not belonging to the AS-C cross-hybridized with the probe. Those that gave the strongest signals were characterized. Sequencing of the cross-hybridizing regions showed that they had no significant homology with the conserved domain, the sequence similarity extending at the most for 37 nucleotides. Although our results do not conclusively disprove the existence of a family of AS-C-like genes, they indicate that the conservation of the domain would be lower than that found for shared motifs in other families of Drosophila developmental genes.     

A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

Background  

Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (Hd P2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa.     

A mitochondrial carrier gene, <Emphasis Type="Italic">CG32103</Emphasis>, is highly expressed in the corpora allata in the fruit fly <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> (Diptera: Drosophilidae)

Here we describe a novel gene that is highly expressed in the corpora allata, an endocrine organ responsible for synthesizing juvenile hormones (JHs), in the fruit fly, Drosophila melanogaster Meigen. We isolated an enhancer-trap line in which the transgene was inserted at the locus CG32103, which encodes a mitochondrial carrier family protein with calcium-binding motifs. RNA in situ hybridization revealed that CG32103 is predominantly expressed in the corpora allata in D. melanogaster larvae. Putative orthologs of CG32103 are conserved in many insect species. Mitochondrial carriers are responsible for transporting metabolites across the inner mitochondrial membrane. Given that both mitochondrial membrane transport and cytoplasmic calcium signaling are important for JH biosynthesis regulation, we speculated that CG32103 represents a new member of the family of JH biosynthesis regulators in insects.     

Comparative analysis of vertebrate <Emphasis Type="Italic">PEPT1</Emphasis> and <Emphasis Type="Italic">PEPT2</Emphasis> genes

The plasma membrane transport proteins belong to SoLute Carrier 15 (SLC15) family and two members of this family have been characterized extensively in higher vertebrates, namely PEPT1 and PEPT2. Despite many efforts have made to define a pharmacophore model for efficient binding and transporting of substrates, there is not a comprehensive study performed to elucidate the evolutionary mechanisms among the SLC15 family members and to statistically evaluate sequence conservation and functional divergence between members. In this study, we compared and contrasted the rates and patterns of molecular evolution of 2 PEPT genes. Phylogenetic tree assembly with all available vertebrate PEPTs suggests that the PEPTs originated by duplications and diverged from a common protein at the base of the eukaryotic tree. Topological structure demonstrates both members share the similar hydrophobic domains (TMDs), which have been constrained by purifying selection. Although both genes show qualitatively similar patterns, their rates of evolution differ significantly due to an increased rate of synonymous substitutions in the structural domains in one copy, suggesting substantial differences in functional constraint on each gene. Site-specific profiles were established by posterior probability analysis revealing significantly divergent regions mainly locate at the hydrophobic region between predicted transmembrane domains 9 and 10 of the proteins. Thus, these results provide the evidence that several amino acid residues with reduced selective constraints are largely responsible for functional divergence between the paralogous PEPTs. These findings may provide a starting point for further experimental verifications.     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family,DLX4

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21–q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989; Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.     

Pax-6 origins – implications from the structure of two coral Pax genes

 Vertebrate Pax-6 and its Drosophila homolog eyeless play central roles in eye specification, although it is not clear if this represents the ancestral role of this gene class. As the most ”primitive” animals with true nervous systems, the Cnidaria may be informative in terms of the evolution of the Pax gene family. For this reason we surveyed the Pax gene complement of a representative of the basal cnidarian class (the Anthozoa), the coral Acropora millepora. cDNAs encoding two coral Pax proteins were isolated. Pax-Aam encoded a protein containing only a paired domain, whereas Pax-Cam also contained a homeodomain clearly related to those in the Pax-6 family. The paired domains in both proteins most resembled the vertebrate Pax-2/5/8 class, but shared several distinctive substitutions. As in most Pax-6 homologs and orthologs, an intron was present in the Pax-Cam locus at a position corresponding to residues 46/47 in the homeodomain. We propose a model for evolution of the Pax family, in which the ancestor of all of the vertebrate Pax genes most resembled Pax-6, and arose via fusion of a Pax-Aam-like gene (encoding only a paired domain) with an anteriorly-expressed homeobox gene resembling the paired -like class. Received: 25 February 1998/Accepted: 23 March 1998