Mitochondrial glutamate carriers from Drosophila melanogaster: Biochemical,evolutionary and modeling studies

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.     

Comparison of the oxidative phosphorylation (OXPHOS) nuclear genes in the genomes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles gambiae

Background

In eukaryotic cells, oxidative phosphorylation (OXPHOS) uses the products of both nuclear and mitochondrial genes to generate cellular ATP. Interspecies comparative analysis of these genes, which appear to be under strong functional constraints, may shed light on the evolutionary mechanisms that act on a set of genes correlated by function and subcellular localization of their products.

Results

We have identified and annotated the Drosophila melanogaster, D. pseudoobscura and Anopheles gambiae orthologs of 78 nuclear genes encoding mitochondrial proteins involved in oxidative phosphorylation by a comparative analysis of their genomic sequences and organization. We have also identified 47 genes in these three dipteran species each of which shares significant sequence homology with one of the above-mentioned OXPHOS orthologs, and which are likely to have originated by duplication during evolution. Gene structure and intron length are essentially conserved in the three species, although gain or loss of introns is common in A. gambiae. In most tissues of D. melanogaster and A. gambiae the expression level of the duplicate gene is much lower than that of the original gene, and in D. melanogaster at least, its expression is almost always strongly testis-biased, in contrast to the soma-biased expression of the parent gene.

Conclusions

Quickly achieving an expression pattern different from the parent genes may be required for new OXPHOS gene duplicates to be maintained in the genome. This may be a general evolutionary mechanism for originating phenotypic changes that could lead to species differentiation.     

Functional constraint underlies 60 million year stasis of Dipteran testis-specific beta-tubulin

How do proteins evolve while maintaining their function? Previous studies find a highly stringent structure/function relationship between the Drosophila melanogaster testis‐specific tubulin β2 and the spermtail axoneme, such that small changes in the β2 protein render it unable to generate a motile axoneme. This raises the question, how does β2 evolve while maintaining its function? To address this question we cloned full‐ and partial‐length β2 sequences from 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. Not a single amino acid difference is coded among them—β2 maintains its function by not evolving. We also performed gene genealogical analyses to determine ortholog/paralog relationships among insect tubulins. We find that the Lepidopteran and Dipteran testis‐specific β‐tubulins are likely orthologs, and surprisingly, despite functioning in the same structure, the Lepidopteran orthologs are evolving rapidly. We argue that differences in tubulin isoform use in the testes cause the Dipteran axoneme to be less evolvable than the Lepidopteran axoneme, which has facilitated the evolution of a unique amino acid synergism in Drosophila and Hirtodrosophilaβ2 that is resistant to change, contributing to its evolutionary stasis.     

Sequence conservation among orthologous vomeronasal type 1 receptor-like (ora) genes does not support the differential tuning hypothesis in Salmonidae

Salmon utilize olfactory cues to guide natal stream homing during spawning migrations. Both inorganic and biogenic chemicals have been proposed as odorants that might be used by salmon during homing. In this study, we used genomic DNA sequence data from nine salmonid species to compare nucleotide identities for orthologous main olfactory receptor (mOR) genes with nucleotide identities for orthologous vomeronasal type 1-like (ora) receptor genes. We found that orthologs for both classes of olfactory receptor genes (mORs and Oras) appear to be highly conserved among species. Our findings do not support the differential tuning hypothesis in Salmonidae, which predicts higher sequence conservation for mORs than ora. We did, however, find convincing evidence for site-specific positive selection acting on paralogous main olfactory receptor genes.     

Identification of a novel insect neuropeptide,CNMa and its receptor

To identify ligands for orphan GPCRs, we searched novel neuropeptide genes in the Drosophila melanogaster genome. Here, we describe CNMa, a novel cyclic neuropeptide that is a highly potent and selective agonist for the orphan GPCR, CG33696 (CNMaR). Phylogenetic analysis revealed that arthropod species have two paralogous CNMaRs, but many taxa retain only one. Drosophila CNMa potently activates CNMaR-2 from Apis mellifera, suggesting both receptors are functional. Although CNMa is conserved in most arthropods, Lepidoptera lack the CNMa gene. However, they retain the CNMaR gene. Bombyx CNMaR showed low sensitivity to Drosophila CNMa, hinting toward the existence of additional CNMaR ligand(s).     

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.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

Preservation of genes involved in sterol metabolism in cholesterol auxotrophs: facts and hypotheses

Background

It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols de novo. However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs): Drosophila melanogaster and Caenorhabditis elegans.

Principal Findings

We have been able to detect bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs). Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s), which keep them under pressure.

Conclusions

By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs.     

Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex

This study investigated the potential of the conserved intron scanning approach to develop molecular markers for genes involved in lignin biosynthesis among members of the Saccharum complex and a distantly related Imperata species. Five intron-flanking primer sets targeting genes encoding five lignin biosynthetic enzymes??phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4-CL), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POX)??were designed based on the sequence analysis between sugarcane expressed sequence tags (ESTs) and Sorghum bicolor orthologs. Nucleotide sequence analyses of the amplicons of PAL, 4-CL and CCoAOMT orthologs revealed the presence of single nucleotide polymorphisms (SNPs) and insertions?Cdeletions (INDELs) in the target introns as well as (CT)-simple sequence repeats (SSRs) in CCoAOMT orthologs. The SSR marker screening against fifty-nine accessions of the Saccharum complex and an Imperata species confirmed that the identified SSR markers were highly polymorphic among Saccharum and Erianthus species. PCR-restriction fragment length polymorphism (PCR?CRFLP) and cleaved amplified polymorphic sequence (CAPS) marker screening of 4-CL and CCoAOMT orthologs developed genus-specific molecular markers that confirmed the intergeneric hybridization status of Saccharum Fiji hybrids. The current study showed that the conserved intron scanning strategy is applicable to multiple copy genes of polyploid monocots. The conserved intron scanning approach provides a novel way of investigating DNA polymorphisms among species within the Saccharum complex and has the potential to help in the development of marker-assisted selection in intergeneric hybrids.     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.     

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.     

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, γ-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ667181–DQ667195.