Revealing the anti-HRP epitope in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

Antibodies are very often used as specific cell and/or tissue markers. An example of this is anti-horseradish peroxidase (HRP), an antibody raised against a plant glycoprotein, which was shown some twenty-five years ago to specifically stain neural tissue in an animal, Drosophila melanogaster. This peculiar finding was later expanded to other invertebrate species including Caenorhabditis elegans, which were also shown to bear anti-HRP epitopes. Initial experiments indicated that the epitopes recognised by anti-HRP in invertebrates are of carbohydrate nature. Indeed, more recent experiments have characterised relevant core α1-3-fucosylated N-glycan structures that act as epitopes in various model and parasitic organisms. Moreover, a number of enzymes required for the synthesis of such structures have been identified. Over the years, medically-relevant roles of these structures have become apparent as regards allergenicity and immunoregulation. Although major advances have been made in understanding of the underlying mechanisms and structures related to the anti-HRP epitope, the in vivo role of the relevant epitopes in neural and other tissues is yet to be resolved. Current understanding of the anti-HRP epitopes synthesis and their relevance is discussed and elaborated.

Nutritional regulation of <Emphasis Type="Italic">ANR1</Emphasis> and other root-expressed MADS-box genes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The ANR1 MADS-box gene in Arabidopsis thaliana (L.) Heynh. has previously been identified as a key regulator of lateral root growth in response to signals from external nitrate (NO₃⁻). We have used quantitative real-time PCR to investigate the responsiveness of ANR1 and 11 other root-expressed MADS-box genes to fluctuations in the supply of N, P and S. ANR1 expression in roots of hydroponically grown Arabidopsis plants was specifically regulated by changes in the N supply, being induced by N deprivation and rapidly repressed by N re-supply. This pattern of N responsiveness differs from the NO₃⁻ -inducibility of ANR1 previously observed in Arabidopsis root cultures [H.M. Zhang and B.G. Forde (1998) Science 279:407–409]. Seven of the other MADS-box genes responded to N in a manner similar to ANR1, but less strongly, while four (AGL12, AGL17, AGL18 and AGL79) were unaffected. Six of the N-regulated genes (ANR1, AGL14, AGL16, AGL19, SOC1 and AGL21) belong to just two clades within the type II MADS-box lineage, while the other two (AGL26 and AGL56) belong to the poorly characterized type I lineage. Only SOC1 was additionally found to respond to changes in the P and S supply, suggesting a possible role in a general response to nutrient stress. Studies with an ANR1 transposon-insertion mutant provided no evidence for regulatory interactions between ANR1 and the other root-expressed MADS-box genes. The implications of the current data for our understanding of the role of ANR1 and other MADS box genes in the nutritional regulation of lateral root growth are discussed.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Association of Misexpression with Sterility in Hybrids of <Emphasis Type="Italic">Drosophila simulans</Emphasis>and <Emphasis Type="Italic">D. mauritiana</Emphasis>

Recent studies have identified genes associated with hybrid sterility and other hybrid dysfunctions, but the consequences of introgressions of these speciation genes are often poorly understood. Previously, we identified a panel of genes that are underexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to pure species. Here, we build on this reverse-genetics approach to demonstrate that the underexpression of at least five of these genes in hybrids is associated with hybrid sterility and that these five genes are coordinately regulated. We map one upstream regulator of these genes to a region previously shown to harbor one or more factors causing hybrid sterility. Finally, we show that the genes underexpressed in hybrids are often highly conserved, as might be predicted for downstream targets of the genetic changes that cause hybrid sterility. This approach integrates forward genetics with reverse genetics to show a proximate consequence of the introgression of particular hybrid sterility-conferring regions between species: underexpression of genes necessary for normal spermatogenesis.[Reviewing Editor: Martin Kreitman]     

Expression of <Emphasis Type="Italic">defective proventriculus</Emphasis> during head capsule development is conserved in <Emphasis Type="Italic">Drosophila</Emphasis> and stalk-eyed flies (<Emphasis Type="Italic">Diopsidae</Emphasis>)

Hypercephaly, in the form of lateral extensions of the head capsule, is observed in several families of Diptera. A particularly exaggerated form is found in Diopsid stalk-eyed flies, in which both eyes and antennae are laterally displaced at the end of stalks. The processes of early development and specification of the head capsule in stalk-eyed flies are similar to those in Drosophila melanogaster. In Drosophila the homeobox gene ocelliless (oc) shows a mediolateral gradient of expression across the region of the eye-antennal imaginal disc that gives rise to the head capsule and specifies the development of different head structures. The genes and developmental mechanisms that subsequently define head shape in Drosophila and produce hypercephaly in stalk-eyed flies remain unclear. To address this, we performed an enhancer trap screen for Drosophila genes expressed in the same region as oc and identified the homeobox gene defective proventriculus (dve). In the eye-antennal imaginal disc, dve is coexpressed with oc in the region that gives rise to the head capsule and is active along the medial edge of the antennal disc and in the first antennal segment. Analyses of dve expression in mutant eye-antennal discs are consistent with it acting downstream of oc in the development of the head capsule. We confirm that orthologues of dve are present in a diverse panel of five stalk-eyed fly species and analyse patterns of dve sequence variation within the clade. Our results indicate that dve expression and sequence are both highly conserved in stalk-eyed flies.M. Carr and I. Hurley contributed equally to this work.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

A Nested Alpha-Amylase Gene in <Emphasis Type="Italic">Drosophila ananassae</Emphasis>

The amylase gene family of Drosophila ananassae consists in seven copies, scattered on several chromosomal arms. We have evidenced that a member of the family, Amy35, lies within an intron of a gene homologous to the CG14696 gene of D. melanogaster. This nested arrangement seems restricted to the D. ananassae subgroup. The nested and the nest genes are encoded on opposite strands. Both are actively transcribed in the midgut at the same time, raising the possibility of interference between their mRNAs. Our data also help to elucidate the history of the Amy family, suggesting that Amy35 arose by duplication and translocation from another ancestral locus, into a formerly short intron, in an ancestor of the subgroup.     

Evolutionary History and Mode of the <Emphasis Type="Italic">amylase</Emphasis> Multigene Family in <Emphasis Type="Italic">Drosophila</Emphasis>

Previous studies indicate that the tandemly repeated members of the amylase (Amy) gene family evolved in a concerted manner in the melanogaster subgroup and in some other species. In this paper, we analyzed all of the 49 active and complete Amy gene sequences in Drosophila, mostly from subgenus Sophophora. Phylogenetic analysis indicated that the two types of diverged Amy genes in the Drosophila montium subgroup and Drosophila ananassae, which are located in distant chromosomal regions from each other, originated independently in different evolutionary lineages of the melanogaster group after the split of the obscura and melanogaster groups. One of the two clusters was lost after duplication in the melanogaster subgroup. Given the time, 24.9 mya, of divergence between the obscura and the melanogaster groups (Russo et al. 1995), the two duplication events were estimated to occur at about 13.96 ± 1.93 and 12.38 ± 1.76 mya in the montium subgroup and D. ananassae, respectively. An accelerated rate of amino acid changes was not observed in either lineage after these gene duplications. However, the G+C contents at the third codon positions (GC3) decreased significantly along one of the two Amy clusters both in the montium subgroup and in D. ananassae right after gene duplication. Furthermore, one of the two types of the Amy genes with a lower GC3 content has lost a specific regulatory element within the montium subgroup species and D. ananassae. While the tandemly repeated members evolved in a concerted manner, the two types of diverged Amy genes in Drosophila experienced frequent gene duplication, gene loss, and divergent evolution following the model of a birth-and-death process.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in <Emphasis Type="Italic">Chenopodium rubrum</Emphasis> differ in expression patterns

FLOWERING LOCUS T (FT) like genes are crucial regulators (both positive and negative) of flowering in angiosperms. We identified two FT homologs in Chenopodium rubrum, a short-day species used as a model plant for the studies of photoperiodic flower induction. We found that CrFTL1 gene was highly inducible by a 12-h dark period, which in turn induced flowering. On the other hand, photoperiodic treatments that did not induce flowering (short dark periods, or a permissive darkness interrupted by a night break) caused only a slight increase in CrFTL1 mRNA level. We demonstrated diurnal oscillation of CrFTL1 expression with peaks in the middle of a light period. The oscillation persisted under constant darkness. Unlike FT homologs in rice and Pharbitis, the CrFTL1 expression under constant darkness was very low. The CrFTL2 gene showed constitutive expression. We suggest that the CrFTL1 gene may play a role as a floral regulator, but the function of CrFTL2 remains unknown.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Gene inactivation mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in the filamentous fungi <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.