Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Genes encoding putative biogenic amine receptors in the parasitic nematode <Emphasis Type="Italic">Brugia </Emphasis><Emphasis Type="Italic">malayi</Emphasis>

Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. The lack of a vaccine against B. malayi, combined with ineffective chemotherapy against the adult has prompted the examination of biogenic amine receptors (BARs) as possible targets for drug discovery. We employed bioinformatics to identify genes encoding putative B. malayi BARs. Surprisingly, the B. malayi genome contains half of the genes predicted to encode BARs in the genomes of free-living nematodes such as Caenorhabditis elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans. The B. malayi genes encode each of the major BAR subclasses, including three serotonin, two dopamine and two tyramine/octopamine receptors and the structure of orthologous BAR genes is conserved. We find that potential G-protein coupling and ligand-specificity of individual BARs may be predicted by phylogenetic comparisons. Our results provide a framework for how G-protein coupled receptors may be targeted for drug development in medically important parasitic nematodes.     

The NemaGENETAG initiative: large scale transposon insertion gene-tagging in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The nematode Caenorhabditis elegans is a widely appreciated, powerful platform in which to study important biological mechanisms related to human health. More than 65% of human disease genes have homologues in the C. elegans genome, and essential aspects of mammalian cell biology, neurobiology and development are faithfully recapitulated in this organism. The EU-funded NemaGENETAG project was initiated with the aim to develop cutting-edge tools and resources that will facilitate modelling of human pathologies in C. elegans, and advance our understanding of animal development and physiology. The main objective of the project involves the generation and evaluation of a large collection of transposon-tagged mutants. In the process of achieving this objective the NemaGENETAG consortium also endeavours to optimize and automate existing transposon-mediated mutagenesis methodologies based on the Mos1 transposable element, in addition to developing alternatives using other transposon systems. The final product of this initiative—a comprehensive collection of transposon-tagged alleles—together with the acquisition of efficient transposon-based tools for mutagenesis and transgenesis in C. elegans, should yield a wealth of information on gene function, immediately relevant to key biological processes and to pharmaceutical research and development.     

<Emphasis Type="Italic">Mariner</Emphasis>-like elements in <Emphasis Type="Italic">Rhynchosciara americana</Emphasis> (Sciaridae) genome: molecular and cytological aspects

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<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.     

Formation of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> biofilms on multiple surfaces on <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Summary A liquid-based assay was used to evaluate the ability of Yersinia pseudotuberculosis to form a bacterial biofilm on the nematode Caenorhabditis elegans. After 3 days of incubation in the liquid assay a biofilm was clearly visible by light microscopy on both the head and vulva region of the worms. At times, the biofilm formation on the vulva appeared to prevent the laying of eggs by the adult hermaphrodite; the eggs would later hatch inside of the worm. One possible explanation for the biofilm formation observed on the vulva may be the increased motion of the cuticle surrounding the vulva when the worm is immersed in a liquid culture. This is the first report of biofilm formation on the vulva of C. elegans.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

The ontogenetic basis for floral diversity in <Emphasis Type="Italic">Agonis</Emphasis>, <Emphasis Type="Italic">Leptospermum</Emphasis> and <Emphasis Type="Italic">Kunzea</Emphasis> (Myrtaceae)

Floral development in three species each of Leptospermum and Kunzea, and one species of Agonis, is described and compared. Differences in the number of stamens and their arrangement in the flower at anthesis are determined by the growth dynamics of the bud. In Leptospermum, early expansion of the bud is predominantly in the axial direction and causes the stamen primordia to be initiated in antepetalous chevrons. In Kunzea, early expansion occurs predominantly in the lateral direction and successive iterations of stamen primordia are inserted alternately at more or less the same level. In both genera, further expansion in the lateral plane spreads the stamens into a ring around the hypanthium. Agonis flexuosa is similar to Leptospermum. Other variable factors include the timing at which stamen initiation commences (earlier in Leptospermum than Kunzea), the duration of stamen initiation (hence the total number of stamens produced – varies within genera), and very late differential expansion that forces stamens into secondary antesepalous groups in A. flexuosa and L. myrsinoides.The authors thank Dr H. Toelken for kindly providing some material and the impetus for this project. This research was supported by Australian Research Council grant AS19131815.     

<Emphasis Type="Italic">Nramp1</Emphasis> deletion does not confer susceptibility to<Emphasis Type="Italic"> Rhodococcus equi</Emphasis> infection in mice

Rhodococcus equi is an intracellular bacterium that causes pneumonia in immunocompromised people and foals. The Nramp1 gene influences susceptibility to a variety of intracellular bacteria (including mycobacterial species), but not to Mycobacterium tuberculosis. In this study, we demonstrate that mice functionally deleted of the Nramp1 gene were not more susceptible to infection with virulent R. equi (ATCC 33701) than wild-type mice. Susceptibility of mice to infection with the intracellular bacterium R. equi is more similar to that of M. tuberculosis than to other intracellular bacteria, including other mycobacteria.     

The<Emphasis Type="Italic"> HY2</Emphasis> gene as an efficient marker for transposon excision in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transposable elements can generate germinal and somatic mutations, and hence represent a powerful tool for the analysis of gene function. Transposons from maize have been adapted to mutagenise the genomes of diverse species. The efficiency of these systems partly relies on the ease with which germinal (i.e. germinally transmitted) or somatic excisions can be detected. Here we describe the use of HY2, a gene that codes for an enzyme involved in the biosynthesis of the phytochrome chromophore, to monitor the excision of a Ds gene-trap element in Arabidopsis thaliana. Taking advantage of the altered germination and de-etiolation behaviour of a Ds -tagged hy2 mutant, we have designed an efficient protocol for the recovery of germinal revertants, making HY2 the most precocious excision marker available, to the best of our knowledge. In addition, HY2 is also useful for generating visible sectors in photosynthetic tissues, thanks to the somatic instability of this mutable hy2 allele.Communicated by M.-A. Grandbastien     

Parasitism <Emphasis Type="Italic">versus</Emphasis> mutualism in the ant-garden parabiosis between <Emphasis Type="Italic">Camponotus femoratus</Emphasis> and <Emphasis Type="Italic">Crematogaster levior</Emphasis>

Ant-gardens represent a special type of association between ants and epiphytes. Frequently, two ant species can share the same nest in a phenomenon known as ‘parabiosis’, but the exact nature (i.e., mutualistic or parasitic) of this interaction is the subject of debate. We thus attempted to clarify the mutual costs and benefits for each partner (ants and plants) in the Crematogaster levior/Camponotus femoratus ant-garden parabiosis. The ants’ response to experimental foliar damage to the epiphytes and to the host tree as well as their behavior and interactions during prey capture were investigated to see if the purported parasitic status of Cr. levior could be demonstrated in either the ant-ant or in the ant-plant interactions. The results show that both species take part in protecting the epiphytes, refuting the role of Cr. levior as a parasite of the ant-garden mutualism. During capture of large prey Ca. femoratus took advantage from the ability of Cr. levior to discover prey; by following Cr. levior trails Ca. femoratus workers discover the prey in turn and usurp them during agonistic interactions. Nevertheless, the trade-off between the costs and benefits of this association seems then to be favorable to both species because it is known that Cr. levior benefits from Ca. femoratus building the common carton nests and furnishing protection from vertebrates. Consequently, parabiosis can then be defined as the only mutualistic association existing between ant species, at least in ant-gardens. Received 31 August 2006 ; revised 8 December 2006 ; accepted 12 December 2006     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.