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     

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.     

Genetic analysis of ryanodine receptor function in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> based on<Emphasis Type="Italic"> unc-68</Emphasis> revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca²⁺-induced Ca²⁺ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca²⁺ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.Communicated by C. P. Hollenberg     

Mechanotransduction: Touch and Feel at the Molecular Level as Modeled in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The survival of an organism depends on its ability to respond to its environment through its senses. The sense of touch is one of the most vital; still, it is the least understood. In the process of touch sensation, a mechanical stimulus is converted into electrical signals. Groundbreaking electrophysiological experiments in organisms ranging from bacteria to mammals have suggested that this conversion may occur through the activation of ion channels that gate in response to mechanical stimuli. However, the molecular identity of these channels has remained elusive for a very long time. Breakthroughs in our understanding of the cellular and molecular mechanisms of touch sensation have come from the analysis of touch-insensitive mutants in model organisms such as Caenorhabditis elegans and Drosophila melanogaster. This review will focus on the elegant genetic, molecular, imaging, and electrophysiological studies that demonstrate that a channel complex composed of two members of the DEG/ENaC gene family of channel subunits (named for the C. elegans degenerins and the related mammalian epithelial amiloride-sensitive Na channel), MEC-4 and MEC-10, and accessory subunits is gated by mechanical forces in touch-sensing neurons from C. elegans. I also report here electrophysiological and behavioral studies employing knockout mice that have recently shown that mammalian homologues of MEC-4, MEC-10, and accessory subunits are needed for normal mechanosensitivity in mouse, suggesting a conserved function for this channel family across species. The C. elegans genome encodes 28 DEG/ENaC channels: I discuss here the global role of DEG/ENaCs in mechanosensation, reporting findings on the role of other three nematode DEG/ENaCs (UNC-8, DEL-1, and UNC-105) in mechanosensitive and stretch-sensitive behaviors. Finally, this review will discuss findings in which members of another family of ion channels, the Transient Receptor Potential channels family, have been implicated in mechanosensitive behaviors in organisms ranging from C. elegans to mammals.     

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.     

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.     

<Emphasis Type="Italic">odd-skipped</Emphasis> homologs function during gut development in<Emphasis Type="Italic"> C. elegans</Emphasis>

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family.Edited by C. Desplan     

<Emphasis Type="Italic">Caenorhabditis elegans gcs-1</Emphasis> Confers Resistance to Arsenic-Induced Oxidative Stress

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type/As(V)>gcs-1/As(V)> wild-type/As(III)>gcs-1/As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.     

Isopentenyl-diphosphate isomerase is essential for viability of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Homozygosity for a mutation in the idi-1 gene of Caenorhabditis elegans results in paralysis during the first larval stage, followed by an arrest of growth and development late in the first larval stage. Apoptotic corpses, which are apparently the result of normal programmed cell death, persist in the arrested larvae. In genetic mosaics, an additional defect becomes evident upon examination with Nomarski optics: cells that are genotypically mutant enlarge, and their cytoplasm becomes dimpled. Electron microscopy indicates that the dimpling reflects an accumulation of many enlarged lysosomes and autophagosomes. The mosaics demonstrate that the lethal mutation acts cell autonomously with respect to this vesicular abnormality and that there is a maternal effect with respect to the time of developmental arrest of mutant progeny. Cloning of the gene reveals that it is the only gene in C. elegans for isopentenyl-diphosphate isomerase, an enzyme that is important for the synthesis of lipophilic molecules, including farnesyl and geranyl diphosphates.     

The invertebrate microtubule-associated protein PTL-1 functions in mechanosensation and development in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

PTL-1, a microtubule-associated protein of the structural MAP2/tau family, is the sole member of this gene family in Caenorhabditis elegans. Sequence analysis of available invertebrate genomes revealed a number of single, putative tau-like genes with high similarity to ptl-1. The ptl-1 gene is expressed in a number of cells, most notably mechanosensory neurons. We examined the role of ptl-1 in C. elegans in adult neurons as well as during development. A ptl-1 knockout strain of worms exhibited an egg-hatching defect, as well as a reduced sensitivity to touch stimuli. In addition, the knockout allele ptl-1(ok621) acts as a dominant enhancer of several temperature-sensitive alleles of mec-7 and mec-12, which code the isoforms of β-tubulin and α-tubulin that together form the unusual 15 protofilament microtubules involved in touch sensation. These results demonstrate for the first time a functional role for this microtubule-associated protein in nematodes and suggest that PTL-1 is involved in mechanosensation as well as some aspect of embryogenesis.     

Public and private mechanisms of life extension in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Model organisms have been widely used to study the ageing phenomenon in order to learn about human ageing. Although the phylogenetic diversity between vertebrates and some of the most commonly used model systems could hardly be greater, several mechanisms of life extension are public (common characteristic in divergent species) and likely share a common ancestry. Dietary restriction, reduced IGF-signaling and, seemingly, reduced ROS-induced damage are the best known mechanisms for extending longevity in a variety of organisms. In this review, we summarize the knowledge of ageing in the nematode Caenorhabditis elegans and compare the mechanisms of life extension with knowledge from other model organisms.     

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.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

Alternative splicing,gene duplication and connectivity in the genetic interaction network of the nematode worm <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

We examined the relationship between gene duplication, alternative splicing, and connectedness in a predicted genetic interaction network using published data from the nematode worm Caenorhabditis elegans. Similar to previous results from mammals, genes belonging to families with only one member ("singletons") were significantly more likely to lack alternative splicing than were members of large multi-gene families. Genes belonging to multi-gene families lacking alternative splicing tended to have higher connectedness in the genetic interaction network than did genes in families that included one or more alternatively spliced members. Moreover, alternatively spliced genes were significantly more likely to interact with other alternatively spliced genes. These results support the hypothesis that certain key proteins with high degrees of network connectedness are subject to selection opposing the occurrence of alternatively spliced forms.     

Nucleosome transactions on the<Emphasis Type="Italic"> Hypocrea jecorina</Emphasis> (<Emphasis Type="Italic"> Trichoderma reesei</Emphasis>) cellulase promoter<Emphasis Type="Italic"> cbh2</Emphasis> associated with cellulase induction

The 5 regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes –1 and –2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome –1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5 regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome –1.Communicated by C. A. M. J. J. van den Hondel     

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">Actinomucor elegans</Emphasis> var. <Emphasis Type="Italic">kuwaitiensis</Emphasis> isolated from the wound of a diabetic patient

The new variety of Actinomucor elegans var. kuwaitiensis, isolated from an open necrotic wound of a diabetic patient is described. This strain differed from the two previously described varieties of A. elegans, A. elegans var. elegans and A. elegans var. meitauzae by nearly 1% or more sequence divergence within D1/D2 regions of 28S rRNA and the ITS region of rRNA genes. Like the other two varieties, no zygospores were observed, however, there was evidence suggesting intersexual diploidy, a feature not described previously in this species. Additionally, A. elegans var. kuwaitiensis was pathogenic to white mice causing 100% mortality within 5 days.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein