Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

The genome of Caenorhabditis elegans.

The physical map of the 100-Mb Caenorhabditis elegans genome consists of 17,500 cosmids and 3500 yeast artificial chromosomes (YACs). A total of 22.5 Mb has been sequenced, with the remainder expected by 1998. A further 15.5 Mb of unfinished sequence is freely available online: because the areas sequenced so far are relatively gene rich, about half the 13,000 genes can now be scanned. More than a quarter of the genes are represented by expressed sequence tags (ESTs). All information pertaining to the genome is publicly available in the ACeDB data base.

Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans.

Substantial progress has been made in understanding the genetic basis of temperature-compensated circadian clocks. Ultradian rhythms, with a period shorter than 24 h, are at least as widespread as circadian rhythms. We have initiated genetic analysis of defecation behavior, which is controlled by an ultradian clock in Caenorhabditis elegans. The defecation motor program is activated every 45 sec, and this rhythm is temperature compensated. We describe mutations in 12 genes that either shorten or lengthen the cycle period. We find that most of these mutations also disrupt temperature compensation, suggesting that this process is an integral part of the clock. These genes open the way for molecular genetic dissection of this ultradian clock.

APX-1 can substitute for its homolog LAG-2 to direct cell interactions throughout Caenorhabditis elegans development.

The homologous LAG-2 and APX-1 membrane proteins are putative signaling ligands in the GLP-1/LIN-12 signal-transduction pathway in Caenorhabditis elegans. Normally, LAG-2 and APX-1 mediate distinct cell interactions. Here, we demonstrate that APX-1, which normally interacts with GLP-1 in the early embryo, can substitute for LAG-2 throughout development. When expressed under control of the lag-2 promoter, an apx-1 cDNA can completely rescue a lag-2 null mutant. To substitute for LAG-2, APX-1 must be able to interact with both GLP-1 and LIN-12 receptors and to mediate a variety of cell interactions during development. Therefore, APX-1 and LAG-2 are essentially equivalent in their ability to influence receptor activity. On the basis of this result, we suggest that the existence of multiple-signaling ligands in the LIN-12/GLP-1 signal transduction pathway does not reflect the evolution of functionally distinct proteins but rather the imposition of distinct controls of gene expression upon functionally similar proteins. Finally, we propose that the specification of distinct cell fates by the LIN-12/GLP-1 signal-transduction pathway relies on activities functioning downstream of the ligand and receptor, rather than on specific ligand-receptor interactions.

Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.

Transgenic Caenorhabditis elegans nematodes have been engineered to express potentially amyloidic human proteins. These animals contain constructs in which the muscle-specific unc-54 promoter/enhancer of C. elegans drives the expression of the appropriate coding regions derived from human cDNA clones. Animals containing constructs expressing the 42-amino acid beta-amyloid peptide (derived from human amyloid precursor protein cDNA) produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies. A subset of these deposits also bind the amyloid-specific dye thioflavin S, indicating that these deposits have the tinctural characteristics of classic amyloid. Co-expression of beta-peptide and transthyretin, a protein implicated in preventing the formation of insoluble beta-amyloid, leads to a dramatic reduction in the number of dye-reactive deposits. These results suggest that this invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.

Identification and characterization of putative transposable DNA elements in solanaceous plants and Caenorhabditis elegans.

Several families of putative transposable elements (TrEs) in both solanaceous plants and Caenorhabditis elegans have been identified by screening the DNA data base for inverted repeated domains present in multiple copies in the genome. The elements are localized within intron and flanking regions of many genes. These elements consist of two inverted repeats flanking sequences ranging from 5 bp to > 500 bp. Identification of multiple elements in which sequence conservation includes both the flanking and internal regions implies that these TrEs are capable of duplicative transposition. Two of the elements were identified in promoter regions of the tomato (Lycoperiscon esculentum) polygalacturonase and potato (Solanum tuberosum) Win1 genes. The element in the polygalacturonase promoter spans a known regulatory region. In both cases, ancestral DNA sequences, which represent potential recombination target sequences prior to insertion of the elements, have been cloned from related species. The sequences of the inverted repeated domains in plants and C. elegans show a high degree of phylogenetic conservation. While frequency of the different elements is variable, some are present in very high copy number. A member of a single C. elegans TrE family is observed approximately once every 20 kb in the genome. The abundance of the described TrEs suggests utility in the genomic analysis of these and related organisms.

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.

Evolution of Outcrossing in Experimental Populations of Caenorhabditis elegans

Caenorhabditis elegans can reproduce exclusively by self-fertilization. Yet, males can be maintained in laboratory populations, a phenomenon that continues to puzzle biologists. In this study we evaluated the role of males in facilitating adaptation to novel environments. For this, we contrasted the evolution of a fitness component exclusive to outcrossing in experimental populations of different mating systems. We introgressed a modifier of outcrossing into a hybrid population derived from several wild isolates to transform the wild-type androdioecious mating system into a dioecious mating system. By genotyping 375 single-nucleotide polymorphisms we show that the two populations had similar standing genetic diversity available for adaptation, despite the occurrence of selection during their derivation. We then performed replicated experimental evolution under the two mating systems from starting conditions of either high or low levels of diversity, under defined environmental conditions of discrete non-overlapping generations, constant density at high population sizes (N = 10(4)), no obvious spatial structure and abundant food resources. During 100 generations measurements of sex ratios and male competitive performance showed: 1) adaptation to the novel environment; 2) directional selection on male frequency under androdioecy; 3) optimal outcrossing rates of 0.5 under androdioecy; 4) the existence of initial inbreeding depression; and finally 5) that the strength of directional selection on male competitive performance does not depend on male frequencies. Taken together, these results suggest that androdioecious males are maintained at intermediate frequencies because outcrossing is adaptive.

CemaT1 is an active transposon within the Caenorhabditis elegans genome

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and T-C transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike T-C transposons in mutator strains of C elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control T-C activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome. (C) 2004 Elsevier B.V. All rights reserved.

Polpa de jambolão (Eugenia jambolana Lam.) fresca e desidratada: características físico-químicas, bioativas e funcionais, efeitos biológicos em Caenorhabditis elegans e uso para produção de frozen yogurt caprino probiótico

This work evaluated the fresh, spray dried (with 10 % of Arabic Gum) and freeze dried jambolan pulp (Eugenia jambolana Lam.) in regard to physicochemical (pH, moisture, water activity, average particle diameter, solubility and color), bioactive [total phenolic content (TPC), monomeric anthocyanin, pronathocyanidin (PA), total elagic acid (TEA), myricetin and cyanidin] and in vitro functionality (antioxidant, antienzymatic and antimicrobial activities]. In addition, the in vivo functionality of jambolan pulp was investigated using the Caenorhabditis elegans model for insulin signaling, longevity and induced neurodegeneration (Alzheimer’s disease and Parkinson’s disease related symptoms). The dried jambolan pulp presented TPC retention (50% to 75%), PA (90% to 98%), TEA (31% to 83%), myricetin (40% to 84%), cyanidin (72% to 84%) and antioxidant activity (15%). The fresh jambolan pulp, the freeze dried pulp and the spray dried jambolan pulp presented high enzymatic inhibitory activity against pancreatic lipase (4,4 to 5,8 mg/mL), alpha-glycosidase (10,3 to 13,8 mg/mL) and alpha-amylase (8,9 to 11,2 mg/mL). They also were active inhibitors against the pathogen S. aureus. The dried jambolan experimental samples were able to increase the expression of several genes linked to the insulin signaling pathways (SIR-2.1, PPTR-1, DAF-16, SOD-3, e CTL) and increased the lifespan in C. elegans (18,07 % - 24,34 %), besides decreasing the amyloid AB1-42 aggregation induced paralysis and MPP+ (1-methyl-4-phenylpyridinium) induced neurodegeneration. Based on that, the jambolan pulp and the spray dried jambolan pulp were further selected for the production of caprine frozen yogurt with the addition of Bifidobacterium animalis subsp. lactis BI-07. The final product were evaluated in regard to their physicochemical (pH, acidity, total solids, protein, total reducing sugars, fat, ashes, overrun, melting test), bioactive (TPC and monomeric anthocyanin, antioxidant activity, probiotic viability and sensory analysis (sensory acceptance). The results showed that samples with probiotic had lowest pH and higher acidity, TPC, anthocyanin and antioxidant activity. It was also observed low overrun (14.2% to 22.6%). vi Samples with probiotic had lower flavor scores. Overall, this research presents the jambolan as a highly functional bioactive-rich fruit with the potential to modulate important biological pathways, extend lifespan and retard the development of neurodegenerative diseases. Jambolan is an underexploited exotic fruit with a high colorant potential and this thesis shows for the first time in the literature important technological, biological and scientific data about this fruit that could be used towards the development of health-oriented food products.

Linking gene expression in the intestine to production of gametes through the phosphate transporter PITR-1 in Caenorhabditis elegans

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane bound transporters and coupled to Na+ transport. Mammalian sodium-dependent Pi co-transporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than 2 decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the C. elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.

From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans.

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.