Elegans Gene Research Articles

A recombineering pipeline for functional genomics applied to Caenorhabditis elegans

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Of Mice and Nematodes

Of Mice and Nematodes

ATP-binding cassette transporter A7 enhances phagocytosis of apoptotic cells and associated ERK signaling in macrophages

The mammalian ATP-binding cassette transporters A1 and A7 (ABCA1 and -A7) show sequence similarity to CED-7, a Caenorhabditis elegans gene that mediates the clearance of apoptotic cells. Using RNA interference or gene targeting, we show that knock down of macrophage ABCA7 but not -A1 results in defective engulfment of apoptotic cells. In response to apoptotic cells, ABCA7 moves to the macrophage cell surface and colocalizes with the low-density lipoprotein receptor–related protein 1 (LRP1) in phagocytic cups. The cell surface localization of ABCA7 and LRP1 is defective in ABCA7-deficient cells. C1q is an opsonin of apoptotic cells that acts via phagocyte LRP1 to induce extracellular signal–regulated kinase (ERK) signaling. We show that ERK signaling is required for phagocytosis of apoptotic cells and that ERK phosphorylation in response to apoptotic cells or C1q is defective in ABCA7-deficient cells. These studies reveal a major role of ABCA7 and not -A1 in the clearance of apoptotic cells and therefore suggest that ABCA7 is an authentic orthologue of CED-7.

From worm genetic networks to complex human diseases

Systematic mapping of genetic interactions for Caenorhabditis elegans genes involved in signaling pathways implicated in human disease reveals a network of 350 interactions. The topology of this network resembles that mapped previously in yeast, reinforcing the idea that similar networks may underlie the genetic basis of complex human disease.

The C. elegans gene dig-1 encodes a giant member of the immunoglobulin superfamily that promotes fasciculation of neuronal processes

The adhesion of growing neurites into appropriate bundles or fascicles is important for the development of correct synaptic connectivity in the nervous system. We describe fasciculation defects of animals with mutations in the C. elegans gene dig-1 and show that dig-1 encodes a giant molecule (13,100 amino acids) of the immunoglobulin superfamily. Five new alleles of dig-1 were isolated in a screen for mutations affecting the morphology or function of several classes of head sensory neurons. Mutants showed process defasciculation of several classes of neurons. Analysis of a temperature-sensitive allele revealed that dig-1 is required during embryogenesis for normal process fasciculation of one class of head sensory neuron. Partial sequencing of two alleles, RNA interference (RNAi) and rescuing experiments showed that dig-1 encodes a giant molecule of the immunoglobulin superfamily. DIG-1 protein contains many domains associated with adhesion, is likely secreted, and has some features of proteoglycans. dig-1 mutants were originally isolated due to their displaced gonads [Thomas, J.H., Stern, M.J., Horvitz, H.R., 1990. Cell interactions coordinate the development of the C. elegans egg-laying system. Cell 62, 1041–52]; thus, dig-1 alleles were also characterized for their effects on gonad placement. Mutant phenotypes suggest that DIG-1 may mediate cell movement as well as process fasciculation and that different regions of the protein may mediate these functions.

Identification of Conserved Pathways of DNA-Damage Response and Radiation Protection by Genome-Wide RNAi

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

The Molecular Identities of the Caenorhabditis elegans Intraflagellar Transport Genes dyf-6, daf-10 and osm-1

The Caenorhabditis elegans genes dyf-6, daf-10, and osm-1 are among the set of genes that affect chemotaxis and the ability of certain sensory neurons to take up fluorescent dyes from the environment. Some genes in this category are known to be required for intraflagellar transport (IFT), which is the bidirectional movement of raft-like particles along the axonemes of cilia and flagella. The cloning of dyf-6, daf-10, and osm-1 are described here. The daf-10 and osm-1 gene products resemble each other and contain WD and WAA repeats. DYF-6, the product of a complex locus, lacks known motifs, but orthologs are present in flies and mammals. Phenotypic analysis of dyf-6 mutants expressing an OSM-6::GFP reporter indicates that the cilia of the amphid and phasmid dendritic endings are foreshortened. Consistent with genetic mosaic analysis, which indicates that dyf-6 functions in neurons of the amphid sensilla, DYF-6::GFP is expressed in amphid and phasmid neurons. Movement of DYF-6::GFP within the ciliated endings of the neurons indicates that DYF-6 is involved in IFT. In addition, IFT can be observed in dauer larvae.

Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc and might require invertebrate-specific factors for the formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and open new avenues to explore O-glycan function in this model organism.

ATP-binding Cassette Transporters Are Required for Efficient RNA Interference inCaenorhabditis elegans

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

Modulated microRNA expression during adult lifespan in Caenorhabditis elegans

MicroRNAs (miRNAs) are small, abundant transcripts that can bind partially homologous target messages to inhibit their translation in animal cells. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signaling and oncogenesis. Little is known, however, of miRNA contributions to aging. We examined the expression of 114 identified Caenorhabditis elegans miRNAs during the adult lifespan and find that 34 miRNAs exhibit changes in expression during adulthood (P<or= 0.05), 31 with more than a twofold level change. The majority of age-regulated miRNAs decline in relative abundance as animals grow older. Expression profiles of developmental timing regulators lin-4 and let-7 miRNAs, as well as conserved muscle miRNA miR-1, show regulation during adulthood. We also used bioinformatic approaches to predict miRNA targets encoded in the C. elegans genome and we highlight candidate miRNA-regulated genes among C. elegans genes previously shown to affect longevity, genes encoding insulin-like ligands, and genes preferentially expressed in C. elegans muscle. Our observations identify miRNAs as potential modulators of age-related decline and suggest a general reduction of message-specific translational inhibition during aging, a previously undescribed feature of C. elegans aging. Since many C. elegans age-regulated miRNAs are conserved across species, our observations identify candidate age-regulating miRNAs in both nematodes and humans.

Generation of cloned transgenic pigs rich in omega-3 fatty acids.

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).

Total Information Awareness for Worm Genetics

A comprehensive genetic interaction map of C. elegans can be derived from a database of information about C. elegans genes, proteins and their interactions, augmented with similar data from Drosophila and yeast.

URI-1 is required for DNA stability inC. elegans

Unconventional prefoldin RPB5 interactor (URI), an evolutionary conserved member of the prefoldin family of molecular chaperones, plays a central role in the regulation of nutrient-sensitive, TOR (target-of-rapamycin)-dependent gene expression programs in yeast. Mammalian URI has been shown to associate with key components of the transcriptional machinery, including RPB5, a shared subunit of all three RNA polymerases, the ATPases TIP48 and TIP49, which are present in various chromatin remodeling complexes, and human PAF1 and parafibromin, which are components of a transcription elongation complex. Here, we provide the first functional characterization of a URI-1 homolog in a multicellular organism and show that the C. elegans gene uri-1 is essential for germ cell proliferation. URI-1-deficient cells exhibit cell cycle arrest and display DNA breaks as evidenced by TUNEL staining and the appearance of HUS-1::GFP foci formation. In addition, uri-1(lf) mutants and uri-1(RNAi) worms show a p53-dependent increase in germline apoptosis. Our findings indicate that URI-1 has an important function in the mitotic and meiotic cell cycles. Furthermore, they imply that URI-1 participates in a pathway(s) that is associated with the suppression of endogenous genotoxic DNA damage and highlight a role for URI-1 in the control of genome integrity.

Cloning and regulation of the vertebrate homologue of lin‐41 that functions as a heterochronic gene in Caenorhabditis elegans

The Caenorhabditis elegans gene lin-41 is the one of the heterochronic genes that regulate the timing of many developmental events. MicroRNA let-7 negatively regulates the expression of lin-41 through RNA-RNA interaction on its 3' untranslated region (UTR). Here, we report the isolation of chick and mouse homologues of lin-41 that encode the RBCC-NHL family protein and their expression patterns. C. elegans lin-41 is one of the RBCC-NHL families and the predicted amino acid sequences of isolated two genes encode the same family proteins. Chick and mouse lin-41 expression was also observed in developing limb buds, branchial arches, and tail buds. The 3'UTRs of the mouse and chick lin-41 genes contain multiple let-7 complementary sites. Using luciferase assay, we showed that lin-41 expression can be regulated through let-7 complementary sites.

A Genome-Wide Map of Conserved MicroRNA Targets in C. elegans

Metazoan miRNAs regulate protein-coding genes by binding the 3' UTR of cognate mRNAs. Identifying targets for the 115 known C. elegans miRNAs is essential for understanding their function. By using a new version of PicTar and sequence alignments of three nematodes, we predict that miRNAs regulate at least 10% of C. elegans genes through conserved interactions. We have developed a new experimental pipeline to assay 3' UTR-mediated posttranscriptional gene regulation via an endogenous reporter expression system amenable to high-throughput cloning, demonstrating the utility of this system using one of the most intensely studied miRNAs, let-7. Our expression analyses uncover several new potential let-7 targets and suggest a new let-7 activity in head muscle and neurons. To explore genome-wide trends in miRNA function, we analyzed functional categories of predicted target genes, finding that one-third of C. elegans miRNAs target gene sets are enriched for specific functional annotations. We have also integrated miRNA target predictions with other functional genomic data from C. elegans. At least 10% of C. elegans genes are predicted miRNA targets, and a number of nematode miRNAs seem to regulate biological processes by targeting functionally related genes. We have also developed and successfully utilized an in vivo system for testing miRNA target predictions in likely endogenous expression domains. The thousands of genome-wide miRNA target predictions for nematodes, humans, and flies are available from the PicTar website and are linked to an accessible graphical network-browsing tool allowing exploration of miRNA target predictions in the context of various functional genomic data resources.

Fluoxetine-Resistance Genes inCaenorhabditis elegansFunction in the Intestine and May Act in Drug Transport

Fluoxetine (Prozac) is one of the most widely prescribed pharmaceuticals, yet important aspects of its mechanism of action remain unknown. We previously reported that fluoxetine and related antidepressants induce nose muscle contraction of C. elegans. We also reported the identification and initial characterization of mutations in seven C. elegans genes that cause defects in this response (Nrf, nose resistant to fluoxetine). Here we present genetic evidence that the known nrf genes can be divided into two subgroups that confer sensitivity to fluoxetine-induced nose contraction by distinct pathways. Using both tissue-specific promoters and genetic mosaic analysis, we show that a gene from one of these classes, nrf-6, functions in the intestine to confer fluoxetine sensitivity. Finally, we molecularly identify nrf-5, another gene in the same class. The NRF-5 protein is homologous to a family of secreted lipid-binding proteins with broad ligand specificity. NRF-5 is expressed in the intestine and is likely secreted into the pseudocoelomic fluid, where it could function to transport fluoxetine. One model that explains these findings is that NRF-5 binds fluoxetine and influences its presentation or availability to in vivo targets.

Studies of Caenorhabditis elegans DAF‐2/insulin signaling reveal targets for pharmacological manipulation of lifespan

SummaryMuch excitement has arisen from the observation that decrements in insulin-like signaling can dramatically extend lifespan in the nematode, Caenorhabditis elegans, and fruitfly, Drosophila melanogaster. In addition, there are tantalizing hints that the IGF-I pathway in mice may have similar effects. In addition to dramatic effects on lifespan, invertebrate insulin-like signaling also promotes changes in stress resistance, metabolism and development. Which, if any, of the various phenotypes of insulin pathway mutants are relevant to longevity? What are the genes that function in collaboration with insulin to prolong lifespan? These questions are at the heart of current research in C. elegans longevity. Two main theories exist as to the mechanism behind insulin's effects on invertebrate longevity. One theory is that insulin programs metabolic parameters that prolong or reduce lifespan. The other theory is that insulin determines the cell's ability to endure oxidative stress from respiration, thereby determining the rate of aging. However, these mechanisms are not mutually exclusive and several studies seem to support a role for both. Here, we review recently published reports investigating the mechanisms behind insulin's dramatic effect on longevity. We also spotlight several C. elegans genes that are now known to interact with insulin signaling to determine lifespan. These insights into pathways affecting invertebrate lifespan may provide a basis for developing strategies for pharmacological manipulation of human lifespan.

Transposons in C. elegans

Transposons are discrete segments of DNA capable of moving through the genome of their host via an RNA intermediate in the case of class I retrotransposon or via a "cut-and-paste" mechanism for class II DNA transposons. Since transposons take advantage of their host's cellular machinery to proliferate in the genome and enter new hosts, transposable elements can be viewed as parasitic or "selfish DNA". However, transposons may have been beneficial for their hosts as genome evolution drivers, thus providing an example of molecular mutualism. Interactions between transposon and C. elegans research were undoubtedly mutualistic, leading to the advent of needed genomic tools to drive C. elegans research while providing insights into the transposition field. Tc1, the first C. elegans transposon to be identified, turned out to be the founding member of a widespread family of mobile elements: the Tc1/mariner superfamily. The investigation into transposition regulation in C. elegans has uncovered an unforeseen link between transposition, genome surveillance and RNA interference. Conversely, transposons were utilized soon after their identification to inactivate and clone genes, providing some of the first molecular identities of C. elegans genes. Recent results suggest that transposons might provide a means to engineer site-directed mutations into the C. elegans genome. This article describes the different transposons present in the C. elegans genome with a specific emphasis on the ones that proved to be mobile under laboratory conditions. Mechanisms and control of transposition are discussed briefly. Some tools based on the use of transposons for C. elegans research are presented at the end of this review.

Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy

A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.

The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis elegans

BackgroundThe nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however.ResultsWe show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects.ConclusionWe have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly.