Regulatory potential of nonautonomous mariner elements and subfamily crosstalk

Two naturally occurring nonautonomous mariner elements were tested in vivo for their ability to down-regulate excision of a target element in the presence of functional mariner transposase. The tested elements were the peach element isolated from Drosophila mauritiana which encodes a transposase that differs from the autonomous element Mos1 in four amino acid replacements, and the DTBZ1 element isolated from D. teissieri which encodes a truncated protein consisting of the first 132 residues at the amino end of the normally 345-residue transposase. We provide evidence that the protein from the peach element does interact to down-regulate wildtype transposase, indicating that at least some nonautonomous elements in natural populations that retain their open reading frame may play a regulatory role. In contrast, our tests reveal at most a weak interaction between transposase from the autonomous Mos1 element and the truncated protein from DTBZ1 and none between Mos1 transposase and that from the distantly related mariner-like element Himar1 identified in the horn fly Haematobia irritans. Hence, the extent of regulatory crosstalk between mariner-like elements may be limited to closely related ones. The evolutionary implications of these results are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tests for parental imprinting in the nematode Caenorhabditis elegans

Summary The mutation him-6(e1423) leads to generalized chromosomal nondisjunction during meiosis in oogenesis and spermatogenesis of C. elegans. As a result, gametes nullisomic or disomic for each of the six chromosomes occur at appreciable frequency. Crosses utilizing marked him-6 strains were used to generate and identify exceptional euploid progeny which had received both homologues of a marked autosome either from the male parent or from the female parent. Examples of all ten possible exceptions were identified and found to be viable and fertile. These results (together with previous data for the X chromosome) indicate that major chromosomal imprinting effects do not occur during gametogenesis in this organism.     

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering

The advent of genome editing techniques based on the clustered regularly interspersed short palindromic repeats (CRISPR)–Cas9 system has revolutionized research in the biological sciences. CRISPR is quickly becoming an indispensible experimental tool for researchers using genetic model organisms, including the nematode Caenorhabditis elegans. Here, we provide an overview of CRISPR-based strategies for genome editing in C. elegans. We focus on practical considerations for successful genome editing, including a discussion of which strategies are best suited to producing different kinds of targeted genome modifications.     

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.     

Identification of Novel Non-autonomous CemaT Transposable Elements and Evidence of their Mobility within the C. elegans Genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152–367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4’s ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.     

Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

A new transmembrane 4 superfamily molecule in the nematode,Caenorhabditis elegans

Transmembrane 4 superfamily (TM4SF) molecules are predominantly mammalian cell surface glycoproteins that are thought to transduce signals mediating cell development, activation, and motility. Analysis of the Genpept sequence database reveals YKK8, a novel member of the TM4SF in the nematode,Caenorhabditis elegans. YKK8 is a putative 27.4-kDa protein encoded by a gene on chromosome III of theC. elegans genome (Wilson et al. [1994]Nature 368:32–38). The assignment of YKK8 to the TM4SF is justified by three criteria: statistical comparison of protein sequences, conserved TM4SF protein sequence motifs, and conserved TM4SF intron/exon boundaries in the genomic sequence. The discovery of a TM4SF molecule in the nematode extends this superfamily to a more primitive branch of the phylogenetic tree and suggests a fundamental role for TM4SF molecules in biology. Correspondence to: M.G. Tomlinson     

Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas

CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.     

Abundance,Distribution, and Mutation Rates of Homopolymeric Nucleotide Runs in the Genome of Caenorhabditis elegans

Homopolymeric nucleotide runs, also called mononucleotide microsatellites, are a ubiquitous, dominant, and mutagenic feature of eukaryotic genomes. A clear understanding of the forces that shape patterns of homopolymer evolution, however, is lacking. We provide a focused investigation of the abundance, chromosomal distribution, and mutation spectra of the four strand-specific homopolymer types (A, T, G, C) 8 bp in the genome of Caenorhabditis elegans. A and T homopolymers vastly outnumber G and C HPs, and the run-length distributions of A and T homopolymers differ significantly from G and C homopolymers. A scanning window analysis of homopolymer chromosomal distribution reveals distinct clusters of homopolymer density in autosome arms that are regions of high recombination in C. elegans. Dramatic biases are detected among closely spaced homopolymers; for instance, we observe 994 A homopolymers immediately followed by a T homopolymer (5 to 3) and only 8 instances of T homopolymers directly followed by an A homopolymer. Empirical homopolymer mutation assays in a set of C. elegans mutation-accumulation lines reveal an 20-fold higher mutation rate for G and C homopolymers compared to A and T homopolymers. Nuclear A and T homopolymers are also found to mutate 100-fold more slowly than mitochondrial A and T homopolymers. This integrative approach yields a total nuclear genome-wide homopolymer mutation rate estimate of 1.6 mutations per genome per generation.Novel sequences are deposited in GenBank under accession numbers AY219759–AY219789.     

CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

Tat-mediated protein delivery in living Caenorhabditis elegans

The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.     

Caenorhabditis elegans contains genes encoding two new members of the Zn-containing alcohol dehydrogenase family

We have characterized two cDNA clones from the nematode Caenorhabditis elegans that display similarity to the alcohol dehydrogenase (ADH) gene family. The nucleotide sequences of these cDNAs predict that they encode Zn-containing long-chain ADH enzymes. Phylogenetic analysis suggests that one is most similar to dimeric class III ADHs found in diverse taxa; the other is most similar to the tetrameric forms of ADH previously described only in fungi. Correspondence to: J.J. Collins     

The nematode Caenorhabditis elegans as a model to study the roles of proteoglycans

The nematode Caenorhabditis elegans is a powerful animal model for exploring the genetic basis of metazoan development. Recent genetic and biochemical studies have revealed that the molecular machinery of glycosaminoglycan (GAG) biosynthesis and modification is highly conserved between C. elegans and mammals. In addition, genetic studies have implicated GAGs in vulval morphogenesis and zygotic cytokinesis. The extensive knowledge of C. elegans biology, including its elucidated cell lineage, together with the completed and well annotated DNA sequence and availability of reverse genetic tools, provide a platform for studying the functions of proteoglycans and their GAG modification. Published in 2003.