RESUMO
Simplicity has made C. elegans pharyngeal development a particularly well-studied subject. Nevertheless, here we add the previously uncharacterized homeobox gene F20D12.6/ceh-19 to the set of transcription factor genes involved. GFP reporter assays revealed that ceh-19 is expressed in three pairs of neurons, the pharyngeal pace-maker neurons MC, the amphid neurons ADF and the phasmid neurons PHA. ceh-19(tm452) mutants are viable and fertile, but grow slightly slower, produce less progeny over a prolonged period, and live longer than the wild type. These phenotypes are likely due to the moderately reduced pharyngeal pumping speed arising from the impairment of MC activity. MC neurons are still born in the ceh-19 mutants but display various morphological defects. ceh-19 expression in MC is completely lost in progeny from animals subject to RNAi for pha-4, which encodes an organ-specifying forkhead transcription factor. CEH-19 is required for the activation in MCs of the excitatory FMRFamide-like neuropeptide-encoding gene flp-2. A regulatory pathway from pha-4 through ceh-19 to flp-2 is thereby defined. The resilience of MC identity in the absence of CEH-19 may reflect the buffering qualities of transcription factor regulatory networks.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Neurônios Motores/fisiologia , Neuropeptídeos/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fertilidade/genética , Expressão Gênica , Longevidade/genética , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Mutação , Neurogênese/genética , Neuropeptídeos/genética , Faringe/inervação , Faringe/fisiologia , Fenótipo , Filogenia , RNA Interferente Pequeno , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal's genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. RESULTS: Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. CONCLUSIONS: Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms.
Assuntos
Caenorhabditis elegans/genética , Perfilação da Expressão Gênica , Fatores de Transcrição/genética , Animais , Éxons/genética , Íntrons/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Espaço-TemporalRESUMO
BACKGROUND: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. RESULTS: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. CONCLUSIONS: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.
Assuntos
Caenorhabditis elegans/genética , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Genoma , Proteínas Luminescentes/metabolismo , Animais , Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genéticaRESUMO
Regulatory transcription factors operate in networks, conferring biological robustness that makes dissection of such gene control processes difficult. The nematode Caenorhabditis elegans is a powerful molecular genetic system that allows the close scrutiny needed to understand these processes in an animal, in vivo. Strikingly lower levels of gene expression were observed when a gfp reporter was inserted into C. elegans transcription factor genes, in their broader genomic context, in comparison to when the reporter was fused to just the promoter regions. The lower level of expression is more consistent with endogenous levels of the gene products, based on independent protein and transcript assays. Through successive precise manipulations of the reporter fusion genes, elements essential for the lower level of expression were localised to the protein-coding region. With a closer focus on four transcription factor genes, the expression of both genes encoding transcriptional activators was found to be restricted by a post-transcriptional mechanism while expression of both genes encoding transcriptional repressors was delimited by transcriptional repression. An element through which the transcriptional repression acts for unc-4 was localised to a 30 base-pair region of a protein-encoding exon, with potentially wider implications for how homeobox genes operate. The hypothesis that the distinction in mechanisms delimiting expression of the two types of transcription factor genes, as observed here, may apply more widely is raised. This leads to observations concerning the implications of these different mechanisms on stochastic noise in gene expression and the consequent significance for developmental decisions in general.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Éxons , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Regiões Promotoras GenéticasRESUMO
Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica/métodos , Longevidade/fisiologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Cromatina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Longevidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. RESULTS: The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. CONCLUSIONS: By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.
Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Vetores Genéticos , Origem de Replicação , Animais , Caenorhabditis elegans/genética , Cromossomos Artificiais Bacterianos , Colífagos , Proteínas de Escherichia coli/genética , Dosagem de Genes , Fusão Gênica , Genes Reporter , Recombinação GenéticaRESUMO
Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.
Assuntos
Envelhecimento/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Regiões Promotoras Genéticas/genética , Proteoma/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Microscopia de Fluorescência , Proteoma/genética , Distribuição TecidualRESUMO
The ryanodine receptor mediates intracellular calcium ion release with excitation of nerve and muscle cells. Ryanodine receptor missense variants cause a number of myopathologies, such as malignant hyperthermia, and have been linked with various neuropathologies, including Alzheimer's disease. We characterized the consequences of ryanodine receptor variants in vivo. Eight Caenorhabditis elegans strains, with ryanodine receptor modifications equivalent to human myopathic RYR1 variants, were generated by genome editing. In humans, these variants are rare and confer sensitivity to the inhalational anaesthetic halothane when heterozygous. Increased sensitivity to halothane was found in both homozygous and heterozygous C. elegans. Close analysis revealed distinct subtle locomotion defects, due to the different single amino acid residue changes, even in the absence of the external triggering agent. Distinct pre- and postsynaptic consequences of the variants were characterized through the responses to cholinergic pharmacological agents. The range of phenotypes reflects the complexity of the regulatory inputs to the ryanodine receptor and the criticality of the calcium ion channel opening properties, in different cell types and with age. Ryanodine receptors with these single amino acid residue changes still function as calcium ion channels, but with altered properties which are likely to have subtle consequences for human carriers of such variants. The long-term consequences of subtly altered calcium ion signalling could be cumulative and may be focussed in the smaller nerve cells rather than the more robust muscle cells. It was important to assess phenotypes in vivo to properly appreciate consequences for a whole organism.
RESUMO
Observation of gene expression in situ provides a direct connection between the genetic information in the genome sequence and the fully determined developmental cell lineage of Caenorhabditis elegans. Green Fluorescent Protein (GFP) reporters have been fused with many C. elegans genes, in large-scale projects, by conventional DNA ligation, PCR stitching, Gateway recombination and recombineering. These reporter gene fusions have then been used in C. elegans transformation either by microinjection or microprojectile bombardment. So far, the developmental distributions of GFP, as driven by the C. elegans DNA to which the reporter gene has been attached, have been determined simply from direct examination of the transgenic strains by epifluorescence microscopy. Automation of GFP expression pattern determination promises improvements in both quality and quantity of this data type, facilitating the handling of such expression pattern data within computer databases. As with the descriptions of the developmental cell lineage and the genome sequence, a complete description of gene expression patterns will provide a vital knowledge framework through which a full understanding of the development of this animal can emerge.
Assuntos
Caenorhabditis elegans/genética , Perfilação da Expressão Gênica/métodos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously. To ensure inclusion of all such elements and provide details of subcellular localization, construction of translational reporters would, preferably, utilize genomic clones, containing the complete locus plus flanking regions and permit seamless insertion of the reporter anywhere within the gene. We have developed such a method based upon lambda Red-mediated recombineering coupled to a robust two-step counter-selection protocol. We have inserted either gfp or cfp precisely at the C-termini of three Caenorhabditis elegans target genes, each located within different fosmid clones, and examined previously with conventional reporter approaches. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporters highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox.
Assuntos
Caenorhabditis elegans/genética , Expressão Gênica , Genes Reporter , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Bacteriófago lambda/genética , Sequência de Bases , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Clonagem Molecular , DNA/genética , Marcação de Genes , Genes de Helmintos , Genoma Helmíntico , Genômica/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Substâncias Luminescentes/análise , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Recombinação GenéticaRESUMO
BACKGROUND: The C. elegans Promoterome is a powerful resource for revealing the regulatory mechanisms by which transcription is controlled pan-genomically. Transcription factors will form the core of any systems biology model of genome control and therefore the promoter activity of Promoterome inserts for C. elegans transcription factor genes was examined, in vivo, with a reporter gene approach. RESULTS: Transgenic C. elegans strains were generated for 366 transcription factor promoter/gfp reporter gene fusions. GFP distributions were determined, and then summarized with reference to developmental stage and cell type. Reliability of these data was demonstrated by comparison to previously described gene product distributions. A detailed consideration of the results for one C. elegans transcription factor gene family, the Six family, comprising ceh-32, ceh-33, ceh-34 and unc-39 illustrates the value of these analyses. The high proportion of Promoterome reporter fusions that drove GFP expression, compared to previous studies, led to the hypothesis that transcription factor genes might be involved in local gene duplication events less frequently than other genes. Comparison of transcription factor genes of C. elegans and Caenorhabditis briggsae was therefore carried out and revealed very few examples of functional gene duplication since the divergence of these species for most, but not all, transcription factor gene families. CONCLUSION: Examining reporter expression patterns for hundreds of promoters informs, and thereby improves, interpretation of this data type. Genes encoding transcription factors involved in intrinsic developmental control processes appear acutely sensitive to changes in gene dosage through local gene duplication, on an evolutionary time scale.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis/genética , Duplicação Gênica , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Genes Reporter , Técnicas Genéticas , Genômica , Proteínas de Fluorescência Verde/metabolismo , Filogenia , Especificidade da EspécieRESUMO
Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Longevidade/genética , Doenças Musculares/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/metabolismo , Humanos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Fenótipo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The forkhead genes encode a family of transcriptional regulators with important roles in the control of animal development. The genomic sequence has revealed Caenorhabditis elegans has 15 forkhead genes of which six had been experimentally characterised previously. The remaining nine have now been investigated by RNAi and reporter gene expression pattern analysis. Two (B0286.5 and F26B1.7) have key developmental roles, four (F26D12.1, K03C7.2, F40H3.4 and C25A1.2) are expressed in, and may function in, the nervous system and three (C29F7.4, C29F7.5 and F26A1.2), which are closely related to each other, do not appear to be essential. Conservation with forkhead genes in the closely related nematode Caenorhabditis briggsae does not appear to correspond completely with demonstrated functionality in C. elegans. Comparisons of expression patterns and RNAi/mutant phenotypes for all 15 C. elegans forkhead genes reveal the potential complexity of functional interactions for this gene family.
Assuntos
Caenorhabditis elegans/genética , Família Multigênica/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Longevity is modulated by a range of conserved genes in eukaryotes, but it is unclear how variation in these genes contributes to the evolution of longevity in nature. Mutations that increase life span in model organisms typically induce trade-offs which lead to a net reduction in fitness, suggesting that such mutations are unlikely to become established in natural populations. However, the fitness consequences of manipulating longevity have rarely been assessed in heterogeneous environments, in which stressful conditions are encountered. Using laboratory selection experiments, we demonstrate that long-lived, stress-resistant Caenorhabditis elegans age-1(hx546) mutants have higher fitness than the wild-type genotype if mixed genotype populations are periodically exposed to high temperatures when food is not limited. We further establish, using stochastic population projection models, that the age-1(hx546) mutant allele can confer a selective advantage if temperature stress is encountered when food availability also varies over time. Our results indicate that heterogeneity in environmental stress may lead to altered allele frequencies over ecological timescales and indirectly drive the evolution of longevity. This has important implications for understanding the evolution of life-history strategies.
RESUMO
Expression pattern data are fundamental to understanding transcriptional regulatory networks and the biological significance of such networks. For Caenorhabditis elegans, expression pattern analysis of transcription factor genes, with cellular resolution, typically involves generation of transcription factor gene/reporter gene fusions. This is followed by the creation of C. elegans strains transgenic for, and determination of expression patterns driven by, these fusions. Physiologically relevant regulatory relationships between transcription factors are both inferred from their expression patterns, in combination with protein-DNA interaction data, and evidenced from alterations of expression patterns when networks are disturbed.
Assuntos
Caenorhabditis elegans/genética , Regulação da Expressão Gênica/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/metabolismo , Redes Reguladoras de Genes/genética , Fatores de Transcrição/metabolismoRESUMO
Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix-turn-helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1.
Assuntos
Caenorhabditis elegans/genética , Interneurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , MasculinoRESUMO
In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ(9) desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ(9) desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ(9) desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Estearoil-CoA Dessaturase/genética , Fatores de Transcrição/genética , Aclimatação , Animais , Temperatura Baixa , Fatores de Transcrição Forkhead , Proteínas de Fluorescência Verde/metabolismo , Insulina/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/genética , Interferência de RNA , Transdução de Sinais , Temperatura , Fatores de TempoRESUMO
A screen was instigated to identify novel protein components of the Caenorhabditis elegans sarcomere. The subcellular localisation of full-length GFP fusion proteins was examined, in transgenic animals, for 62 essentially uncharacterized genes thought to be expressed within bodywall muscle cells. Three genes, T03G6.3, C46G7.2 and K04A8.6, were identified for further study. K04A8.6::GFP only displayed a regular sarcomeric distribution sporadically. However, C46G7.2::GFP localised to the centre of A-bands and dense bodies and T03G6.3::GFP localised in the I-band, of the bodywall muscle sarcomeres, consistently. This success with such a small screen suggests that there are further minor components of the C. elegans sarcomere yet to be discovered. Fluorescence Recovery After Photobleaching (FRAP) was applied to live transgenic individuals to assess the mobility of T03G6.3 and C46G7.2 and other well-known constituents of the sarcomere in vivo. Proteins associated with the thin filaments showed dynamic exchange whilst those associated with thick filaments appeared more static. This is the first demonstration that there are sarcomeric proteins in C. elegans muscle cells in dynamic exchange and that the rates of exchange in vivo correspond in general terms with observations in other experimental systems.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Sarcômeros/genética , Sarcômeros/metabolismo , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Genes de Helmintos , Proteínas de Fluorescência Verde , Microscopia Confocal , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sarcômeros/ultraestruturaRESUMO
INTRODUCTION: As in yeast, flies and mammals, over-expression of the Caenorhabditis elegans sirtuin gene sir-2.1 leads to extension of lifespan and deletion of the gene shortens lifespan. The sir-2.1 gene, however, is located in an operon, an organization not taken into account in previous studies of this gene's expression. MATERIALS AND METHODS: Recombineering allowed insertion of both a mCherry and a gfp reporter gene precisely at the end of the two protein-coding regions of the 4.5kb sir-2.1 operon within a 29.3kb genomic DNA fosmid clone. RESULTS AND DISCUSSION: In C. elegans transgenic for this recombineered fosmid, with abundant food, the sir-2.1::mCherry distribution indicated that sir-2.1 is indeed expressed in the hypodermis and many nerve cells, as previously described, but also in the intestine and in muscles. This broader expression of sir-2.1, which would fit with an expectation that SIR2.1 function in influencing lifespan might be required in most cell types, arises from transcription starting with the gene upstream of sir-2.1 in the operon. Importantly, the expression of both genes in the operon increases upon starvation, this induction also depending on the operon promoter. Furthermore, SIR-2.1::mCherry undergoes a dynamic subcellular relocalization through starvation.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Dieta , Expressão Gênica , Sirtuínas/genética , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/fisiologia , Restrição Calórica , Privação de Alimentos/fisiologia , Proteínas de Fluorescência Verde/genética , Mucosa Intestinal/metabolismo , Longevidade , Músculos/metabolismo , Óperon/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Sirtuínas/fisiologiaRESUMO
The ability of an animal to locomote through its environment depends crucially on the interplay between its active endogenous control and the physics of its interactions with the environment. The nematode worm Caenorhabditis elegans serves as an ideal model system for studying the respective roles of neural control and biomechanics, as well as the interaction between them. With only 302 neurons in a hard-wired neural circuit, the worm's apparent anatomical simplicity belies its behavioural complexity. Indeed, C. elegans exhibits a rich repertoire of complex behaviors, the majority of which are mediated by its adaptive undulatory locomotion. The conventional wisdom is that two kinematically distinct C. elegans locomotion behaviors-swimming in liquids and crawling on dense gel-like media-correspond to distinct locomotory gaits. Here we analyze the worm's motion through a series of different media and reveal a smooth transition from swimming to crawling, marked by a linear relationship between key locomotion metrics. These results point to a single locomotory gait, governed by the same underlying control mechanism. We further show that environmental forces play only a small role in determining the shape of the worm, placing conditions on the minimal pattern of internal forces driving locomotion.