Isolation and mapping of DNA probes within the linkage group I gene cluster of Caenorhabditis elegans

We have isolated probes for DNA polymorphisms across the linkage group I gene cluster in Caenorhabditis elegans, using Tc1-linkage selection. The probes detect strain polymorphism between the wild-type strains of var. Bristol and var. Bergerac. As a result of mapping the sites hP4, hP5, hP6, hP7, hP9, and sPl, more than 1000 kilobases (kb) of cloned cosmid DNA has been positioned on the genetic map. We found there is more DNA per map unit in the center of the gene cluster than expected on the basis of the genomic average. Furthermore, the amount is not constant across the entire region but reaches a peak in the hP9 unc-13 interval. To find the coding regions, we examined DNA cross-homology between two species, Caenorhabditis elegans and Caenorhabditis briggsae. Approximately one-third of the DNA in the hP5 hP9 interval was examined for coding regions and 21 sequences were identified within 318 kb of DNA.     

Temporal dynamics and linkage disequilibrium in natural Caenorhabditis elegans populations

Caenorhabditis elegans is a major laboratory model system yet a newcomer to the field of population genetics, and relatively little is known of its biology in the wild. Recent studies of natural populations at a single time point revealed strong spatial population structure and suggested that these populations may be very dynamic. We have therefore studied several natural C. elegans populations over time and genotyped them at polymorphic microsatellite loci. While some populations appear to be genetically stable over the course of observation, others seem to go extinct, with full replacement of multilocus genotypes upon regrowth. The frequency of heterozygotes indicates that outcrossing occurs at a mean frequency of 1.7% and is variable between populations. However, in genetically stable populations, linkage disequilibrium between different chromosomes can be maintained over several years at a level much higher than expected from the heterozygote frequency. C. elegans seems to follow metapopulation dynamics, and the maintenance of linkage disequilibrium despite a low yet significant level of outcrossing suggests that selection may act against the progeny of outcrossings.     

An evidence-based approach to identify aging-related genes in Caenorhabditis elegans

Background

Extensive studies have been carried out on Caenorhabditis elegans as a model organism to elucidate mechanisms of aging and the effects of perturbing known aging-related genes on lifespan and behavior. This research has generated large amounts of experimental data that is increasingly difficult to integrate and analyze with existing databases and domain knowledge. To address this challenge, we demonstrate a scalable and effective approach for automatic evidence gathering and evaluation that leverages existing experimental data and literature-curated facts to identify genes involved in aging and lifespan regulation in C. elegans.

Results

We developed a semantic knowledge base for aging by integrating data about C. elegans genes from WormBase with data about 2005 human and model organism genes from GenAge and 149 genes from GenDR, and with the Bio2RDF network of linked data for the life sciences. Using HyQue (a Semantic Web tool for hypothesis-based querying and evaluation) to interrogate this knowledge base, we examined 48,231 C. elegans genes for their role in modulating lifespan and aging. HyQue identified 24 novel but well-supported candidate aging-related genes for further experimental validation.

Conclusions

We use semantic technologies to discover candidate aging genes whose effects on lifespan are not yet well understood. Our customized HyQue system, the aging research knowledge base it operates over, and HyQue evaluations of all C. elegans genes are freely available at http://hyque.semanticscience.org.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0469-4) contains supplementary material, which is available to authorized users.     

秀丽隐杆线虫抗铜突变体的筛选及SNP定位

铜在有机体代谢过程中发挥着重要作用, 但过量可产生毒害效应。文章以秀丽隐杆线虫(Caenorhabditis elegans)为模式生物, 寻找多细胞生物中铜代谢调节的关键基因。采用甲基磺酸乙酯(EMS)诱变秀丽隐杆线虫, 通过100 000个杂合基因组的筛选得到两个抗铜突变体ms1和ms2。在筛选培养基上野生型停止发育, 而抗铜突变体则可发育到成虫, 且抗铜性状能稳定遗传。与N2的回交实验表明, ms1的抗铜表型可能由单基因隐性突变导致, ms2的抗铜表型消失, 可能是由多基因突变引起。以CB4856和ms1作为亲本, 构建了F2群, 经SNP定位, 确定ms1突变位点位于染色体II(LGII)上, 进一步对LGII染色体上的8个SNP标记进行分析, 将ms1的突变位点定位在LGII:-6附近。秀丽隐杆线虫抗铜突变体ms1的筛选和定位可为深入研究线虫铜代谢及调控的分子机制提供实验依据。     

A tissue-specific approach to the analysis of metabolic changes in Caenorhabditis elegans

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.     

Combined linkage disequilibrium and linkage mapping: Bayesian multilocus approach

Quantitative trait loci (QTL) affecting the phenotype of interest can be detected using linkage analysis (LA), linkage disequilibrium (LD) mapping or a combination of both (LDLA). The LA approach uses information from recombination events within the observed pedigree and LD mapping from the historical recombinations within the unobserved pedigree. We propose the Bayesian variable selection approach for combined LDLA analysis for single-nucleotide polymorphism (SNP) data. The novel approach uses both sources of information simultaneously as is commonly done in plant and animal genetics, but it makes fewer assumptions about population demography than previous LDLA methods. This differs from approaches in human genetics, where LDLA methods use LA information conditional on LD information or the other way round. We argue that the multilocus LDLA model is more powerful for the detection of phenotype–genotype associations than single-locus LDLA analysis. To illustrate the performance of the Bayesian multilocus LDLA method, we analyzed simulation replicates based on real SNP genotype data from small three-generational CEPH families and compared the results with commonly used quantitative transmission disequilibrium test (QTDT). This paper is intended to be conceptual in the sense that it is not meant to be a practical method for analyzing high-density SNP data, which is more common. Our aim was to test whether this approach can function in principle.     

Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans

One approach to understanding behavior is to define the cellular components of neuronal circuits that control behavior. In the nematode Caenorhabditis elegans, neuronal circuits have been delineated based on patterns of synaptic connectivity derived from ultrastructural analysis. Individual cellular components of these anatomically defined circuits have previously been characterized on the sensory and motor neuron levels. In contrast, interneuron function has only been addressed to a limited extent. We describe here several classes of interneurons (AIY, AIZ, and RIB) that modulate locomotory behavior in C. elegans. Using mutant analysis as well as microsurgical mapping techniques, we found that the AIY neuron class serves to tonically modulate reversal frequency of animals in various sensory environments via the repression of the activity of a bistable switch composed of defined command interneurons. Furthermore, we show that the presentation of defined sensory modalities induces specific alterations in reversal behavior and that the AIY interneuron class mediates this alteration in locomotory behavior. We also found that the AIZ and RIB interneuron classes process odorsensory information in parallel to the AIY interneuron class. AIY, AIZ, and RIB are the first interneurons directly implicated in chemosensory signaling. Our neuronal mapping studies provide the framework for further genetic and functional dissections of neuronal circuits in C. elegans.     

Species differentiation in Caenorhabditis briggsae and Caenorhabditis elegans

Identification of five laboratory strains (1-5) of putative Caenorhabditis briggsae was undertaken. Examination of the male bursal ray arrangement, mating tests with males of Caenorhabditis elegans, malate dehydrogenase zymograms, and SDS polyacrylamide electrophoresis demonstrated that strain 4 was C. briggsae and the others were C. elegans.     

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival—but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

SNP detection and genetic mapping of porcine genes encoding enzymes in hepatic metabolic pathways and evaluation of linkage with carcass traits

We have previously identified and mapped porcine expressed sequence tags (ESTs) derived from genes that are preferentially expressed in liver. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) in porcine genes encoding enzymes in hepatic metabolic pathways and use the SNPs for mapping. Furthermore, these genes, which are involved in utilization and partitioning of nutrients, were examined for their effects on carcass and meat quality traits by linkage analyses. In total, 100 ESTs were screened for SNPs by single strand conformation polymorphism analyses across a diverse panel of animals with a 36% success rate. Twelve of 36 polymorphic loci segregated in a three-generation Duroc x Berlin Miniature Pig (F2) resource population, the DUMI resource population, and were genetically mapped. Interval mapping of the corresponding chromosomes was performed to verify mapping of the genes within quantitative trait loci (QTL) regions detected in this resource population. QTL with genome-wide significance were detected in the vicinity of GNMT, ESTL147 and HGD. These loci therefore are positional candidate genes.     

Globins in Caenorhabditis elegans

Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB-1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions.     

Mechanotransduction in Caenorhabditis elegans

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabiditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.