Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Résumé

À l'inverse de la situation des vertébrés et de la majorité des insectes, chez qui un gène unique code pour l'acétylcholinestérase (AChE), quatre gènes d'AChE ont été clones et caractérisés chez Caenorhabditis elegans. Le gène ace-1 (localisé sur le chromosome X) et le gène ace-2 (chromosome I) assurent respectivement l'expression de l'AChE dans les tissus musculaire (ace-1) et nerveux (ace-2). Les gènes ace-x et ace-y ne sont séparés que de quelques centaines de paires de bases sur le chromosome II et leur rôle est pour l'instant inconnu.     

单核细胞增生李斯特菌对秀丽隐杆线虫致病性的研究

单核细胞增生李斯特菌(Listeria monocytogenes,Lm)是李斯特菌病的病原细菌。用Lm野生株EGDe、弱毒株ΔprfA、毒力回复株+prfA和高毒株+prfA^*喂饲模式生物秀丽隐杆线虫N2,并以线虫的良好食源大肠埃希菌OP50以及非致病的无害李斯特菌（Listeria innocua）作为对照,检测Lm对线虫发育周期、寿命和产卵数的影响。结果显示：当以无害李斯特菌、Lm野生株以及PrfA突变株为食时,线虫的产卵数虽有所下降,但线虫不仅能够正常产卵,而且其发育周期和寿命均较以OP50为食时显著延长（P≤0.05）;线虫体表和消化道中均可检测到大量李斯特菌,但粪便中的活菌数极少。以上结果说明Lm不能杀死秀丽隐杆线虫,对线虫也没有显著致病性,不适合作为研究Lm致病机制的模型;Lm可在线虫体表和消化道存在,暗示Lm可借助线虫在土壤环境中生存和传播。     

Formaldehyde mutagenesis in the nematode Caenorhabditis elegans

We have found that formaldehyde is capable of inducing mutations in the nematode Caenorhabditis elegans. 4 concentrations of formaldehyde were tested. At a concentration of 1%, formaldehyde is lethal to the nematode, and 0.01% formaldehyde did not induce any mutations in approx. 60 000 tested chromosomes. 2 concentrations of formaldehyde, 0.1% and 0.07%, were found to be mutagenic, inducing both point mutations and deficiencies in the unc-22 region of linkage group IV.4 of the point mutations have been demonstrated to be alleles of the unc-22 gene and have been mapped within the locus. 2 of the putative deficiencies have been confirmed. Each spans the unc-22 gene and at least 2 other genes in the region. A rough estimate of the forward mutation frequency using 0.1% formaldehyde in this region is 3 × 10⁻⁵, while for 0.07% the frequency is 2 × 10⁻⁴.     

Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster

There is a rich diversity of paucimannose N-glycans in worms and flies, and these may play a role in the survival of these organisms. Although paucimannose N-glycans are not expressed in vertebrates, complex N-glycans may take over some of the functions of paucimannose N-glycans. Identification of the target proteins of β-1,2-N-acetylglucosaminyltransferase I (GnTI) in worms and flies and elucidation of their functions may thus lead to a better understanding of the role of GnTI-dependent glycoproteins in the survival/longevity of both invertebrates and vertebrates.     

Kynurenine hydroxylase mutants of the nematode Caenorhabditis elegans

Summary The relation of intestinal autofluorescence to tryptophan catabolism in the free-living nematode Caenorhabditis elegans has been investigated. L-Kynurenine hydroxylase (EC. 1.14.13.9) activity has been detected in normal (wild-type) individuals. Mutants in the gene flu-1 which are characterized by an altered autofluorescence of the intestine cells, i.e., more intense than wild type and bluish purple instead of light blue have also been examined. They show a markedly reduced activity of kynurenine hydroxylase. The finding supports the previously proposed model for altered fluorescence based on chromatographic identification of tryptophan catabolites present.     

线虫体内新piRNA研究

PIWI-interacting RNAs(piRNA)是一类內源性小RNA,负责抵御转座子和转基因对基因组的入侵.已发现1.6万多种piRNA,在piRNA上游存在保守序列,根据上游序列特征可以预测新的piRNA.将线虫同步化培养至L4时期,分别提取野生和prg-1突变样本中的小RNA,并对其进行高通量测序.基于piRNA上游保守序列特征,在野生线虫L4时期中,发现了967种新piRNA,这些新piRNA在prg-1突变后表达消失.新piRNA的基因座集中分布在四号染色体的2个piRNA簇内,首位碱基以U为主.与已发表的成虫发育时期的PRG-1免疫共沉淀数据比对,发现有153种piRNA存在于与PRG-1免疫沉淀的数据中.同时还发现一些只在野生线虫中表达的non-21nt小RNA,它们与已知piRNA的基因座相同,推测这些non-21nt小RNA可能是其piRNA前体加工的产物.总之,通过小RNA测序,在线虫中发现了一些新的piRNA.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

秀丽隐杆线虫模型在抗衰老研究中的应用

随着人口老龄化问题的凸显,衰老相关的研究越来越被重视。秀丽隐杆线虫（Caenorhabditis elegans）是抗衰老研究领域中非常重要的生物模型,具有生命周期短、易于培养和观察等优点,但与其他哺乳动物模型相比仍有一些局限性,如DNA甲基化的缺乏等。本文主要综述了秀丽隐杆线虫模型在抗衰老研究和药物筛选中的应用,包括抗衰老药物对线虫寿命和抗性的测定与评估、药物筛选以及健康衰老研究中的应用,并概括了该模型的优势和局限性,为秀丽隐杆线虫模型在抗衰老研究中的应用提供理论依据。     

秀丽隐杆线虫模型在阿尔茨海默病研究中的应用

阿尔茨海默病（Alzheimer’s disease,AD）是一种与年龄相关的神经退行性疾病,该疾病的病理特征为老年斑（SPs）和神经原纤维缠结（NFTs）的存在。目前,阿尔茨海默病患病率呈现逐年上升的趋势,寻找完全治愈或延缓阿尔茨海默症发展的有效疗法和药物迫在眉睫。秀丽隐杆线虫（Caenorhabditis elegans）的基因和神经元功能与人类具有高度同源性,可作为研究阿尔茨海默病发病机制研究的较好模型。本文综述AD的发病机制假说、秀丽隐杆线虫AD模型以及线虫模型在AD治疗中的应用进展,旨在为后续研究AD提供理论参考。     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(−) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(−) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(−) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(−), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(−) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frameshift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD. Received: 10 June 1998 / Accepted: 21 July 1998     

Protein analysis of an individual Caenorhabditis elegans single-cell embryo by capillary electrophoresis

We present a simple one-dimensional electrophoretic map of the expressed proteins in a Caenorhabditis elegans embryo. The embryo was taken from an adult nematode, injected into a 50-μm I.D. capillary, and lysed. The proteins were fluorescently labeled and then separated by capillary electrophoresis and detected by laser-induced fluorescence. Over 20 components were resolved in the 22-min separation. The dynamic range was outstanding for this separation, noise in the baseline was less than 0.01% the amplitude of the largest component.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

Heritable determinants of male fertilization success in the nematode Caenorhabditis elegans

Background  

Sperm competition is a driving force in the evolution of male sperm characteristics in many species. In the nematode Caenorhabditis elegans, larger male sperm evolve under experimentally increased sperm competition and larger male sperm outcompete smaller hermaphrodite sperm for fertilization within the hermaphrodite reproductive tract. To further elucidate the relative importance of sperm-related traits that contribute to differential reproductive success among males, we quantified within- and among-strain variation in sperm traits (size, rate of production, number transferred, competitive ability) for seven male genetic backgrounds known previously to differ with respect to some sperm traits. We also quantified male mating ability in assays for rates of courtship and successful copulation, and then assessed the roles of these pre- and post-mating traits in first- and second-male fertilization success.     

A plethora of intercellular signals during Caenorhabditis elegans development

Reproducible cell-cell interactions contribute to the invariance of Caenorhabditis elegans development and allow high resolution study of molecular mechanisms of intercellular signaling. A number of new cell interactions have been discovered in the past year. The power of nematode molecular genetics has been increased through several technical advances and the genome project, and these new approaches are now being successfully applied both to familiar and new signaling mechanisms.