Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.

BACKGROUND: In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive. RESULTS: Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype. CONCLUSIONS: RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens

<p>Abstract</p> <p>Background</p> <p>The Ahringer <it>C. elegans </it>RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1) mis-annotation (the clone with the retired gene name should be remapped to the actual target gene); 2) nonspecific PCR amplification; 3) cross-RNAi; 4) mis-operation such as sample loading error, <it>etc</it>.</p> <p>Results</p> <p>Here we performed a reliability analysis on the Ahringer <it>C. elegans </it>RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3%) of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54%) bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs). The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (<url>http://biocompute.bmi.ac.cn/CelRNAi/</url>) was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies.</p> <p>Conclusions</p> <p>Because of the potential unreliability of the Ahringer <it>C. elegans </it>RNAi feeding library, we strongly suggest the user examine the reliability information of the bacterial strains in the CelRNAi database before performing RNAi experiments, as well as the post-RNAi experiment analysis.</p

Human immunodeficiency virus type I-specific CD8+ T cell subset abnormalities in chronic infection persist through effective antiretroviral therapy

Background: Effective highly active antiretroviral therapy (HAART) reduces human immunodeficiency virus (HIV) replication, restores CD4 +T lymphocyte counts and greatly reduces the incidence of opportunistic infections. While this demonstrates improved generalized immune function, rapid rebound to pre-treatment viral replication levels following treatment interruption indicates little improvement in immune control of HIV replication. The extent to which HAART can normalize HIV-specific CD8 +T cell function over time in individuals with chronic infection remains an important unresolved issue. In this study, we evaluated the magnitude, general specificity and character of HIV specific CD8 +T cell responses at four time points across 2-9 years in 2 groups of chronically infected individuals separated on the basis of either effective antiretroviral suppression or ongoing replication of HIV.Methods: Peripheral blood mononuclear cells (PBMC) were stimulated with overlapping 15mer peptides spanning HIV Gag, Pol, Env and Nef proteins. Cells producing interferon-γ (IFN-γ) or interleukin-2 (IL-2) were enumerated by ELISPOT and phenotyped by flow cytometry.Results and Conclusions: The magnitude of the HIV-specific CD8 +T cell response ranged from < .01 to approximately 1.0% of PBMC and was significantly greater in the group with detectable viral replication. Stronger responses reflected higher numbers of CD8 +CD45RA -effector memory cells producing IFN-γ, but not IL-2. Magnitude, general specificity and character of the HIV-specific CD8 +T cell response changed little over the study period. While antiretroviral suppression of HIV in chronic infection reduces HIV-specific CD8 +T cell response magnitude in the short term, it had no significant effect on response character over periods up to 9 years

High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor® as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5–8 megabase sub-chromosomal region

LEM-3 – A LEM Domain Containing Nuclease Involved in the DNA Damage Response in C. elegans

The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage – possibly by promoting the reorganization of damaged chromatin

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo

Duplication and Retention Biases of Essential and Non-Essential Genes Revealed by Systematic Knockdown Analyses

When a duplicate gene has no apparent loss-of-function phenotype, it is commonly considered that the phenotype has been masked as a result of functional redundancy with the remaining paralog. This is supported by indirect evidence showing that multi-copy genes show loss-of-function phenotypes less often than single-copy genes and by direct tests of phenotype masking using select gene sets. Here we take a systematic genome-wide RNA interference approach to assess phenotype masking in paralog pairs in the Caenorhabditis elegans genome. Remarkably, in contrast to expectations, we find that phenotype masking makes only a minor contribution to the low knockdown phenotype rate for duplicate genes. Instead, we find that non-essential genes are highly over-represented among duplicates, leading to a low observed loss-of-function phenotype rate. We further find that duplicate pairs derived from essential and non-essential genes have contrasting evolutionary dynamics: whereas non-essential genes are both more often successfully duplicated (fixed) and lost, essential genes are less often duplicated but upon successful duplication are maintained over longer periods. We expect the fundamental evolutionary duplication dynamics presented here to be broadly applicableclose9

Nociceptin Induces Hypophagia in the Perifornical and Lateral Hypothalamic Area

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ’s target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu