suppressor screen c elegans

suppressor screen c elegans

2006) may also be helpful. In this screen, let-60 worms were mutagenized by EMS and scored for suppression of the let-60 multivulval (Muv) mutant phenotype. lin-35 is a bit unusual, however, in that inactivation of both chromosomal copies of lin-35 does not lead to a vulval cell-lineage defect. stream In this article, however, the suppressor phenotype is the result of knocking down gene expression with RNAi. The article can certainly be used as a teaching aid in isolation, and instructors may want to apply the C.R.E.A.T.E. This approach is called a “suppressor screen.”. To better determine how sax-7 and MAPK regulate locomotion, I performed a screen to isolate additional genetic suppressors for rab-3;sax-7 locomotion defects using chemical mutagenesis. Tissue-specific transcription factors may also recruit proteins that alter chromatin structure, thereby making the promoter and enhancer more or less accessible to other transcription factors. Polley and Fay use the model genetic organism Caenorhabditis elegans to discover how the retinoblastoma (Rb) protein and other gene products can affect intestinal function. How might you interpret these data? Characterization of sirtuin inhibitors in nematodes expressing a muscular dystrophy protein reveals muscle cell and behavioral protection by specific sirtinol analogues. One such second mutant gene was identified as slr-2, encoding a protein containing a zinc-finger domain typically found in transcription factors. In addition, Polley and Fay found suppressors that did not fit into any of these categories. 2005). Both the original light switch and the dimmer switch operate in the same circuit and therefore control the same outcome (phenotype). What conclusions can you draw about the minimal number of mechanisms of suppression by comparing the data shown in figure 1, B–D, to figure 1A in Polley and Fay (2012)? We used the Plate Runner HD® (Trophos, France), a 96/384-well plate motorized device able to collect fluorescence at resolutions ranging from 1024x1024 px (1 px is 7.4 µm) to 8192x8192 px (1 px is 1 µm). To transcribe a gene in a multi-cellular organism, tissue-specific transcription factors bind to DNA sequences, such as enhancers, and recruit general transcription factors and RNA polymerase to the promoter of the gene. WormBase (http://www.wormbase.org), a carefully maintained database of published knowledge about C. elegans genes, including what is known about orthologs of those genes in other organisms, was used to sort the suppressor genes into three functional classes: (1) genes involved in ribosome production; (2) genes already known to suppress a synMuv phenotype; and (3) genes encoding prohibitins, proteins that have multiple functions including cell cycle regulation and mitochondrial function. Polley and Fay’s solution was to add a wild-type copy of lin-35 to the double-mutant worms on an extrachromosomal array. Here, we describe a 96-well plate assay that illustrates how high resolution imaging may be used in day-to-day research as well as RNAi and drug screens. setTimeout(function(){var a=document.createElement("script"); Group 2: Describe the data shown in figure 1A in the accompanying article. Polley and Fay (2012) again harnessed the power of a genetic approach to identify other genes involved in intestinal function by isolating genes that, when downregulated, allow the survival of lin-35; slr-2 double-mutant worms. 2000). Group 5: How did Polley and Fay assess the mechanism by which the prohibitins suppress L1 lethality? Could you say that G148 treatment phenocopies the suppressor phenotype? It is important to note that the alleles of lin-35 and slr-2 used by Polley and Fay are null alleles—versions of each gene that make no functional protein product: the lin-35 allele is a G→A base substitution that produces an in-frame stop codon; the slr-2 allele alters an mRNA splice site that produces aberrantly spliced transcripts with premature stop codons (Kirienko et al. Group 3: For what purpose did Polley and Fay produce the double- and triple-mutant animals indicated in figure 1, B–D, in the accompanying article? << /Length 5 0 R /Filter /FlateDecode >> Drug screening is now ongoing using this assay. See Hodgkin (2005) for more details about suppressors. One reasonable division of material for discussion is the following: Group 1: Diagram the reverse genetic screen and provide an answer to question 1 below. The array also carries another gene tagged with green fluorescent protein (GFP). Pasco MY, Rotili D, Altucci L, Farina F, Rouleau GA, Mai A, Néri C. (2010). Similar strategies may be used in the development of a variety of suppressor screens. %��������� "+Math.floor(new Date().getTime()/3600000); One classic example is the let-60 (gf) suppressor screen carried out in recent years by the Han lab. What would you measure to produce quantitative data? 2002). Genetics instructors are encouraged to provide this primer article to students concurrently with Polley and Fay (2012). Subscribe for updates to the WBG by email or RSS. If reduction or knockdown of expression of gene X allows the lin-35; slr-2 double mutants to survive past the early larval stage, then gene X would be a suppressor of lin-35; slr-2-induced lethality. Kirienko and co-workers demonstrated that LIN-35 and SLR-2 have redundant functions in the intestine because intestinal deficiency is seen only in double-mutant animals (another synthetic phenotype). At 46 hours, FUDR (0.1 mg/ml) is added to prevent egg-laying and hatching. (2008) that describes the forward genetic screen that identified additional lin-35 synthetic phenotypes and the genes that confer those phenotypes, followed by Polley and Fay (2012). The reverse genetic (RNAi) screen is the centerpiece technique of the article, followed by the initial characterization of suppressors identified by RNAi. If you were a graduate student charged with choosing to study further a single suppressor gene identified in this screen, which gene would you choose? Polley and Fay fed each bacterial strain to lin-35; slr-2 mutant worms and screened their offspring for growth beyond the L1 stage. ... Suppressor screen. These genes are thus termed “suppressors of the lin-35; slr-2 synthetic lethal phenotype.” Remember that suppression is seen only when the product of the suppressor gene is downregulated or missing. A previous pilot genetic suppressor screen uncovered the role of the mitogen-activated protein kinase (MAPK) pathway in regulating locomotion. By undertaking RNAi of a suppressor gene in worm strains that contain the lin-35; slr-2 mutations plus a null allele of another known suppressor, Polley and Fay determined whether reductions of suppressor proteins have additive effects. Groups 7 and 8: Address question 8 below, choosing different genes. Your email address will not be displayed and will never be shared or distributed. Our members work to advance knowledge in the basic mechanisms of inheritance, from the molecular to the population level. Day-3 adults are immobilized using 2,3-butanedione monoxime (0.1 M, 300 µl/well). Julie Ahringer and co-workers designed an RNAi library—a set of bacterial strains each expressing a different double-stranded RNA molecule, one strand of which is complementary to mRNA encoded by a single C. elegans gene (Fraser et al. David Fay’s laboratory has been addressing several questions. Genet. a.src=document.location.protocol+"//script.crazyegg.com/pages/scripts/0042/1390.js? This kind of suppressor screen is therefore a way to identify genes whose products function together in the same pathway or in compensatory pathways. The article provides an opportunity to discuss the utility of transgenic organisms and extrachromosomal arrays (used in worms) as opposed to artificial chromosomes (used in yeast) or plasmids (more familiar to students). Be sure to include in your answer the cellular role of lin-35 and the cellular effect of a lin-35 null allele. 4 0 obj Available freely online through the author-supported open access option. Genes that have not been studied in detail are labeled with a clone number, e.g., W07E6.2. In the case of lin-35, the associated lineage defect leads to an increase in the number of cells that are programmed to become part of the egg-laying structure, termed the vulva. Describe a possible mechanism of suppression of just one or two lin-35 synthetic phenotypes (from figure 4 in the accompanying article). �'�|���l������SȰo#����ztj"m�����I��T���N���ѥHء4�x. 1A). Polley and Fay identified genes that act as suppressors in pathways that are different from the lin-35; slr-2 cellular circuit, as well as several that might be in the same functional circuit. Thus Polley and Fay placed several GFP-positive worms on 16,757 agar plates, each plate containing a bacterial strain expressing a different double-stranded RNA. Many core genetics concepts are reinforced well with data in figures 1–4 in the accompanying article by Polley and Fay. We do not retain these email addresses. The authors employ an elegant and effective reverse genetic approach that identifies gene products that function in the same cellular pathways as the Rb protein. Some of the progeny of the microinjected worm will incorporate this DNA into their nuclei in the form of an extrachromosomal array—many end-to-end copies of the added gene(s)—that is not in a chromosome.

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