Powell-Coffman JA et al. (1997) Worm Breeder's Gazette "A C. elegans Dioxin Receptor"

Wood WB, Powell-Coffman JA

[Worm Breeder's Gazette, 1997]

The vertebrate aryl hydrocarbon receptor (AHR; a.k.a. the dioxin receptor) mediates the teratogenic and carcinogenic effects of certain environmental contaminants. AHR ligands include benzo(a)pyrene, the primary carcinogen in cigarette smoke, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous man-made pollutant. Unliganded AHR resides in the cytoplasm in a complex with the 90-kDa heat shock protein (HSP90). Upon binding ligand, AHR translocates to the nucleus, dissociates from HSP90, and dimerizes with the AHR nuclear translocator protein (ARNT). Both AHR and ARNT belong to a class of regulatory proteins that contain a basic-helix-loop-helix DNA binding motif and a PAS domain (also found in Drosophila period and single-minded). The AHR-ARNT heterodimer binds specific promoter sequences to regulate transcription. (1) In mice AHR function is required for the proper development of the immune system and liver, but the presumptive endogenous ligand(s) is unknown. (2) Until recently, it had not been possible to study AHR signaling in a model system amenable to genetic analysis, as no invertebrate orthologues of AHR or ARNT had been reported. We have cloned the C. elegans homologues of AHR and ARNT. CeAHR may be the egl-33 gene; it is included in the 15kb genomic fragment that rescues egl-33 (ct315). (3) CeARNT maps to the right of egl-33 on LGI. We have expressed these gene products in rabbit reticulocyte lysates. Initial gel electrophoretic shift experiments demonstrate that CeAHR and CeARNT interact to bind the mammalian AHR-ARNT enhancer site. Competition experiments with wild-type and a mutant form of the enhancer site confirm that this binding is specific. These data suggest that we have cloned true C. elegans orthologues of AHR and ARNT. We are also collaborating with C. Bradfield and colleagues at the University of Wisconsin to determine the binding affinity of CeAHR for TCDD. To identify the cellular decisions regulated by the AHR signaling complex in C. elegans, we are screening for Tc1-induced deletions in CeAHR and CeARNT, and we are examining the expression patterns of these two genes. RNA blots indicate that CeAHR and CeARNT are coordinately expressed and are most abundant during embryogenesis and the first larval stage. We have constructed lacZ and GFP reporter genes and have detected expression of CeAHR::lacZ as early as the 30-cell stage. At the 300-cell stage, it is expressed in approximately 20% of the cells. As morphogenesis begins, most cells cease to express CeAHR::lacZ, and at hatching, it is detectable in a subset of neuronal and hypodermal cells. These experiments will establish the foundation for further genetic analysis of the AHR signaling pathway. 1. Hankinson, O. (1995) Annu. Rev. Pharmacol. Toxicol. 35: 307-34; Whitlock, J. P. (1993) Chem. Res. Toxicol. 6: 754-763; Bock, K. W. (1993) Rev. Phys. Biochem. Pharmacol. 125: 1-42 2. Fernandez-Salguero, P. et al. (1995) Science 268: 722-726 3. Powell-Coffman, J. A. and B. Wood. 1996 West Coast Worm Mtg.

Winge P et al. (1994) Worm Breeder's Gazette "R-ras 1 and R-ras 2 (TC21) Homologs."

Gobel V, Friend S, Winge P, Fleming JT

[Worm Breeder's Gazette, 1994]

R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with bc1-2, the human homolog to ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.

Fabio Piano et al. (2001) Worm Breeder's Gazette "On the reproducibility of large-scale - RNAi screens"

Aaron Schetter, Fabio Piano, Ken Kemphues, Kris Gunsalus, Valerie Reinke, Stuart Kim, Diane Morton

[Worm Breeder's Gazette, 2001]

RNAi is being used routinely to determine loss-of-function phenotypes and recently large-scale RNAi analyses have been reported (1,2,3). Although there is no question about the value of this approach in functional genomics, there has been little opportunity to evaluate reproducibility of these results. We are engaged in RNAi analysis of a set of 762 genes that are differentially expressed in the germline as compared to the soma (4 -- "Germline"), and have reached a point in our analysis that allows us to look at the issue of reproducibility. We have compared the RNAi results of genes in our set that were also analyzed by either Fraser et al. (1 -- Chromosome 1 set "C1") or Gonczy et al. (2 -- Chromosome 3 set "C3"). In making the comparison we have taken into account the different operational definition of "embryonic lethal" used by the three groups. In the C3 study, lethal was scored only if there were fewer than 10 surviving larva on the test plate, or roughly 90% lethal. In our screen and the C1 screen the percent survival was determined for each test. To minimize the contribution of false positives from our set, in our comparison with the C1 set we defined our genes as "embryonic lethal" if at least 30% of the embryos did not hatch, but included all lethals defined by Fraser et al. (> 10%). For our comparison with the C3 set, we used a more restrictive definition of "embryonic lethal" that required that 90% of the embryos did not hatch. (This means that in Table 1, five genes from our screen that gave lethality between 30-90% were included in the not lethal category; one of these was scored as lethal by Gonczy et al.). We have analyzed 149 genes from the germline set that overlap with the C1 set and 132 genes that overlap with the C3 set. The table below shows the number of genes scored as embryonic lethal (EL) or not embryonic lethal (NL) in each study. (Note that these comparisons do not include data from our published collection of ovary-expressed cDNAs.) Table 1. Comparing RNAi analysis of the same genes in different studies. Germline Chromosome 1 Germline Chromosome 3 NL (117) EL (32) NL (97) EL (35) NL (104) 100 4 NL (89) 87 2 EL (45) 17 28 EL (43) 10 33 Overall, the degree of reproducibility is high. The concordance between our results and the published results was 86% with C1 (128/149 genes) and 90% with C3 (120/132). However, we scored a larger number of genes as giving rise to embryonic lethal phenotypes than the other studies did. What does this mean? One possibility is that we are generating a large number of false positives (God forbid!). The other interpretation is that there is a fairly high frequency of false negatives in each screen (4-8% in our screen (2/45; 4/49); 22% in the C3 screen (10/45); and 35% (17/49) in the C1 screen). It is no surprise that the different methods used by the three groups resulted in slightly different outcomes and we can only speculate on which methodological variation contributed most. In comparing our methods to those used in the C3 study we note that our two groups used different primer pairs for each gene; that we tested genes individually while they tested genes in pairs; and that the operational definition of "embryonic lethal" differed. Considering the latter two differences, we speculate that even with pools of two, the competition noted by Gonczy et al. in dsRNA pools could reduce levels of lethality below the 90% cutoff. The major difference between our approach and the C1 approach is feeding vs. injection, raising the possibility that for some genes feeding may be a less effective means of administering dsRNA. Whatever the basis for the difference, these comparisons indicate that genes scored as "non-lethal" in any single study may show an embryonic lethal RNAi phenotype when reanalyzed. It therefore seems useful to have more than one pass at analyzing C. elegans genes via RNAi. We are indebted to P. Gonczy for very useful comments. Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408 , 325-330. Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408 , 331-336. Piano, F., Schetter, A. J., Mangone, M., Stein, L. and Kemphues, K. J. (2000). RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr Biol 10 , 1619-1622. Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S. et al. (2000). A global profile of germline gene expression in C. elegans. Mol Cell 6 , 605-616.