[
Worm Breeder's Gazette,
1995]
Correlation of the Genetic and Phvsical maps within the
rol-3 region of LGV (left) W. Bradley Barbazuk and David L. Baillie Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC. CANADA, VSA 1 S6 In an attempt to identify the physical location of the
rol-3 (V) gene we have begun the systematic germ-line transformation rescue of lethal mutations within zones 16-18 of LGV (JOHNSEN and BAILLIE, GENETICS 129: 735-?52). Using H. Kagawa's placement of
unc-68 (WBG 12(4)) as a starting point we began to inject cosmids to the right of C46C6 into N2 hermaphrodites. In doing so we were able to obtain a battery of strains each carrying cosmids singly, or multiply, in stable arrays which were then used as mini-duplications in subsequent crosses to lethal bearing strains. In total we used eight cosmid bearing strains and one YAC bearing strain in the rescue of 13 lethal mutations including
rol-3 (Table 1). Our germ-line transformation rescue data has enabled us to split up and order some of the lethal mutations within the zone 16-18 gene clusters. (Figure 1). We would like to thank C. Mello and J. Priess for providing a strain carrying an array composed of ZK307, F25G6 and FOlH8, as well as providing purified DNA for many of the cosmids injected.
[
Worm Breeder's Gazette,
1990]
To identify the control regions of the C. elegans gut-specific esterase gene (
ges-1), we are using a variety of approaches including a comparison of the upstream control regions of
ges-1 and the homologous esterase gene from C. briggsae (cloned and sequenced from the Baillie library by Brian Kennedy and Fran Allen). For this approach to be valid, we needed to show that the esterase gene from one species is properly expressed in the other species. It was easy to show that the C. briggsae esterase is expressed in the gut of C. elegans by transforming the C. briggsae gene into a
ges-1(0) strain of C. elegans and then staining the transformants for esterase. We did this experiment using both transient transformation and extrachromosomal heritable transformation. To show that the C. elegans properly expressed in C. briggsae, we needed to make a strain of C. briggsae that was transformed with the C. elegans tried the
rol-6 dominant mutant gene and followed the transformation procedure of Mello et al. (WBG 11-1:18 and WBG 11-3:12). Roller strains of C. briggsae were easily obtained (the rachis of C. briggsae is even easier to hit than that of C. elegans). When the
ges-1 gene was used in a cotransformation with the
rol-6 gene all of the esterase expression was confined to the guts of the transformants i.e. the transformants looked exactly like wildtype C. briggsae. We then used isoelectric focusing gels on extracts of the transformed C. briggsae followed by staining of the gel for esterase activity to show that the C. elegans indeed being expressed. Thus, the C. elegans expressed in C. briggsae and it is expressed only in the gut. A deleted construct of
ges-1 that contains only 521 base pairs 5' from the translation initiation codon is expressed in the pharynx muscles of C. elegans (E. Aamodt, M. Chung and J. McGhee, manuscript submitted). When this deleted
ges-1 gene was transformed into C. briggsae, the transformants also express the esterase gene in their pharynx. In other words, not only is the intact gene properly expressed but the ectopic expression of the deleted
ges-1 is identical between the two species. Thus, both
ges-1 and the mutant
rol-6 genes are properly expressed in C. briggsae. Cotransformation of C. briggsae using the
rol-6 marker gene should be useful to others who wish to see if their favorite C. elegans gene works in C. briggsae.
[
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.