[
Worm Breeder's Gazette,
1994]
Expression studies of the putative Caenorhabditis elegans cyclin A and B genes Monique A. Kreutzer, James P. Richards and Karen L. Bennett, University of Missouri-Columbia, Columbia, Missouri 65212. We have cloned putative A, B1 and B2 cyclins from C. elegans. Cyclin B1 and B2 are single copy genes while cyclin A belongs to a multigene family. Cyclin A and B2 each correspond to single transcripts while cyclin B1 recognizes three transcripts. The C. elegans temperature sensitive germline defective mutants
fem-1,
fem-3 gf) and
glp4 were used to analyze cyclin germline expression. When hybridizing C. elegans cyclin A or B2 to the mutant RNAs that produce no oocytes, the cyclin A and B2 messages are significantly reduced. When normal numbers of oocytes are produced, the cyclin A and B2 messages are at wild type levels. Therefore, the cyclin A and B2 transcripts appear to be primarily maternal with some somatic component. Consistent with a somatic component, cyclin A and B2 cDNAs cross-hybridize to Ascans intestinal poly A+ RNA. Hybridization of the cyclin B1 cDNA to these germline defective RNAs suggest that the cyclin B1 transcripts are differentially expressed in the germline. The two larger cyclin B1 transcripts appear to be primarily maternal and the smallest transcript appears to be sperm- specific. The observation that two polyadenylation signals are present in the cyclin B1 3'UTR, as well as results using cyclin B1 probes from specific regions of the B1 3'UTR, suggests that two of the three cyclin B1 transcripts are produced by polyadenylation a~ different sites. There is precedence in mice and flies for producing tissue-specific transcripts by different use of the cyclin B 3'UTRs. It has been shown in mouse that cyclin B1 hybridizes to four differentially expressed transcripts, one of which is testis- specific. Two of these mouse transcripts differ in the choice of polyadenylation sites and therefore in the lengths of their 3'UTRs (Chapman and Wolgemuth (1992) MoL Repro.Dev. 33, 259-269). In Drosophila, the cyclin B cDNA detects two female-specific transcripts that are made by splicing of a region of the cyclin B 3'UTR (Dalby and Glover (1992) Development 115, 989-997). In summary, our initial findings show that the C. elegans cyclin As and Bs are highly expressed in the germline and also suggest that the three cyclin B1 transcripts are differentially expressed in oocytes and sperm.
[
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,
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.
[
Worm Breeder's Gazette,
2002]
Heat-shock has traditionally been used as a means to obtain males, but particular worm strains will not tolerate this process. This problem may be overcome through genetic means by introducing a him mutation but this involves additional steps and may complicate later experiments. In an effort to obtain males from strains that are not amenable to heat-shock, we turned to RNA mediated interference as an approach. We made and tested a
him-14 feeding construct (many thanks to Yuji Kohara for the
him-14 cDNA and Lisa Timmons and Andy Fire for the L4440 vector) to see if males could be generated. Hermaphrodites that consume HT115(DE3) bacteria producing
him-14 dsRNA consistently produce a low but significant number of males. Interestingly, the males appear among the progeny at the end of the brood. We typically see 5-7% males in the last 100 progeny from three pooled hermaphrodites at 20degC. Our best results are obtained by feeding L4 hermaphrodites for about 50-55 hours and then transferring these worms to a fresh RNAi plate and inspecting the progeny from the end of the brood for males. We did not observe an increase in the percentage of males if the RNAi feeding was carried over additional generations. To test if the effect persists once worms are removed from the dsRNA-producing bacteria, hermaphrodite siblings of RNAi-induced males were transferred to OP50 bacterial lawns. These animals threw fewer than 1% males (from a pooled total of 12 bulk-inspected hermaphrodite broods) in the first generation and none in the subsequent generation. We have also confirmed that males produced by
him-14 RNAi can sire cross progeny. Finally, we did not see evidence of embryonic or larval lethality. We stress that feeding
him-14 dsRNA to produce males is effective and reversible, but not efficient. We have not successfully obtained sufficient numbers of males from strains that are somewhat unhealthy or that have a reduced brood size. Since males appear at the end of a normal brood, low brood size may hinder the production of males by this approach. Furthermore, we found that the bacteria used to produce males must be freshly grown from a healthy plate colony and used immediately. Ultimately, other genes may be more useful for this purpose. Indeed, large scale RNAi screens have identified many genes that give Him phenotypes when targeted by RNAi (e.g., Fraser et al ., 2000). Theresa Stiernagle kindly agreed to distribute GC363, the HT115 (DE3) bacterial strain carrying pGC8 [
him-14 partial cDNA in the Fire L4440 vector]. Many thanks to David Greenstein for discussions that led to this experiment. These results were also described in a March, 2001 note to the C. elegans newsgroup.