[
Worm Breeder's Gazette,
1979]
We have now completed a study of some 90 roller mutants. The accompanying table indicates the genes which can mutate to give a roller phenotype. Several new genes have been identified and several mapping errors were found.
e187, a widely used roller allele of
dpy-2, is the canonical allele of another closely linked gene
rol-6. The E8 E26 strain usually used for complementation testing at the
dpy-2 locus is in fact a triple mutant containing a non-visible allele of the rol- 6 gene, which is where the confusion among these mutants arose. unc- 76 is closer to the cluster on chromosome V than previously indicated. It is within 0.3 map units of
rol-4, which is located 2.9 + .8 map units to the right of
unc-42. The new position of
unc-76 was checked by mapping against
dpy-11.We have found that the genes fall into five different classes with regard to phenotypic characteristics: left roller (LR), right roller (RR), left squat (LS), right squat (RS), and left dumpy roller (LDR). Mutants of genes in these groups show remarkably distinctive phenotypic differences in terms of roller handedness, time of onset of phenotype, presence or absence of a dumpy phenotype, and effects on cuticle morphology. All roller alleles of these genes possess a helically twisted cuticle. Some genes contain nonroller alleles and the cuticles of these mutants are found to be straight. Other types of cuticle aberrations are listed in the table. The most bizarre phenotypes are manifest by the squats which are rollers as heterozygotes but straight non-rollers as homozygotes. The right squat homozygotes roll only during the L3 and dauer stages and not as L4's or adults except when the L4 or adult arises from the dauer. This latter finding indicates that adults derived from L3's and dauers are not equivalent, at least in some respects. Dominant or semi-dominant mutations have been detected in five genes:
sqt-1,
sqt-3, positions of these genes are indicated below. [See Figure 1] [See Figure 2] [See Figure 3]
[
Worm Breeder's Gazette,
1997]
Neuropeptides form the largest class of neuroactive substances, yet as a group their roles in nervous system function and development are still poorly understood. Only the FMRFamides and related neuropeptides (FaRPs) have been intensely studied in C. elegans. However, in Aplysia californica, over 40 neuropeptides have been identified and characterized. This suggests that many C. elegans neuropeptides have not yet been discovered. With the near completion of the C. elegans genome sequencing project, reverse genetic techniques can be applied to identify C. elegans neuropeptide genes. We are using FASTA and BLAST to search with previously identified neuropeptide sequences (stored in GENBANK) against the C. elegans genome sequence. We expect the products of neuropeptide genes to fit a prepropeptide model: they should contain a putative signal peptide region, followed by interrupted multiple repeats of the same or similar neuropeptide. Additionally, the predicted neuropeptides should be flanked by pairs of basic amino acids (KR, RR, KK, RK) or less frequently by monobasic residues (R or K) which are preferred sites of endoproteolytic cleavage. Potential C. elegans neuropeptide gene compatible with the prepropeptide model are then selected for molecular and genetic analysis. We are currently analyzing C. elegans genes encoding neuropeptides similar to Aplysia buccalin (C. elegans C01C4.1) and myomodulin (C. elegans T24D8.3, T24D8.4, T24D8.5 and T01B6.4). (See below.) Even though the predicted C. elegans neuropeptide sequences are not strikingly similar to the Aplysia neuropeptides, there is strong internal homology between neuropeptides within the same C. elegans genes. We are creating reporter constructs by fusing the promoter region of potential neuropeptide genes to the GFP gene using Fire laboratory vectors. We are injecting these reporter constructs to assess cellular expression patterns of these myomodulin-like and buccalin-like genes. We think that it is likely that the GENEFINDER predicted genes of T24D8.3, .4 and .5 are exons of a single gene. We will use RT-PCR to determine mRNA splicing patterns. Our long range goals include creating mutations in these genes and determining the function of these and other putative C. elegans neuropeptides. Identity is indicated with a colon, gaps with underlining. SLSMLRLG____ Aplysia Myomodulin A :MA:G:::LRPG T24D8.5 :MAYG:Q:FRPG # :IALG:S:FRPG :MAIG:A:MRPG T24D8.4 :IAIG:A:FRPG T24D8.3 N:LVG:Y:FRIG T01B6.4 DPNVDPYSYLPSVG Aplysia Buccalin _VNL::N:FRM:F: C01C4.1 ___M:ANAFRM:F: # ___M::NAFRM:F:
[
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.