[
Worm Breeder's Gazette,
1992]
We are interested in the control of cell cycle in C. elegans and wish to use genetics to describe the various genes and their parts in the regulation of cell division. We started with
cm4 g2 ,a
cdc2 cDNA homolog from C. Martin, B. Waterston, J. Sulston et al.. We determined the complete cDNA sequence of the gene from this clone and a homologous clone selected from B. Barstead cDNA library: the protein is 72% identical (89% homologous) to human
cdc2 .We mapped it on center II (Y24H1 ),then narrowed its position to about 20 kb on the physical map (thanks to Alan Coulson et al.). Connection to the genetic map was achieved by testing the presence of our gene in four deficiencies of the area, generated by B. Herman and provided by M. Edgley. We used PCR on single embryos, a marvellous technique developed by Barstead, Schrankc and Waterston, which in our hands works routinely on 80-cell embryos. Our
cdc2 gene appears to be present in these four deficiencies, so it lies in a region that includes three known genes: a maternal effect lethal
zyg-9 which affects the first embryonic mitosis, a zygotic lethal let 252 and a haplo-insufficient locus.
zyg-9 looked like a good candidate from its phenotype. In addition four putative polymorphic alleles have been recovered, three mutator-induced by Phil Carter and Bob Edgar, one psoralen induced by Julie Ahringer. Using PCR we showed however that none of the four is structurally rearranged in the transcribed
cdc2 region. We are now analyzing the alleles by genomic Southern, and trying to rescue the three known functions in the area by transformation with the cosmids. We are also interested in other
cdc2 -1ikegenes -another atypical PSTAVRE (like
cm4 g2 )containing gene has been studied in Paul Sternberg's- but is there a "true" PSTAIRE containing gene? What are the functions, inter-relations, specificities or redundancies of this set of genes ?
[
Worm Breeder's Gazette,
1988]
The behavioral phenotype of
unc-24 worms is characterized by severely kinked L1 worms that do not wriggle more than one quarter body length in either forward or backward directions, whereas worms from the L2 stage onward toward adults are only slightly kinked in motion and make progress similar to N2 worms. These observations appear to indicate that the
unc-24 gene may be affecting the embryonic motoneurones (DAs, DBs and DDs). Reconstruction of three different alleles of
unc-24 have not been consistent. The canonical allele (
e138) reconstruction shows DA motoneurones to be lacking commissures to the dorsal musculature. A Tc1 allele (
e2386) reconstruction contains one DA motoneurone with the same aberration but another with normal morphology. The P32 induced allele (
e927) reconstruction presents DA motoneurones with normal morphologies, but the commissures of all D motoneurones (DAs, DBs and DDs) proceed toward the dorsal cord on the opposite side to the wild type condition. To clear up this ambiguity, a reconstruction of an L1 from, the canonical strain is being completed. The
unc-24 gene has been isolated. A 5.6kb BamHI Tc1 containing fragment found in an allele (
e2386) isolated from the TR679 strain that also shifts to a 4.0kb BamHI fragment in revertants and Bristol strains was cloned into pUC 8. A 1.0kb non-Tc1 containing fragment was isolated from this plasmid by BamHI and EcoRV digestion and was used as a probe to screen the Coulson and Sulston cosmid library. Two true positives were identified that mapped to a contig containing the
vit-6 gene on linkage group IV. Fortunately, a larger cosmid in this contig which covered both newly identified cosmids had been used as an in situ hybridization probe by Rita Fishpool and Donna Albertson and showed binding to chromosome IV. The same 1.0kb non-Tc1 fragment was used to isolate 16 lambda clones from the 2a cDNA library of Julie Ahringer. The lambda inserts range in size from 0.6kb to 2.6kb. The largest of these was used as a probe and showed polymorphic band shifts on the genomic Southerns composed of different alleles of unc- 24. This insert is being sequenced.
[
Worm Breeder's Gazette,
1987]
Benomyl and other benzimidazole carbamates interfere with the polymerization of microtubules. In the presence of benomyl, wild-type C. elegans grows slowly, is severely uncoordinated, and has fewer neuronal processes in the ventral cord. Mutations conferring resistance to this drug map to one gene,
ben-1, on chromosome III. Virtually all of the EMS-induced and the TR679-derived alleles result in a temperature-dependent dominant drug resistance.
ben-1 animals have no visible phenotype when grown in the absence of benomyl. Since benomyl resistance is conferred by mutations in -tubulin genes in Aspergillus, Saccharomyces, and Physarum, we have performed low stringency hybridizations of -tubulin sequences (provided by Linda Gremke and Joe Culotti) to DNA isolated from
ben-1 mutants. DNA from two of the TR679 derived strains contains an insertion of approximately 2.1 kb in one of the -tubulin homologous restriction fragments. We have cloned a 7.6 EcoRI fragment from wild-type C. elegans that corresponds to this -tubulin. The -tubulin homology maps to the right end of the fragment, as depicted below. The left end of this fragment contains a repetitive DNA element (a non-Tc1 homologous sequence present in approximately 30 copies in C. elegans). The two transposon insertions are localized to different sites in the 2.1 kb BglII fragment. Although these are clearly not Tc1 insertions, we have not yet identified which, if any, of the characterized transposons are associated with the
ben-1 mutations. Preliminary transcription analysis suggests that the
ben-1 mRNA is abundant in wild-type animals. (Consistent with this observation, we have been successful in isolating a highly homologous clone from Julie Ahringer's cDNA library.) The transcript is approximately 3 kb and the direction of transcription is from left to right as shown. Molecular analysis has provided some insights into the mechanism of benomyl resistance. One of our
ben-1 alleles (derived from TR679) is actually a deletion of the 2.1 and 1.4 kb restriction fragments. Since this deletion allele is dominant, it appears that a critical level of
ben-1 gene product is required for sensitivity to benomyl. Genetic and molecular experiments directed towards confirming this hypothesis are underway.
[
Worm Breeder's Gazette,
1990]
Using the method reported previously (WBG 11, #1, p.67) with several modification, I have prepared amplified cDNA from single embryos at various stages (every 2 hr after 1st cleavage). The attempt for subtractive hybridization (ibid.) was not productive due to its trickiness, so I performed differential screening of a cDNA library. The set of amplified cDNA were labeled and probed Julie Ahlinger's embryonic cDNA library. I expected a big difference in the pattern of positive clones between far different stages, but there was not a big difference. This is probably because many (most?) of the cDNA clones have artificial rearrangement due to insufficient methylation of EcoRI sites during library construction: many of such rearranged clones carry parts of abundant cDNA. However, by comparing the signals in autoradiograms very carefully, several clones seemed to be differentially expressed. To examine these, aliquots of the amplified cDNA were dot-blotted onto strips of nylon membrane and then hybridized with [32P]-labeled cDNA insert. The results were quite encouraging. For example, clone 4-3 gave a very strong signal at 1. 5hr embryo (1.5hr after 1st cleavage), a weak signal at 3.5hr embryo and virtually no signal at 0hr and other stages. Clone 4-1 also gave a strong signal at 7.5hr embryo. A control clone 1-1 which did not show differential expression gave strong signals at similar level at all stages. These hybridization patterns were essentially reproducible in another experiment using amplified cDNA from another series of embryos. Clones 4-1 and 4-3 were mapped near
zyg-11(LGII) and
ceh-16 (LGIII) by probing the YAC filters. Now I am trying to identify the cell lineage in which these genes are expressed using the protocol for in situ hybridization by David Greenstein (personal communication and WBG 11 #3 p.78). Homologous clones to clones 4-1 and 1-1 appeared in the library at the ratio of 8 (0.016%) and 35 (0.07%) out of 50,000 pfu, respectively. Much less abundant clones could be analyzed in this assay. So, I picked up 16 low abundant cDNA clones at random and probed the test strips with them. Out of them one clone gave a signal only at 0hr embryo, one clone at 5.5hr embryo, three clones at early stages (0 and 1.5hr embryo) and other clones showed a similar result to that of clone 1-1 more or less. Being encouraged by these results, attempts to characterize all clones of a cDNA library which is normalized to some extent are in progress toward construction of a 'true' gene library of C. elegans. For this purpose, I wish to know more about the validity of the test strips, so, please send me cDNA clones which contain their 3' ends (N.B. By my method up to 1 Kb from 3'-end is amplified). I should probe the test strips with them and give the results back to the senders.
[
Worm Breeder's Gazette,
1988]
Analysis of
unc-13 cDNA clones The identification of
unc-13 gene has been described in the last WBG. Since then we have looked for the cDNA clone in 'Ahringer cDNA library 4: N2 mating population', using the polymorphic DNA fragment as a probe, and have found some positives all of which have about 3 kb- long inserts. This is consistent with the result of Northern analysis of the transcript using cosmid clone as a probe although we have not detected the transcript on Northern blot of total RNA using purified polymorphic DNA and cDNA as probes for unknown reason, but probably due to a low copy number of the transcript and/or a large size of the gene. We are now working on Northern analysis of the gene transcript using much more purified mRNA. In order to confirm that the cDNA clones derived from the
unc-13 gene, we hybridized cDNA to the genomic Southern blot as well as to the cosmid and lambda genomic clone DNA encompassing
unc-13 region. All of these clones hybridized to the polymorphic fragment on the unc- 13 mutant genome and have been localized on the restriction map. At least three different transcripts (more and more coming up) have been identified so far, as shown below in Figure 1. These clones do precisely overlap each other in the region of polymorphic band (shown by a vertical arrow in Figure 1) of
unc-13 mutant and encoded in the segment of about 35 kb in length (What a huge gene for only 3 kb mRNAs!). Sequencing one of the cDNAs The whole insert of one of the cDNA clones, C2, was sequenced. The cDNA clone has 2541 bp-long insert which encodes a full 5' region including noncoding stretch of 51 bp in length, but terminates at EcoRI site inside of coding region because there is neither stop codon nor AT-rich 3' noncoding segment. By searching NBRF library we found that amino-terminal half of 'C2 product' is homologous (50%) to protein kinase C (PKC), but not in the carboxy-terminal half of PKC which has a kinase activity. This might mean that 'C2 product' is similar to the regulatory domain of PKC but does not have a kinase activity. It is interesting that the amino-terminal half homologous to PKC contains the typical cysteine-repeating region. The function of the segment in PKC is not known, but the positions of cysteine residues is almost perfectly conserved in DNA-binding domain of steroid hormone receptors and so on (Fig. 2), suggesting that C2 product may be under a similar regulation to PKC in the signal transduction process, and may presumably regulate gene expression of some other genes through DNA-binding. This possibility remains to be elucidated. I am now on the way to finish sequencing a bit of C-terminus of 'C2 transcript' by sequencing of genomic DNA as well as analyzing some other transcripts. I would like to thank Julie Ahringer in Judith Kimble's lab for providing the cDNA library.
[
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.