Yochem JJ et al. (1987) Worm Breeder's Gazette "sequence of the glutathione s-transferase P subunit of C elegans, or a step towards the complete sequence of the C. elegans genome."

Weston K, Yochem JJ, Greenwald IS

[Worm Breeder's Gazette, 1987]

In the process of completing the sequence of lin-12, we inadvertently obtained the sequence of the adjacent gene on the 3' side of lin-12. It is oriented in the opposite direction relative to lin-12. (We have data indicating that lin-12 runs 5' to 3' with respect to the genetic map.) The sequence presented below is a composite of genomic and cDNA sequence data; the cDNAs were obtained from a library kindly provided by Julie Ahringer and Judith Kimble. The predicted product is 40% identical to rat liver glutathione S- transferase P subunit (Suguoka et al., NAR 13: 6049, 1985); the degree of homology is higher if conservative substitutions are taken into account. Identical amino acids are boxed. The data indicate that there is a splice joining an exon (277-end) to a 5' exon(s). [See Figure 1]

Livingstone D (1989) Worm Breeder's Gazette "some more unc-31 sequence."

Livingstone D

[Worm Breeder's Gazette, 1989]

I am continuing the molecular work on the unc-31 gene started by Roger Hoskins. Roger isolated a 17.5kb genomic clone in lambda2001 which he showed was capable of transformation rescue of unc-31 mutants. He also partially sequenced a 4.5kb fragment contained within this clone which covers some of the polymorphisms associated with mutant alleles of the gene. [See Figure 1] I have made subclones of this phage in lambda2001 and have used them in an attempt to narrow down the position of the gene by transformation rescue. A phage containing the 13.5kb XhoI fragment seems to rescue although this result has still to be confirmed. No rescue has been obtained with phage containing the 13.5kb SacI fragment or the 10.5kb SacI-XhoI fragment. I am currently sequencing the 13.5kb rescuing fragment. Several open reading frames have been found which could be spliced together inframe. Some of these have been confirmed by sequencing partial cDNA clones isolated from Julie Ahringers mixed stage cDNA library. The gene covers more than 8kb of genomic DNA. The 5' end is near or beyond the SacI site and the 3' end is beyond the end of the 4.5kb HindIII fragment. No homology has been found between predicted amino acid sequences and protein sequences present in the PIR and Swissprot databases.

Galas S et al. (1992) Worm Breeder's Gazette "A Quest for cdc-2-like Function in C. elegans"

Peuch CL, Doree M, Ferraz C, Thierry-Mieg D, Devault A, Galas S

[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 ?

Gengyo-Ando K et al. (1988) Worm Breeder's Gazette "head piece of paramyosin and progress of tropomyosin gene cloning."

Kagawa H, Sugimoto K, Gengyo-Ando K

[Worm Breeder's Gazette, 1988]

Complete nucleotide sequence data of paramyosin gene, unc-15 will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X08068. By the friendly information from St Louis, Larry Shriefer and Bob Waterston, a head piece of paramyosin was extended a little bit. Finally, the gene is composed of 10 exons encoding a protein of 866 amino acid with molecular mass of 99,890. The gene had no TATA-box and promoter like sequences at 5' upstream as other muscle genes of the worm. Manuscript entitled 'The paramyosin gene (unc-15) of Caenorhabditis r cloning, nucleotide sequence and models for thick filament assembly' collaboration with Andrew D. McLachlan, Sydney Brenner and Jonathan Karn was submitted to J. Mol. Biol. cDNA clone of tropomyosin gene was cloned from a cDNA library (48hr sample) which was constructed by Julie Ahringer and Judith Kimble by using a tropomyosin gene fragment of C. elegans as a probe. The fragment of probe was cloned by screening an exon expression plasmid library (1985 C. elegans meeting abstract) with anti-tropomyosin antibody. Cloned 1.6kb fragment encoded the 6th to 241th amino acid residue of tropomyosin. The amino acid sequence was very conserved. To clone a complete fragment of the gene of genomic lambdoid phage library (kindly supplied by Claudia Cummins and Phil Anderson) was screened by using 1.6kb of cDNA clone as a probe. Ten positive clones were processed and mapped on the cosmid library by Alan Coulson and John Sulston. Unfortunately, all clones was contained the identical sequence to other part of unrelated cDNA (Worm gazette, Vol. 10, No. 1) which located the upstream of the cDNA clone of tropomyosin. The junction sequence of the both fragment was as follows. We are not sure why tropomyosin gene was not contained in the genomic library. [See Figure 1] Tropomyosin gene will be cloned from a genomic library which will construct with the genomic restriction fragment of the correct size. Original gene of cloned fragment of other part leaves in question.

Nawrocki LW et al. (1988) Worm Breeder's Gazette "characterisationa and cloning of unc-24."

Thomson JN, Nawrocki LW, Southgate E

[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.

Driscoll MA et al. (1987) Worm Breeder's Gazette "cloning of the ben-1 gene."

Reilly E, Bergholz E, Chalfie M, Driscoll MA

[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.

Donkin SG et al. (1993) Worm Breeder's Gazette "C. elegans as a Soil Toxicity Test Organism"

Donkin SG, Dusenbery DB

[Worm Breeder's Gazette, 1993]

We have developed a new procedure for recovering nematodes from soils in an efficient and non-destructive manner, which has permitted the development of a short-term soil toxicity test using nematodes as toxicity indicators. The procedure involves gentle centrifugation through a colloidal silica suspension (Ludox AM or Ludox HS-40, both available from E. I. Du Pont de Nemours & Co., Wilmington, DE), which allows the soil to settle out while floating the nematodes on top of the suspension. The nematodes are recovered with great efficiency (~90%) and are unharmed by the recovery process. Short-term lethality tests (24-hour exposure) were performed with several metals (Cd, Cu, Hg, Ni, Pb, and Zn) in several different characterized soils, and replicable results were obtained, allowing concentration-response curves to be generated and LC(50)s to be estimated. It was found that the presence of soil generally decreased the toxicity of the metals to nematodes when compared to tests done without soil present. This is presumably due to the sorption of the metal ions to the soil particles, which lowered their availability to the nematodes. Comparison between nematode lethality results and data published for earthworms exposed to metals in soil revealed that, in most cases, the nematode is at least as sensitive as the earthworm in terms of its response to toxic metals in soil. The earthworm test, which is currently the standard animal soil toxicity test, has the disadvantage of taking two weeks to perform, while the nematode test can be done in 24 hours. Some correlations between soil or metal properties and the resulting lethal effects were obtained, while other properties showed no significant correlation with toxic effects. This suggests that, as other studies have indicated, the bioavailability of metals in soils cannot be reliably predicted by considering only a few sample conditions. Soils are complex and heterogeneous systems, and there are many physico-chemical processes that may occur within them and which may influence the resulting bioavailability of a contaminant. The C. elegans soil toxicity test may provide the means to identify some of the more important environmental characteristics that influence contaminant bioavailability.

Kohara Y (1990) Worm Breeder's Gazette "Differential Screening of a cDNA library using cDNA probes amplified from individual embryos at various stages"

Kohara Y

[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.

Lee RYN et al. (1995) Worm Breeder's Gazette "eat-4 Is a Member of An Ancient Membrane-Associated Transporter Protein Family: NERD."

Lee RYN, Avery L

[Worm Breeder's Gazette, 1995]

The M3's are inhibitory motor neurons necessary for thepharynx to repolarize quickly after excitation so that bacteria can betrapped efficiently (WBG 13(4): 72). M3 probably does this by releasingglutamate onto pharyngeal muscle to open glutamate-gated Cl-channels(ibid). We are interested in understanding glutamatergic neurotransmissionby taking advantage of mutants that affect M3 function.We know that eat-4 affects the function of M3 and possibly otherglutamatergic neurons. We also know that the defect of eat-4 is upstreamfrom pharyngeal muscle and therefore probably in the M3 neuron itself(ibid). Since eat-4 does not appear to affect behavior known to bemediated by neurotransmitters other than glutamate (e.g., GABA, ACh), wesuspect that eat-4 may be necessary for either the production orconcentration of glutamate in glutamatergic neurons. We previouslylocalized eat-4 to the cosmid ZK512 (WBG 12(5): 65). We have since shownthat a 7 kb fragment rescues, and we have rescued eat-4 with a genomic-cDNA fusion. We fused 2.4 kb of genomic sequence from ZK512 in frame to acDNA yk32h2 that was cloned and partially sequenced by Yoji Kohara. yk32h2is identical to zk512.6 (found in ACEDB) except in two exons. yk32h2 isnearly full length, but may lack a few hundred bases at the 5' end.A sequence database search of EAT-4 produced severalinteresting hits, including three putative genes found on LGIII (part ofthis has been reported by the sequence consortium, in ACEDB). The tablebelow shows percent sequence identity in pairwise comparisons. Members ofthis putative gene superfamily are found in organisms from mammals tobacteria suggesting an ancient ancestry. EAT-4 is most similar (45%identical) to a neuronal specific sodium-dependent inorganic phosphatecotransporter (BNPI) found in rat brain. BNPI has been shown to have iontransporter activity but its biological function is not clear (Ni et al,PNAS 91: 5607).All members of this superfamily appear to be membrane-associatedproteins based on Kyte-Doolittle hydrophobicity analysis. All of theeukaryotic members have very similar hydrophobicity profiles, each havingthe potential to span the lipid bilayer six times. This similarity at thelevel of secondary structure further suggests the possibility that theyare related. The fact that three of the members are sodium-phosphatetransporters (BNPI, and rabbit and human Na-Pi-I) and that all of themseem to be membrane proteins suggests that this family may represent a newclass of transporter proteins. We propose to call them the NERD (neural,eat-4 and renal-derived) transporters.eat-4 is probably necessary for all glutamatergic neurotransmissionin worms (WBG 13(4): 72). How does a NERD do that? Our first model is thatEAT-4 may be a phosphate transporter (like BNPI), necessary formaintaining a high intracellular phosphate concentration in order tostimulate phosphate-activated glutaminase, an enzyme that synthesizesglutamate. (This model was suggested by Robert Edwards.) Alternatively,perhaps EAT-4 is a vesicular glutamate transporter that concentratesglutamate in synaptic vesicles.

Maruyama IN (1988) Worm Breeder's Gazette "a novel protein encoded in unc-13."

Maruyama IN

[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.