Eakin N et al. (1998) Worm Breeder's Gazette "A sordid collection of sorted worms"

Eakin N, Fitch DHA

[Worm Breeder's Gazette, 1998]

We have started a simple, web-based database for strains of nematodes maintained in a number of different laboratories as well as at the CGC. It can be reached via Leon Avery's C. elegans WWW Server (via the link to Other nematodes) or directly at:

Sulston JE et al. (1982) Worm Breeder's Gazette "the cell lineage of C elegans."

Schierenberg E, White JG, Thomson JN, Sulston JE

[Worm Breeder's Gazette, 1982]

The embryonic cell lineage is complete and all cells have been identified. In the accompanying chart, copies of the published post- embryonic lineages have been added in order to illustrate the fates of the blast cells. Embryonic time scale (20 C) runs from first cleavage to hatching. Note change of time scale at hatching. The postembryonic lineages are those of the hermaphrodite except for four cells which divide only in the malen and link precursors which give rise to bilaterally symmetrical groups of cells. In order to make a fully connected chart, trim one time scale from each panel and paste together. [See Figure 1] {Linkage maps excluded due to poor reproducibility. } [See Figures 2-13]

Waring DA (1993) Worm Breeder's Gazette "ncl-1 to the Rescue"

Waring DA

[Worm Breeder's Gazette, 1993]

Mutations in the gene ncl-1 cause many cells in the worm to have unusually large nucleoli. This phenotype and the fact that ncl-1 acts cell autonomously has allowed the ncl-1 gene to be used as a marker for mosaic analysis by various groups. The traditional method for obtaining genetic mosaics, has been to make a strain with mutant alleles of the gene(s) of interest on the chromosomes, and wild type alleles on a free duplication (which is lost during mitosis at a relatively low frequency). Using appropriate marker mutations which are also covered by the same duplication one can identify animals which have lost the free duplication in a subset of cells. When ncl-1 is used as a marker, it is possible to look at an individual cell, under Nomarski optics, and determine its genotype (whether or not it contains the duplication) by examination of the nucleolus. Thus using ncl-1 as a marker can allow single-cell resolution in mosaic analysis. This technique requires that the gene of interest be linked genetically to the ncl-1 gene, and any other markers. This is a severe limitation, as most genes are not on chromosome three. At least two groups have gotten around this problem by artificially linking the genes that they wanted to study to ncl-1 .Hunter and Wood (Nature 1992) did this by linking two duplications together using x-rays thus creating one large free duplication. This of course makes all genes which are covered by this duplication pseudo-linked. Leung-Hagensteijn et al. (Cell 71;289) generated a strain in which the cloned unc-5 gene had been integrated onto the duplication sDp3 which carries ncl-1 . In order that ncl-1 might be even more readily used as a marker for mosaic analysis of other genes I have begun an attempt to clone the.ncl-1 gene. Mapping data from Chisolm, and from Kenyon, placed ncl-1 between the genes lin-39 and mab-5 By first injecting pools and then individual cosmids I have rescued ncl-1 (e1942 )with the cosmid C33C3 (from England). It should now be possible to artificially link ncl-1 to any cloned gene. Extrachromosomal arrays of injected DNA apparently behave like free duplications. By generating an extrachromosomal array containing the gene of interest, ncl-1 and any other cloned marker genes or gene fusions, one should be able to use this array in the same manner that free duplications have been used. It remains to be seen if such an array is lost at an appropriate rate, and if the mosaism that has been observed with extrachromosomal arrays is due solely to mitotic loss or rather to mosaic expression. It should also be possible to integrate the cloned ncl-1 gene onto a free duplication which covers a gene of interest, and in this way link the two genes, in much the same way that Leung-Hagensteijn et al. linked ncl-1 and unc-5 .In this case it would not be necessary to clone the gene of interest if it is covered by a free duplication.

Link CD et al. (1992) Worm Breeder's Gazette "Looking for Glycosylation Mutants"

Enyeart M, Link CD

[Worm Breeder's Gazette, 1992]

Although we have generated a large collection of mutants (designated srf) that have novel surface lectin-binding properties, it is not clear if any of these mutants have intrinsic changes in glycosylation. Surface radio-labeling and lectin blot experiments suggest that these mutants bind lectins not because they have novel glycoproteins, but because normally cuticle-internal glycoproteins are being "unmasked". We have devised a screen to more directly identify glycosylation mutants by looking for extragenic suppressors of srf mutants. Suppressor mutations could theoretically block srf mutant lectin binding by removing, modifying, or masking the relevant cuticle glycoprotein(s). Among these suppressors should be mutants that fail to bind lectins because of changes in the carbohydrate component of the relevant cuticle glycoprotein. srf-5 (ct115)animals show highly penetrant ectopic surface binding of the lectins SBA and WGA, but are otherwise wild-type in morphology and behavior. We screened the progeny of EMS-mutagenized srf-5 animals for variants that did not bind the lectin SBA using a modification of our original biotinylated lectin/ avidinHRP protocol, and identified 9 independent suppressor mutations. These mutations define at least 4 complementation groups. Although we have not yet biochemically characterized these mutants, it is interesting to note that 3 of the 9 suppressors still show ectopic binding of WGA. This phenotype would be expected for mutations that block the addition of N-acetyl galactosamine (recognized by SBA) but not N-acetyl glucosamine (recognized by WGA) to glycoproteins. Because we are strongly biased in believing that C. elegans does not glycosylate proteins so the Link lab can find lectin-binding mutants, our hope is that if we do identify glycosylation mutants, they will have other phenotypes that will give us insights into the biological role of glycosylation. We are therefore encouraged that 4 of our original isolates also show an Unc phenotype.

Yasuda H et al. (1987) Worm Breeder's Gazette "screening C elegans expression libraries with nerve specific anit-tubulin antibodies."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1987]

C. elegans, like many other multicellular organisms, has multiple tubulin genes. Linda Gremke has shown that there are at least 4 and 5 alpha tubulin genes in the nematode, based on homology to the chicken tubulin gene probes(1). Similarly, it has been shown that purified tubulin from the nematode can be resolved in to at least 10 isotypes on isoelectric focusing, including 2 major alpha, 2 major , and about 6 minor alpha and isotypes(2). We have screened C. elegans cDNA and genomic expression libraries (kindly provided by Bob Barstead and Bob Waterston), with a set of anti-tubulin monoclonal antibodies, which preferentially stain neurons cytochemically(2). In the present screen 44 clones have been identified, which test positive on rescreening. The insert size of these clones range from 0.6 to 1.7 kb. Among these 29 clones are specific to anti- tubulin, and 14 are specific to anti-alpha tubulin antibodies. Our attention has been drawn to the problem of false positives in screening expression libraries with antibodies(e.g. C. Link and W. Wood, WBG Nov. 1986, and Eric Aamodt, personal communication). These workers have found E. coli DNA in the 'positive' clones, as a major artefact. Affinity purification of antibodies with the fusion protein, may not preclude the inclusion of false positives (Eric Aamodt, personal communication) either. Therefore, we plan to subclone the inserts in an appropriate vector, and determine the nucleotide sequence of the inserts to distinguish the true from the false positives. This is possible for the tubulin clones, as the nucleotide sequence of tubulin variants is available, including that of C. elegans tubulin(1). We plan to use this strategy for other genes for which we have collected 'positives' using expression libraries, including N-CAM like antigen, and N- CADHERIN like antigen of C. elegans, as these genes have conserved sequences, and their nucleotide sequence data are available.

Stone S et al. (1991) Worm Breeder's Gazette "CLONING DAF-10 AND OSM-1."

Shaw JE, Stone S

[Worm Breeder's Gazette, 1991]

We are interested in studying the molecular genetics of sensory neuron development. Two genes previously implicated in sensilla development, particularly in the development of sensory cilia, are daf- 10(IV) and osm-1(X) (Perkins et. al. 1986 Dev. Biol. 117, 456487). Mutant animals have a variety of ciliary defects. However, the major defect appears to be abnormally short axonemal projections and (at least for osm-1) an assembly of ectopic doublet microtubules which constitute cilia-like posterior projections. These animals have various behavioral abnormalities which presumably result from a failure of the sensory cilia to project into the environment (i.e. daf, osm, che etc.). To understand how these genes function, we have begun a molecular characterization of the two loci. We obtained from Don Riddle's lab alleles osm-1(m530), 8), and daf-10(m534), which were generated in RW7097. Spontaneous revertants were identified and, they and the mutants from which they were derived were outcrossed. DNA samples from outcrossed mutants, revertants, and recombinants were isolated for Southern analysis. For each gene, a single Tc1- containing EcoRI fragment which co-segregated with the mutant phenotype was identified. These EcoRI fragments were cloned and their identities verified. DNA fragments flanking the Tc1 elements were used to screen a genomic library provided by Chris Link. From these experiments we identified three overlapping lambda clones for osm-1 and a single lambda clone which hybridized to the daf-10 probe. The osm-1 and daf-10 lambda clones were sent to John Sulston and Alan Coulson at the MRC for assignment on the physical map. Both genes landed in large contigs. The daf-10 clone was placed within approximately 20kb and to the right of fem-3, and the osm-1 clone was placed within about 100kb and to the right of mtl-1. In addition fragments of the genomic clones were used to identify hybridizing cDNA clones from a library provided by Stuart Kim. The osm-1 and daf-10 probes each identified cDNAs. We have begun sequencing these clones and using them as probes for northern analyses.

Krause MW et al. (1986) Worm Breeder's Gazette "are leader sequences spliced onto actin mRNAs ?"

Hirsh DI, Krause MW

[Worm Breeder's Gazette, 1986]

We have made gene-specific oligonucleotides to the 5' untranslated regions of actin mRNAs in order to map the transcriptional start sites of the four genes by primer extension. Primer extension dideoxy sequencing of actin gene 4 (X) mRNA confirmed earlier Sl-mapping results of a -41 to -43 transcriptional start site relative to the initiator AUG. The primer extension sequence was identical to the genomic sequence found upstream of actin gene 4. Primer extensions using actin gene 1 and/or 3 (V) and gene 2 (V) oligonucleotides yielded a unique 20 nucleotide sequence. This 20 nucleotide sequence is not the same as any of the genomic sequences adjacent to the primers used suggesting a splicing event during message maturation. This was not unexpected as each of these genespecific oligonucleotides abuts a consensus (genes 1 and 3) or near consensus (gene 2) TnCAG splice acceptor site. Actin genes 1 and 2 are oriented in opposite directions with their 5' ends toward each other. m e 20 nucleotide sequence which was derived from primer extensions is not in the 6 kb region containing actin genes 1 and 2. In order to find the source of this unique 5' sequence on the actin mRNAs, an oligonucleotide homologous to the 20 nucleotide sequence was synthesized and used to probe Southern blots of genomic DNA digested with several enzymes. m e results suggest that the 20 nucleotide sequence is part of a 900 bp tandem repeat with a repeat copy number greater than 10. We have screened a C. MBL4 library (provided by Chris Link) confirming the repetitive nature of the 20 nucleotide sequence and identifying phage of interest. One possible interpretation of these results is that the 5' ends of actin 1,2 and 3 mRNAs arise by splicing leaders that are transcribed from a tandem repeat elsewhere in the genome. Precedents for splicing mRNA leader sequences derived from a separate RNA molecule exist in trypanosomes, mouse hepatitis virus and flu virus. Alternatively, the primer entension sequence from actin 1,2 and 3 mRNAs is a giant artifact. We are currently trying to decide between these possibilities.

Ohshima S et al. (1989) Worm Breeder's Gazette "a presumed gene for G protein alpha subunit of C elegans."

Ohshima YM, Park J-H, Tashiro S, Tani T, Ohshima S

[Worm Breeder's Gazette, 1989]

As an approach to analyze neuronal signal transduction in C. elegans, we attempted to characterize a gene for guanine nucleotide- binding regulatory protein (G protein). Rat cDNA probes for Gsalpha and Goalpha, described by Itoh et al., Proc. Natl. Acad. Sci. USA. 83, 3776-3780 (1986), revealed a few bands on a Southern blot of C. elegans N2 total DNA digested with HindIII, EcoRI or XbaI. A single clone was isolated from N2 genomic library in lambdaEMBL-4 from Dr. C. Link, and another from a library in lambda2001 from Dr. A. Coulson, by screening with the 1.1kb EcoRI fragment corresponding to the C- terminal 2/3 of rat Gsalpha (pGI11L). The two clones are similar and probably overlapping. The former clone (lambdaEGLI-9) produces hybridizing fragments on a Southern blot comparable to those from total DNA, and it was further characterized. A 0.8kb XbaI fragment, which hybridizes strongly with the 11L probe, was sequenced. It carries a sequence potentially coding for 48 amino acid sequence which is highly homologous to that found at the C-terminus of the rat Gsalpha (see below). Fourty-four amino acids out of 48 are identical between the two sequences. The sequence 5' to this region and within the XbaI fragment does not show significant homology with the probe, and seems to be an intron. Another fragment, which hybridizes to the rat Gsalpha cDNA fragment corresponding to the N-terminal 1/3 (pGI11S), resides 5' to the sequenced XbaI fragment. Therefore, this clone may carry the entire gene coding for a Gsalpha subunit. We are further analyzing this clone, and also trying to clone a corresponding cDNA from the library of Dr. B. Barstead. Screening the lambda2001 genomic library with the N-terminal cDNA probe produced seven positive clones. A few of these were partially sequenced, but none revealed a putative coding region for Gsalpha. Synthetic oligonucleotide probes for the conserved GTPase or guanine-binding regions revealed several bands on a Southern blot of C. elegans total DNA. We are also screening genomic clones with these oligonucleotide probes. [See Figure 1]

Zarkower D et al. (1991) Worm Breeder's Gazette "Fingering tra-"

Hodgkin JA, Zarkower D

[Worm Breeder's Gazette, 1991]

The tra-1 gene acts as a genetic switch controlling sexual differentiation in C. elegans. Loss-of-function mutations in tra-1 cause XX animals to develop as males, while gain-of-function mutations transform both XO and XX animals into fertile females. A small contig was identified as likely to contain at least part of the tra-1 gene based on the detection by one cosmid of rearrangements cosegregating with several tra-1 mutations (WBG vol. 10, #2). We have examined transcription from the tra-1 region by northern analysis and have detected two groups of transcripts. One correlates precisely with the region delineated by the rearrangements, while the second is encoded by the region immediately adjacent to it. We have focussed so far on the first group of transcripts, but it will be important to establish what role, if any, the adjacent transcripts have in tra-1 function. There are at least three transcripts within the region, all of low abundance. One is about 1.5 kb and is present only during L2. A second RNA is about 5 kb and present during all hermaphrodite stages and in adult males. The third is shorter than 1 kb and has been detected only in adult males. We have not yet examined larval males. In addition there may be one or two other, less reproducibly detected, transcripts from the region. We have isolated cDNAs corresponding to the L2 RNA both from the Kim library and by PCR. The mature message is spliced from a primary transcript of more than 11 kb and can encode a protein of at least 340 amino acids (the 5' end has not yet been unambiguously determined) which includes two C-terminal ''zinc-finger'' motifs. The fingers are 45% identical to those of human GL1, a gene amplified in glioblastoma tumors, while the non-finger region (which is serine-rich) shows no similarity to other proteins in the databases. After the second finger there is a 'link' sequence and a cysteine at the correct position to be part of a third finger, so it is likely that at least one other transcript encodes a protein containing additional zinc fingers. Indeed, preliminary data suggest that the 5 kb message contains additional sequences in the region of the fingers. We are currently isolating cDNA clones of the 5 kb and male-specific messages.

Li WQ et al. (1988) Worm Breeder's Gazette "update on transposon tagging in Minnesota."

Li WQ, Shaw JE, Herman RK, Starich TA

[Worm Breeder's Gazette, 1988]

As reported in the last WBG, Southern blots of EcoRI/PstI digests made from a TR679-derived unc-33 mutant showed two extra Tc4- containing bands (Tc4 is itself cut by PstI), and the bands were eliminated by reversion of the unc-33 mutation. We cloned one of these bands, a 1.7 kb fragment, and then used EcoRI plus XbaI to release a 0.6 kb unique worm sequence. When this 0.6 kb fragment was used to probe Southern blots of EcoRI digests, the unc-33 mutant DNA showed a 6.2 kb band, whereas N2 and several independent revertants of unc-33 showed a 4.2 kb band. The 0.6 kb probe was then used to screen an N2 genomic library (from Chris Link). A positive lambda recombinant phage was identified, and a 4.2 kb EcoRI fragment was subcloned from it. This fragment as probe identified the expected EcoRI bands from unc-33 strains, revertants and N2. In addition, it identified two EcoRI bands, 3.1 kb and 4.6 kb, from an unc-33 mutant kindly sent to us by Ed Hedgecock. Ed also sent us a revertant of his mutant; it has only the normal 4.2 kb EcoRI band. Thus the two mutants have different insertions in the same 4.2 kb EcoRI fragment. We have not yet determined the identity of the second insertion. In our effort to clone unc-3, we have identified an extra Tc1- containing BamHI fragment that is associated with an unc-3 mutation derived from TR679. Revertants of this allele in a largely-TR674 genetic background have recently been obtained and arise at a frequency in the range of 10+E-3-10+E-4. The revertants should now help determine if the Tc1-containing BamHI fragment we have focused on is responsible for the unc-3 mutation. In the last WBG we noted that we are generating a collection of TR679-derived mutants that are defective in the uptake of FITC. We now have a total of nine mutations: three on LGI, two on LGII, one on LGV, two on X, and one as yet unassigned to a linkage group. Mutations assigned to genes include a previously described daf-10 allele, a che-2 X allele, and a mutation that defines a new gene on X, which we propose calling ftc-1, located near and to the right of dpy-6.