Plenefisch JD et al. (1994) Worm Breeder's Gazette "Cloning mua-3: Some Observations on the New Molecular Era"

Plenefisch JD, Hedgecock EM

[Worm Breeder's Gazette, 1994]

Cloning mua-3: some observations on the new Molecular Era John Plenefisch and Edward Hedgecock, Dept. of Biology, Johns Hopkins University, Baltimore MD 21218

Kipreos ET et al. (1992) Worm Breeder's Gazette "Cloning of the C. elegans Tumor Suppressor Genes lin-19 and lin-23"

Hedgecock EM, Kipreos ET

[Worm Breeder's Gazette, 1992]

Cloning of the C. elegans tumor suppressor genes lin-l9 and lin-23. Edward T. Kipreos and Edward M. Hedgecock Department of Biology, Johns Hopkins University, Baltimore, MD 21218

Blumenthal TE et al. (1993) Worm Breeder's Gazette "The C. elegans Cleavage and Polyadenylation Signal"

Fields CA, White O, Blumenthal TE

[Worm Breeder's Gazette, 1993]

THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Gu GQ et al. (1994) Worm Breeder's Gazette "mec-5 Encodes a Collagen Needed For Touch Sensitivity."

William CM, Chalfie M, Gu GQ

[Worm Breeder's Gazette, 1994]

mec-5 Encodes a Collagen Needed for Touch Sensitivity Guoqiang Gu, Chris William, and Marty Chalfie, Department of Biological Sciences, Columbia University in the City of New York, NY 10027 mec-5 is one of the genes required for mechanosensitivity in C. elegans. Although the touch receptor neurons do not function, they appear morphologically normal in mec-5 mutants. Peanut-lectin binding to the touch cell mantle, however, is absent (M. Chalfie and E. Hedgecock, unpublished data). By transformation rescue, we identified a 6.5 kb BamHI-Kpnl fragment in cosmid E03G2 which completely rescues the mec-5 phenotype. We isolated a 1.2 kb full length cDNA (Northern blots show a 1.2 kb mRNA) from the Chris Martin cDNA library. The cDNA sequence encodes a collagen. The protein (329 aa) has a 20 amino acid putative signal sequence at the N- terminus and 69 Gly-X-Y repeats starting at aa 63. These collagen repeats are followed by 60 additional amino acids. Because collagens are often glycosylated on hydroxyproline residues, the loss of MEC-5 may explain the lack of peanut- lectin binding. To verify that this collagen gene is mec-S, we probed genomic Southern blots with the 6.5 kb fragment and found that the 6.5 kb fragment was deleted in two mec-5 strains, the mut-2 derived strain TU828 and the Y-ray strain TU1031. We are now sequencing other mec-5 mutations and characterizing the expression pattern.

Harfe BD et al. (1994) Worm Breeder's Gazette "ceh-24, a New NK Class Homeodomain Gene."

Okkema PG, Fire A, Harfe BD

[Worm Breeder's Gazette, 1994]

ceh-24, a new NK class homeodomain gene Brian Harfe 1, 2, Pete Okkema 1, and Andy Fire 1 1 Carnegie Institution of Washington, Department of Embryology, 2 Biology Graduate Program, The Johns Hopkins University The NK class of homeodomain proteins are a set of regulatory factors with a broad evolutionary distribution and a variety of expression patterns. Analysis of pharyngeal myosin regulation led to the identification of a member of this family, ceh-22, which appears to mediate aspects of pharyngeal gene expression (Okkema and Fire, Development 120, 2175). To determine the extent of the NK homeodomain family in C. elegans we have used a degenerate primer based PCR strategy. Among several candidates from the initial PCR, we have so far characterized one gene, denoted ceh-24. The CEH-24 homeodomain falls into the NK class, but is closer to diverse members of the NK family seen in other species than to CEH-22. In addition to the homeodomain, CEH-24 contains an acid activation domain C-terminal to the homeodomain. ceh-24 maps (thanks to Alan Coulson) to chromosome V just left of him-5.

Consortium TCeGS (1992) Worm Breeder's Gazette "Update on cDNA Sequencing"

Consortium TCeGS

[Worm Breeder's Gazette, 1992]

This is another interim report on the state of the cDNA sequencing and mapping project that we are carrying out alongside the genomic sequencing effort. We have now sequenced 1222 of the 1743 clones in Chris Martin's library. Of these 27 appear to be duplicates in the strong sense that their sequence looks identical and they come from neighboring micro-titre wells that were most likely cross-contaminated. The following results therefore come from the 1195 left after removing these duplicates. Remember that we are only producing a single gel reading from each clone (5' end of the cDNA insert). Similarities: There are 373 (31%) with BLASTX scores to the public protein databases greater than 100, which is a high enough threshold that these similarities are very likely to be meaningful. A list of these is given on the following pages. [Figures excluded due to low reproducability] Trans-spliced leaders: 166 sequences (14%) had trans-spliced leaders at the 5' end, 149 to Sl1 and 17 to SL2 . Repeat isolations: A number of genes clearly have been cloned more than once. We can unambiguously detect this only when the 5' ends of the inserts overlap, which happens for 334 sequences that fall into 130 overlap groups. This is not out of line with Chris Martin's estimate of repeat frequency. It leaves us with 991 distinct sequences of which 861 are represented only once. However it is likely that there are additional repeats that we don't see this way because we have different parts of the same gene. Hits to previously determined worm sequences: There were exact matches to 13 previously determined C. elegans sequences in the public databases, and to 3 of the 44 predicted genes on the first 4 genomic cosmids that we have sequenced. The latter figure allows us to generate an estimate of (44/3)*991 = 14,500 genes in the worm, but the 95% confidence limits for this are 6,000 to 50,000, so we need more data to make this a useful approach to estimating gene density. Mapping: we are now in the process of mapping all the clones using the YAC "polytene" filters. This data will be in ACEDB and will also be provided if you ask for a clone. All the sequences, similarity and mapping data will be in ACEDB, and any clone is available along with more detailed data from the St. Louis lab.

Shakes DC (1993) Worm Breeder's Gazette "Call for Antibody Information"

Shakes DC

[Worm Breeder's Gazette, 1993]

As some of you are already aware, Academic Press has asked Henry Epstein and myself to edit a volume of Methods in Cell Biology devoted to C. elegans. One of the useful appendices which we would like to include is a list of available antibodies and protocols for using them. If you have generated an antibody in your laboratory which you would like to see listed in a methods volume on C. elegans, please contact me as soon as possible. Incidentally, if you would like to make your antibodies widely available but are nervous about being swamped with requests, you might considered contributing your monoclonal antibodies to the Developmental Studies Hybridoma Bank sponsored by the National Institute of Child Health and Human Development. They are providing the wonderful service of growing up monoclonals and making the cells or supernatants available to researchers at reduced fees. I know from personal experience that their anti-tubulin antibody works well for C. elegans, and it is considerably cheaper than commercial products. Contact Dr. Thomas August, Johns Hopkins University at (410) 955-3985 for more information about receiving a catalog or making contributions. I have the impression that they are not anxious to handle hundreds of worm specific antibodies, but that they are interested in handling a limited set of the most widely used C. elegans specific antibodies as well as antibodies which are known to cross-react with other species.

Estevez M et al. (1990) Worm Breeder's Gazette "daf-4, a Gene Controlling Dauer Larva Development, Encodes a Serine/Threonine Kinase"

Estevez M, Albert PS, Riddle DL

[Worm Breeder's Gazette, 1990]

Loss-of-function mutations in daf-4 confer a dauer-constitutive phenotype. Two transposon-tagged alleles of daf-4 have been isolated, and Southern blots probed with the labeled transposon sequence revealed a novel restriction fragment in the mutant DNA that was absent in non-mutant DNA. A transposon-containing fragment was cloned, and sequences flanking the transposon were used to screen Bob Barstead's cDNA library and Chris Link's genomic library. Four overlapping genomic clones and five cDNA clones were isolated. Microinjection of one of the genomic clones into the germline of daf-4 mutants transforms their progeny to wild type. Approximately 1 kb of contiguous cDNA sequence has been determined, and the deduced amino acid sequence shows 30% identity to the C. elegans brane receptor kinase (Georgi et al., 1990, Cell 61: 635) over a 222 amino acid region in the catalytic domain. The deduced amino acid sequence also shares 37% identity with PSK-J3 ( from HeLa cells), a member of the cdc2/CDC28 kinase family over a 107 amino acid region in the catalytic domain. Since genetic interactions suggest that daf-4 acts downstream of the daf-1 receptor, we propose that it mediates the second step in a kinase cascade that normally acts to prevent dauer larva development, and permit growth.

Chan S et al. (1995) Worm Breeder's Gazette "unc-40 Encodes a Protein Related to the Human DCC (Deleted Colorectal Cancer) Protein."

Su M-W, Culotti JG, Hedgecock EM, Chan S

[Worm Breeder's Gazette, 1995]

unc-40 ENCODES A PROTEIN RELATED TO THE HUMAN DCC (DELETED COLORECTAL CANCER) PROTEIN. Shirley Chan, Ming-Wan Su, and Joe Culotti Samuel Lunenfeld Research Institute of Mt. Sinai Hospital Toronto, Canada Ed Hedgecock Department of Biology Johns Hopkins University Baltimore, MD unc-40 is required for circumferential pioneer migrations of axon growth cones and mesodermal cells on the basal surface of the epidermis of C. elegans. Although unc-40 mutations primarily affect migrations oriented towards the ventral side, they also affect dorsally-oriented migrations (although to a lesser extent). The unc-40 gene was cloned by mapping unc-40 mutations relative to DNA polymorphisms followed by transformation rescue. We have determined what we believe may be the entire coding region of this gene comprising 4 kb of sequence. From amino to carboxy terminus, the predicted UNC40 protein has 4 immunoglobulin domains, 6 fibronectin type III domains, a single transmembrane domain and an intracellular domain of 309 amino acids. The overall domain structure of the predicted UNC-40 protein is very similar to the human DCC (deleted colorectal cancer) protein which was initially (incorrectly?) implicated in a fraction of human colorectal cancers. More recently, a DCC-related gene has been cloned from chicken (Jost Vielmetter and William Dryer, Cal Tech, personal communication) called neogenin. neogenin is not the chicken DCC gene, but is related to it. The fibronectin and immunoglobulin domains of UNC-40 are clearly more related to DCC and Neogenin than they are to similar domains in other proteins. Interestingly, the Neogenin protein is expressed in neurons as they extend axons in the developing chick nervous system. We are currently attempting several methods for examining the expression pattern of the UNC-40 protein.