Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA

Hresko MC et al. (1994) Worm Breeder's Gazette "Shoot First, Ask Questions Later"

Shrimankar PV, Hresko MC, Waterston RH

[Worm Breeder's Gazette, 1994]

Shoot First, Ask Questions Later M.C Hresko, P.V. Shrimankar and R.H. Waterston. Washington Univ. Sch. of Med., St. Louis, MO 63110. coutu@sequencer.wustl.edu and pvs@elegans.wustl.edu

Janke DL et al. (1996) Worm Breeder's Gazette "A Cosmid Transgenic Library for C. elegans- An Update"

Franz NW, Janke DL, Rose AM, O'Neil NJ, Schein JE, Baillie DL

[Worm Breeder's Gazette, 1996]

As reported at the 1995 International Worm Meeting, our labs are generating a large number of transgenic C. elegans strains which have been transformed with cosmid DNA sequenced by the C.elegans Genome Sequencing Project (a joint U.S./U.K. effort headed by Drs. R. H. Waterston at

Otsuka AJ (1990) Worm Breeder's Gazette "ISOLATION AND CHARACTERIZATION OF UNC-104 CDNA CLONES"

Otsuka AJ

[Worm Breeder's Gazette, 1990]

Although the unc-104 fragments isolated from an Arhinger-Kimble library were instrumental in obtaining approximately 4 kb of the 6 kb mRNA sequence, it was desirable to obtain full length cDNA clones to both complete the cDNA sequence and to produce the recombinant protein product in bacteria. The discrepancy between the abundance of unc-104 cDNA clones in the Arhinger-Kimble library (approximately 1 per 10,000) and in the Kim-Horvitz (none in 200,000) and Thacker-Capecci libraries (none in 100,000), led us to screen the Barstead-Waterston and Schauer-Wood libraries. The abundance of cDNA clones in these additional libraries suggests that an insufficient number of clones was screened in the cases of the Kim and Thacker libraries. cDNA clones were isolated from 2.6 million phage (1.4 million phage screened from the Barstead-Waterston bank, 0.6 million the Schauer- Wood early embryonic and him-8 embryonic libraries). The probe was the 5'-most cDNA fragment obtained from the library. The results are shown below: [See Figure 1] The two cDNA clones from the Barstead library overlap and cover a region of 4.7 kb. DNA sequencing of the cDNA clones is currently in progress.

Waterston RH (1977) Worm Breeder's Gazette "R73 - a correction."

Waterston RH

[Worm Breeder's Gazette, 1977]

The strain R73 has previously been described as a revertant of unc- 54(e569) (Epstein, et al., J. Mol. Biol. (1974) 90:291-300; Mackenzie and Epstein, Nematode workshop, 1977). However, the fine structure as described by Mackenzie in Woods Hole strongly resembled the muscle of putative missense alleles of unc-15 (Waterston, et al., J. Mol. Biol. in press) and not that of any of the other mutants so far isolated and examined (Waterston, unpublished). Accordingly, R73 was obtained from H. Epstein and further genetic studies carried out. R73 fails to complement unc-15(e1214), with R73/e1214 heterozygotes resembling R73 in their muscle structure by polarizing microscopy. This defect in muscle structure maps close to dpy-5 I, and three factor crosses indicate that the mutation(s) lies near or to the right of unc-13. No evidence for a mutation(s) in the unc-54 gene was found. Thus R73 is apparently a partial revertant of an unc-15 allele, probably e73 and not unc-54(e569) (Where the error in bookkeeping occurred is unclear), and the large paracrystalline arrays, presumably of paramyosin, remain characteristic only of presumptive missense alleles of unc-15.

(1976) Worm Breeder's Gazette

[Worm Breeder's Gazette, 1976]

Robert Wyman (Department of Biology, Yale University) reports that Theodore Bullock (University of California at San Diego, La Jolla, California) has isolated a 4.1-meter long nematode from a 3.8-meter whale. The specimen is preserved, and Dr. Bullock is searching for a taxonomist interested in taking the beast. Want Ad: Does anyone know a way to cause a large population of males to release their sperm into the medium? CONTEST REMINDER: Don t forget that bottle of champagne from S. Ward and D. Hirsh promised to whoever scores the most progeny from a single hermaphrodite (see Newsletter, Vol. 1, # 2 for details). Deadline for submission of entries to either judge is February 23rd.

Matheson C et al. (1992) Worm Breeder's Gazette "The Molecular Analysis of unc-60"

Wakarchuk MF, McKim KS, Baillie DL, Matheson C

[Worm Breeder's Gazette, 1992]

We have been cloning and sequencing the muscle gene unc-60 ,located on the left end of LGV. The genetic organization of the unc-60 region has been described previously by K. McKim (McKim et al. ,1988). Transformation rescue localized unc-60 to a deleted 22 kb cosmid, F53E2 (M. Wakarchuk, WBG Vol. 11, #1). A restriction map of the cosmid has been constructed. Various subcloned fragments spanning the entire cosmid were used to screen a lambda Zap cDNA library (a gift from R. Barstead and R. Waterston) resulting in the isolation of cDNA clones representing two coding regions. Sequencing of cDNA and genomic clones of one of the coding regions has revealed that at least two cofilin/destrin like proteins are produced through alternate splicing. Each transcript has 5 exons, sharing only the first which is untranslated except for a single methionine residue. Cofilin and destrin are actin binding proteins which facilitate actin polymerization and/or depolymerization. This function is consistent with the unc-60 mutant phenotype ie. disorganized thin filaments (Waterston et al., 1980). The only lethal allele of unc-60 ,s1586 ,was studied using PCR and Southern blot analysis and was shown to contain an approximately 500 bp deletion which affects both transcripts. Currently, a 10 kb BamH1 fragment, which contains the coding regions for both cofilin/destrin like proteins, is being isolated for microinjections in an attempt to rescue unc-60 .As well, PCR products from homozygous unc-60 EMS induced mutants are being sequenced to identify their molecular nature.

Kagawa H et al. (1990) Worm Breeder's Gazette "CHROMOSOME MAPPING OF mec-1 AND tmy-1, TROPOMYOSIN GENE"

Kagawa H, Coulson AR, Chalfie M, Sugimoto K, Ando H

[Worm Breeder's Gazette, 1990]

To clone the kra-1 gene which might encode a nicotinic receptor associated NMDA receptor-like ion channel (Hideki Ando and Hiroaki Kagawa, in this issue), mec-1 gene was chosen as the starting point for chromosome walking. 11 lambda clones crossreacted with 5 unique fragments from 2 Tc1 induced mec-1 worms were mapped on the chromosome map LGV as was shown in the bottom. One of the clones; HK#9.1 was sensibly located on the position which was calculated by the average relation value between genetic and physical map. We started to RFLP experiment on gamma ray induced mutants to confirm the position of the mec-1 gene by using candidate cosmid clones. International cooperation will accelerate to finish the jig saw puzzle. Tropomyosin gene cloning was nearly finished but still continuing by some unlucky (The worm gazette Vol.10 #3 1988). Any genomic library do not contained tropomyosin gene by some reason of sequence similarity to lambdoid phage. We have already cloned two genomic restriction fragments which contained 6/8 exons. By screening YAC library with one restriction fragment as a probe, 'tmy-1', tropomyosin gene was mapped on the left of unc-54 LGI. The result was independently confirmed with a different approach by Larry Shriefer and Bob Waterston (in this issue). They are doing experiment on searching a candidate mutant of tropomyosin gene defect. Full length cDNA clone was also obtained from new cDNA library (kindly supplied by Barsted and Waterston). Complete cloning business shows the reason why the gene was not packed in a phage particle. [See Figure 1]

Charest DL et al. (1987) Worm Breeder's Gazette "intragenic mapping of unc-26 IV."

Charest DL, Baillie DL

[Worm Breeder's Gazette, 1987]

Individuals homozygous for an unc-26 mutation are severely paralyzed even though the muscle appears normal (Waterston et al. 1980). Because of the interesting nature of this phenotype, we have undertaken the study of the unc-26 gene. unc-26 is located on the right arm of LGIV, but lies outside the intensively studied unc-22 gene cluster region. A partial fine structure map of the unc-26 alleles has been constructed. Presently, there are eleven alleles available for our study: e176, e205, e314, e345, e429, e446, e568, and e1048 were isolated from 0.05 M EMS mutagenesis (Brenner 1974), while e1196 and m2 were induced with ICR 191 (Riddle and Baillie) and DES (D. Riddle) respectively. e1710 was isolated in our laboratory in a mut-4 background (kindly supplied by D. Moerman) and probably resulted from a Tc1 insertion. The method used for fine structure analysis was modified from Moerman and Baillie (1979). Triple mutant hermaphrodites of the genotype unc-22 ssed to unc-26(y)/+ males. The resulting heteroallelic hermaphrodites, unc-22 unc-26(Y) +, self-fertilized and the second generation was screened for exceptional individuals. Figure 1 shows the relative positions of the alleles tested so far. The largest recombinational map distance is 0.025 map units. The remaining alleles are now being positioned on the map. The alleles display variations in the severity of their paralysis. For this reason we are investigating the effect of each mutation in the hemizygous state over the deficiencies sDf21 and nDf27 (from R. Horvitz). The informational suppressor sup-7 (Waterston 1980) is being used to identify alleles within the coding region. Also, work is in progress to isolate the Tc1 flanking sequences in e1710. Please note that we would appreciate receiving any unc-26 alleles not listed above. [See Figure 1]

Amaar YG et al. (1990) Worm Breeder's Gazette "Cloning of histidyl-tRNA synthetase in C."

Amaar YG, Baillie DL

[Worm Breeder's Gazette, 1990]

We have serendipitously recovered a 420 base pair EcoRI cDNA fragment from a C. elegans cDNA library (obtained from B. Barstead and B. Waterston) which probably encodes a portion of the C. elegans histidyl-tRNA synthetase. This fragment was sequenced; and a 250 nucleotide sequence of this fragment was translated and the predicted peptide was used to search EMBL 22 and SWISS-PROT 13 data banks, a significant match with histidyl-tRNA synthetase is found. Our peptide sequence when compared with human histidyl-tRNA synthetase showed a 50% amino acid identity and a 85% similarity when conserved changes are taken into consideration. The 420 bp cDNA fragment was used as a probe to screen the C. elegans cDNA library to obtain a larger cDNA fragment. This resulted in the recovery of a 1.4 kb cDNA fragment that could represent the entire coding region. Both the E. coli and human histidyl-tRNA synthetase coding regions are composed of 424 and 503 codons, respectively (1,2). Using the 420 basepair cDNA fragment as a probe, a YAC filter provided by B. Waterston was used to map this gene. It is found to fall between mec-3 and lin-3 within the deficiency sDf2 on LGIV. We assume that null mutations in this gene will be lethal. The region in which the gene maps is thought to be 70% saturated for essential gene mutations (3). Thus, it is very likely that we already possess mutations in this gene. The gene lies within a 5.5 kb EcoRI genomic DNA fragment, and the gene is likely present in a single copy. We will attempt to identify the lethal which corresponds to this gene by using germ line transformation with the appropriate cosmid.