Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Musset-Bilal F, Schwarzbauer JE, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Townsend SR et al. (1994) Worm Breeder's Gazette "C. elegans Molecular Genetics and Long PCR."

Finelli AL, Savage C, Xie T, Padgett RW, Townsend SR

[Worm Breeder's Gazette, 1994]

C. elegans Molecular Genetics and Long PCR Scott R. Townsend, Cathy Savage, Alyce L. Finelli, Ting Xie, and Richard W. Padgett, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855

Dhillon HS et al. (1994) Worm Breeder's Gazette "More Degenerins in the Worm?"

Dhillon HS, Driscoll MA

[Worm Breeder's Gazette, 1994]

More degenerins in the worm? Harbinder Singh Dhillon and Monica Driscoll. Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes lane, Piscataway, N.J. 08855

Wilkinson HA et al. (1994) Worm Breeder's Gazette "Reciprocal Changes in Expression of lin-12 and lag-2 Prior to Commitment of Z1.ppp and Z4.aaa."

Greenwald IS, Wilkinson HA, Fitzgerald K

[Worm Breeder's Gazette, 1994]

Reciprocal changes in expression of lin-12 and lag-2 prior to commitment of Z1.ppp and Z4.aaa. Hilary A. Wilkinson*, Kevin Fitzgerald and Iva Greenwald, HHMI and Dept. of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; *Current address: Merck Research Laboratories, Rahway, NJ 07065

Ishii N et al. (1988) Worm Breeder's Gazette "partial sequence of the unc-6 gene."

Ishii N, Stern BD, Culotti JG, Hedgecock EM

[Worm Breeder's Gazette, 1988]

In the previous issue (WBG 10(1) p.35), we reported cloning a small DNA fragment containing the Tc1 insertion which caused the unc-6 ( rh1035) mutation. Since then, we have isolated an 11.5 kb EcoRI fragment including this region from N2 DNA (NJ#14, EMBL4 vector) Alan Coulson has kindly positioned this fragment on the MRC genome map. We have now sequenced both the original 1.25 kb XbaI fragment of unc-6( rh1035) DNA (plasmid NJ#6) and a 2.7 kb HindIII fragment of N2 DNA subcloned from phage NJ#14. The 2.7 kb HindIII fragment has five exons which encode a hypothetical protein fragment of 360 residues. This fragment is not closely related to any protein in the current PIR database The last exon in the 2.7 kb HindIII fragment is followed by an intron which continues beyond the fragment. The first exon is preceded by a non-coding region which may be part of an intron or the unc-6 promoter region. In fact, a possible 21 residue signal sequence with an initiator methionine occurs in the first exon shortly upstream of the Tc1::rh1035 insertion site. TATA and CCAAT motifs occur about 90 and 120 bp upstream of the proposed initiator codon. The decanucleotide sequence TTTGCCTCTC is tandemly repeated about 200 bp upstream of this codon. [See Figure 1]

Driscoll MA et al. (1992) Worm Breeder's Gazette "A Hot-Spot for Mutations in a Cys-rich Domain of mec-4 Includes a Site Which,When Mutated, Can Suppress a Dominant Death-Inducing Mutation in Cis"

Driscoll MA, Hong K

[Worm Breeder's Gazette, 1992]

A Hot-spot for Mutations in a Cys-rich Domain of mec-4 Includes a Site Which, When Mutated, Can Suppregg a Dominant Death inducing Mutation in Cis Kyonsoo Hong and Monica Driscoll. Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, N.J. 08855 (908) 463-5193

Steven RM et al. (1990) Worm Breeder's Gazette "Molecular cloning of unc-73."

Steven RM, Culotti JG

[Worm Breeder's Gazette, 1990]

Unc-73 affects the longitudinal growth of certain neurons and the excretory cell as well as certain longitudinal cell migrations on the nematode epidermis (Hedgecock et al., Development 100, 365-382, 1987). We previously isolated unc-73(ev454) on strain RW7097 and identified a Tc1 on a 5 kb HindIII which cosegregated with this mutation in 3 factor crosses (CSH abstracts, 1989, p. 60). We have now cloned this 5 kb Tc1-containing HindIII fragment, excised the Tc1 with EcoRV, and used the flanking sequence to probe Southern blots of DNAs from unc-73( ev454) and from two independently derived spontaneous revertants of unc-73(ev454). As expected, this probe hybridized to a 5 kb HindIII fragment on the unc-73(ev454) lane and to a 3.4 kb HindIII fragment on the revertant lanes. No polymorphisms were observed in DNA from two EMS-induced alleles of unc-73 (e936 and rh40). We have obtained approximately 250 nucleotides of sequence from both ends of the 5 kb HindIII fragment. One of these ends comprises a GC rich open reading frame which fails to show any significant homology to anything in the NBRF database. We have also identified about a dozen phages from an EMBL-3 N2 genomic library (Nishiwaki and Miwa) which hybridize to our Tc1-flanking probe, but we have not yet characterized any of these. We hope that these will allow Coulson and company to identify the unc- 73-containing contig.

Hung M-S et al. (1995) Worm Breeder's Gazette "vab-8 May Encode a Protein with Kex2 Cleavage Sites."

Hung M-S, Way JC

[Worm Breeder's Gazette, 1995]

vab-8 may encode a protein with Kex2 cleavage sites Ming-shiu Hung and Jeff Way. Dept. of Biology, Nelson Labs, Rutgers University, Piscataway NJ 08855. We have identified and sequenced a ~5kb DNA fragment that rescues the withered tail phenotype of vab-8(e1017), and isolated some corresponding, incomplete cDNAs. vab-8 is adjacent to myo-3 and these two genes are transcribed towards each other. Our current interpretation of the sequence suggests that the vab-8 protein is 571 amino acids and contains 20 sites where two or three arginine and/or lysine residues are adjacent. Such pairs of basic amino acids are the target for the Kex2 protease, which is located in the Golgi and which cleaves secreted proteins. In addition, there may be a 18 aa hydrophobic stretch at the N-terminus, followed by a His-Lys sequence that also might be a cleavage site. The C terminal 300 amino acids are confirmed by our cDNAs, but the putative N-terminus has been identified only by sequence-gazing and still needs to be confirmed. vab-8 mutants show a withered tail phenotype as a result of a failure of the posterior migration of the CAN neuron. In addition, some alleles cause an anterior over-migration of the HSN cell (Manser and Wood, Devel. Genetics 11: 49-64). At a low frequency, extra mec-3-expressing PLM cells are observed (Way et al., Dev. Dynamics 194: 289-302), which could be due to a failure in sensing the anterior-posterior direction leading to an asymmetric cell division defect. Bruce Wightman and Gian Garriga (personal communication) have also shown that vab-8 is allelic to unc-107, which has an axon guidance defect, and that the extension of many axons is defective vab-8(-). Only events in the anterior-posterior direction are affected in this mutant. Thus, vab-8 may be part of a directional information system that would be complementary to the unc-5/unc-6 system for the dorsal-ventral direction.

Savage C et al. (1994) Worm Breeder's Gazette "More tolloid Homologs in the Worm."

Padgett RW, Finelli AL, Savage C, Cho SH

[Worm Breeder's Gazette, 1994]

More tolloid homologs in the worm Cathy Savage, Seo Hee Cho, Alyce L. Finelli, and Richard W. Padgett. Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855. Members of the tolloid/BMP-1 family of metalloproteases are thought to interact with extracellular growth factors to regulate a variety of developmental events. In particular, the Drosophila gene tolloid interacts with decapentaplegic (dpp), a member of the TGF-,B superfamily. Similarly, vertebrate BMP- 1 co-purifies with other BMPs (bone morphogenetic proteins) that are related TGF-,B-like ligands. Proteins in the tolloid/BMP-l family share a similar structure: they contain a predicted metalloprotease domain; EGF repeats; and CUB repeats, which are also found in blood complement proteins. These proteases are thought to interact with other proteins through the EGF and CUB repeats, and to regulate their activity through proteolysis. We have previously reported sequence analysis of the first C. elegans member of this metalloprotease family (WBG 12(4), p.30), which we have called toh-l (tollisk). Since then, the genome sequencing consortium has identified four more predicted ORFs with sequence similarity to tolloid. We have therefore isolated and sequenced cDNAs for these genes, to determine whether these new genes encode true members of the tolloid/BMP-1 family. One of the putative homologs displayed no protease homology. The predicted structures of the other three homologs are summarized below. Two of these, on the cosmids COSDl 1 and F42A10, contain related metalloprotease domains, but none of the characteristic protein interaction motifs of the tolloid/BMP-l family. Only one homolog, on the cosmid R151, was found to contain an EGF repeat and a CUB repeat. We therefore propose the name toh-2 for this gene. toh-2 represents a novel member of this family, because it contains repeats (TSP repeats) found in thrombospondin, an extracellular matrix protein. The presence of these two tolloid homologs in the worm raises several interesting questions: (1) Do these homologs act together in a TGF-,B-like signal transduction pathway, in separate pathways, or combinatorially with other, as yet unidentified, tolloid-like proteins? (2) What roles do these genes have in nematode development? (3) What is the function of the toh-2 thrombospondin repeats? Are there homologs in other organisms that also contain these repeats?

Benian GM et al. (1984) Worm Breeder's Gazette "an unc-22 transcript."

Moerman DG, Benian GM, Waterston RH

[Worm Breeder's Gazette, 1984]

We have some preliminary results from our attempts to identify mRNA from unc-22. Probing Northerns with various lambda clones from the unc-22 region reveals 2 transcripts. One is about 1.6kb in size and hybridizes only to our left-most clone, which only partially spans the region defined by the insertions. We assume this message is the product of a gene adjacent to unc-22. A second transcript that hybridizes to the entire set of clones spanning the insertion sites is very large--much larger in fact, than the 6.1kb unc-54 mRNA. The message is somewhat degraded but we do see a definite band. It is present in RNA prepared from mixed cultures and is at roughly 2-10% of the level of unc-54 message. Our reasons for believing that this large transcript is the product of unc-22 are threefold: (1) Tc1 insertions over a 20kb distance can disrupt unc-22 function, which is at least consistent with the large message seen. (2) We have also compared message levels for this presumed unc-22 transcript in N2 and unc-22(s32), an amber allele, and the s32 signal is greatly reduced relative to the signal from N2 (see Brown et al., Gene 20:139-144, 1982). (3) Finally, we have used single-stranded M13 clones of opposite orientation from the central region of unc-22 to probe Northerns of N2 and s32 RNA. One M13 clone hybridizes strongly to the large transcript from N2 but only weakly to RNA from s32, while the probe of opposite orientation does not hybridize to anything in either lane. From this last result we predict that unc-22 is transcribed from right to left on the genetic map. (This is a reversal from what we said at the 1984 GSA meeting). Recent sequencing results show that this direction yields the only continuous open reading frame. A preliminary computer search of DNA and protein sequence libraries using this open reading frame sequence did not reveal any homologous sequences to unc-22.