Waring DA et al. (1991) Worm Breeder's Gazette "pal-1, a Homeobox Gene that Regulates a Homeobox Gene"

Waring DA, Kenyon CJ

[Worm Breeder's Gazette, 1991]

pal-1 is a gene required for the formation of rays by the V6 cell in males. As we have described previously, pal-1 effectively prevents the T cell from inducing V6 to generate alae and, thereby, allows V6 to generate rays. In the absence of the T cell (following laser ablation), pal-1 is not required for V6 ray formation. Previously we presented evidence that pal-1 positively activates mab- 5. Supporting this model we have found that pal-1 is suppressed by the mab-5 gain of function allele e1751 (formerly lin-21). In unc-79( e1061) 91) 51); 90) males, only 5/100 sides had fewer than the wild- type number of rays in the fan; and in those cases, it appeared that V6 generated a few rays but not the wild-type number (V6 makes no rays at all on 85% of the sides in pal-1(e2091); 90)). The e1751 lesion is a duplication of a region of DNA containing mab-5. As Salser and Kenyon describe in an abstract in this gazette, it seems likely that the e1751 phenotype is the result of inappropriate transcription of mab-5. The fact that pal1+ activity is not required in the presence of the e1751 mutation suggests that pal-1 turns on mab- 5 at the level of transcription. Whether or not it does so directly is unknown. At last report we had narrowed the search for pal-1 to a 14 kb clone that carried the homeobox ceh-3. Now we have a 5.5 kb, ceh-3 containing clone, which rescues the pal-1 phenotype completely. We have found 2 independent cDNA clones which are each 1.3 kb (from Chris Martin's miracle library). One is just a few bases longer than the other. The 5.5 kb clone that rescues pal-1 contains about 1 kb upstream of the 5' end of the cDNAs and about 400 basepairs downstream of the 3' end of the cDNAs. For this reason we believe that ceh-3 is pal-1 (although we have not yet ruled out the alternative that there is a second gene on this clone). These cDNAs encode a ~250aa protein that contains the ceh-3 homeodomain. As Burglin and Ruvkin showed, this homeodomain is homologous to the caudal gene of Drosophila. Both cDNAs have part of what appears to be a trans-spliced leader sequence (the longer has 10 bases homologous to the splice-leader), so we believe these cDNAs are virtually full length. We have found no other conserved domains of any known significance, but we have found one curious homology to the mab-5 gene. In the mab-5 cloning paper, Costa et al. pointed out a serine rich region followed by a poly-alanine stretch. Interestingly, pal-1 has a very similar region, which includes an identical stretch of 9 amino acids SAAAAAAAN (the San Box). Such poly-amino acid stretches are common in homeodomain proteins, but their function is unknown. While this homology may be insignificant, the fact that both pal-1 and mab-5 act in V6 to promote ray formation makes this homology mildly but not profoundly tantalizing. Finally, to determine if pal-1 acts directly on mab-5 we are looking for pal-1 binding sites in the vicinity of mab-5. (In principle, if pal-1 binds we have made a pal-1/glutatione-S-transferase fusion protein (glu-pal) and expressed it in E. coli. Our results are very preliminary; but we have been able to purify this fusion protein, and we have seen sequence specific binding (as judged by band shifts) to an oligo-nucleotide probe known to be bound in vitro by engrailed and several other homeodomain proteins (E. Martin-Blanco and T Kornberg, personal communication). We hope to be able to use the glu-pal fusion to identify authentic pal-1 binding sites near mab-5.

Thacker CM et al. (1994) Worm Breeder's Gazette "Identifying Mutations in the bli-4 Gene, a Member of the Kex2-like Gene Family"

Srayko MA, Thacker CM, Rose AM

[Worm Breeder's Gazette, 1994]

We are studying the role of two genes that encode serine endoproteases that belong to the Kex2 -likefamily of prohormone convertases. These two genes include bli-4 and one identified as a sequenced cDNA designated cm1 Ob9 (Waterston et al., 1992, Nature Genet 1:114-123). Genetic analysis of bli-4 has enabled us to group mutant alleles into three classes (Peters et al, 1991, Genetics 129:95-102). The first mutant class includes the only viable mutation e937 which results in the blistering of the adult cuticle. Class II mutants include 11 lethal alleles which arrest development in late embryogenesis prior to hatching. These 11 alleles fail to complement e937 .Class III mutants comprise 2 lethal alleles that complement e937 yet fail to complement the class II lethal alleles. Class III animals predominantly arrest as early L1 larvae, although some lethality in late embryogenesis is also observed. The bli-4 gene was originally cloned in our laboratory by Ken Peters. The gene encodes at least three different proteins with extensive sequence similarity to the Kex2 -likefamily of serine endopeptidases. The three isoforms are produced by alternative splicing at the 3'-end of the gene as depicted in the figure of the gene structure below: So far we have been able to identify molecular lesions in three bli-4 alleles, e937 and two class II lethals. The e937 mutation is a 3.2kb deletion that removes the exon unique to blisterin A (Peters and Rose, W.B G. 11:28, 1991) resulting in the complete abrogation of expression of this product as demonstrated by northern analysis. Presumably, blisterin A is required for the production or maintenance of the adult cuticle. The class II mutant, h1 O10is a spontaneous allele isolated in a precomplementation screen for bli-4 alleles in a mut-6 background. The Tc1 insertion has been sequenced and found to reside in exon 9, 3' of the protease domain. A second class II lethal allele, q508 was found to contain a 366bp deletion that removes the splice acceptor and 33 nucleotides from exon 12. q508 was isolated by Lisa Kadyk in Judith Kimble's laboratory. Both of the lesions identified in the class II lethal alleles occur within the first 12 exons that are common to all of the bli-4 transcripts These results suggest that all of the class II alleles contain mutations either within this common region or in regulatory elements that control the expression of the gene, resulting in the loss of activity of all isoforms. We believe that the class III mutants are the result of mutations within the exons unique to either or both blisterin B and C, suggesting that these alternate isoforms have an essential function for early nematode development. We are currently attempting to identify the molecular lesions in the remaining class II and III lethal alleles using PCR-heteroduplexing and direct sequencing of PCR products. We have also initiated studies on a second gene which was originally identified by the random cDNA sequencing project (Waterston et al., 1992, Nature Genet. 1:114-123). The cDNA cm1 Ob9 has been mapped to the YAC clone Y51C6 in a region of chromosome III that is actively being sequenced by the genome sequencing group. Although the sequence of the gene that encodes cm1 Ob9 is not on the current ACEDB, we anticipate that it will be available in the next update. We have sequenced most of the cDNA and have determined that it represents a partial clone which does not include the protease domain. However, the sequence generated suggests that the cDNA encodes a product that most closely resembles Drosophila and vertebrate furin, a member of the Kex2 -likefamily. We are in the process of characterizing this gene in more detail by isolating cDNA clones that are more complete at the 5'-end. We would like to take this opportunity to thank Patty Wohldmann for forwarding cm1 Ob9 to us, and especially Lisa Kadyk in Judith Kimble's lab for the q508 allele. This work was supported by grants from the MRC (Canada) and the B.C. Health Foundation.

Ogura K-i et al. (1996) Worm Breeder's Gazette "The unc-14 gene is expressed in neurons with a pattern similar to that of the unc-51 gene."

Shirakawa M, Ohshima YM, Ogura K-i, Barnes TM, Hekimi S

[Worm Breeder's Gazette, 1996]

The unc-51 gene is required for axonal elongation in C. elegans and encodes a predicted serine / threonine kinase of 856 amino acids ( Ogura et al., Genes Dev. 8, 2389-2400, 1994 ). Recently, we obtained 16 cDNA clones whose products can interact with UNC-51 in a yeast two-hybrid system. One of them was an unc-51 C-terminal cDNA, and four others encoding a novel protein of 665 amino acids possibly corresponded to the unc-14 gene ( WBG, 14, No.1, 51, 1995 ) We have now extensively examined the ability of various cosmids and their subfragments to rescue unc-14(e57) mutation. The results revealed that the rescuing activity resides in the 5.2kb Hind III - Sal I fragment that encodes the N-terminal half of the novel protein. Furthermore, the mutation sites of the six unc-14 mutants were determined, and all of them were found to have nonsense mutations near the N-terminus of the protein. All these mutations are located in the 5.2 kb Hind III - Sal I fragment. Therefore, we conclude that this novel protein is UNC-14. The C-terminal half of the protein does not seem to be required for the rescue of an unc-14 mutation. The genomic sequence analysis revealed that the unc-14 gene has ten exons. To analyze the expression pattern of unc-14, we constructed an unc-14::lacZ fusion gene which was capable of rescuing unc-14(e57). This fusion gene is expressed in almost all neurons at all stages, especially in cell bodies and axons. The expression pattern is very similar to that of an unc-51::lac Z fusion gene, suggesting that UNC-14 directly interacts with UNC-51 in neurons, possibly in axons. We are also examining the expression of an unc-14::GFP fusion. Does UNC-14 interact with UNC-51 as a substrate or a regulator ? To examine these possibilities, the interacting sites were analyzed by using a yeast two-hybrid system. A region near the N-terminus of UNC-14 ( amino acid residues 200 - 381 ) interacts with a C-terminal region of UNC-51 ( 455 - 856, outside of the kinase domain ) in the two-hybrid system. The same C-terminal region of UNC-51 interacts with itself. We think that UNC-51 acts as a homodimer and that UNC-14 is a regulator of UNC-51 kinase. We plan to examine the direct interactions between UNC-14 and UNC-51, or UNC-51 and UNC-51 by using recombinant proteins produced by E. coli., and the effect of an unc-51 mutation for the binding.

Ohshima YM et al. (1992) Worm Breeder's Gazette "gsa-1 Gene Update: C. elegans Gene for G Protein a(s) Subunit"

Ohshima YM, Park J-H, Tani T

[Worm Breeder's Gazette, 1992]

We obtained a C. elegans genomic clone, which potentially encoded amino acids highly homologous to the C-terminus of rat G protein a(s) subunit, by using a cDNA fragment of rat Gsa as a probe (WBG 11, No 1, 33, 1989). From this clone (lEGL1-9), we subcloned a 13 kb Hind III fragment, and sequenced the hybridizing regions. The results revealed several regions encoding putative Gsa amino acid sequences. We repeatedly screened a cDNA library of Barstead and Waterston using a genomic probe but without success. Then we synthesized several oligonucleotides corresponding to the presumed exons, and used them to isolate cDNA fragments by reverse transcription of poly (A) RNA and PCR. From the sequences of these cDNAs and the genomic clone, we predict a protein of 375 amino acids as shown below. This predicted protein has amino acid identify of 66% overall, and about 90% in conserved regions, with that of rat Gsa. The homologies with Goa, Gpa-1 ,Gpa-2 and Gpa-3 of C. elegans (Silva & Plasterk, J. Mol. Biol. 215. 483487,1990; Lochrie et al., Cell Regulation 2,135-154,1991) are lower (34-40%) and those with other mammalian Ga(Gi, Go, Gt) subunits are also lower(40-42%). Therefore, we conclude that gsa-1 encodes a Gsa protein of C. elegans. Recently, we have screened an embryonic cDNA library of A. Fire (WBG 12, No. 2, 16, 1992) and obtained several positive clones. They are being analyzed. The gsa-1 gene probably consists of 8 exons and 7 introns extending over a 6kb region. It was mapped to a cosmid R06A10 near the left end of chromosome 1, by hybridization with YAC filters and with several candidate cosmid clones. In the region of genetic map which may correspond to cosmid R06A10 ,several mutations had been mapped such as bli-3 ,let-362 ,unc-35 ,egl-30 and lin-6 .We are examining whether one of these mutations resides in gsa-1 .We are also trying to construct gsa-1 /lacZchimeric genes in several ways to probe expression pattern of gsa-1 .[See Figure]

Plasterk RHA et al. (2000) Worm Breeder's Gazette "sgs-1, a gene encoding an adenylyl cyclase involved in Gas signalling, is an essential gene"

Korswagen HC, Plasterk RHA, Moorman C

[Worm Breeder's Gazette, 2000]

Heterotrimeric G proteins are an important class of signal-transducing molecules that couple seven transmembrane receptors to several intracellular effectors. In C. elegans, one homologue of each of the 4 mammalian classes of Ga subunits (Gai/o, Gaq, Ga12 and Gas) has been found.

Spieth J et al. (1984) Worm Breeder's Gazette "the yolk protein gene family of C elegans: YP6, the gene for the precursor to yp115 and yp88."

Spieth J, Blumenthal TE

[Worm Breeder's Gazette, 1984]

Four yolk proteins have been identified in C. elegans: Two closely related proteins of approximately 170,000 daltons (yp170A and yp170B), and two other polypeptides of 115,000 daltons (yp115) and 88,000 daltons (yp88) (Sharrock, 1983. Develo. Biol, 96, 182-188). yp115 and yp88 have been shown to be derived from a 180,000 dalton precursor ( Sharrock, 1984, J. Mol. Biol 174, 419-431). We recently have shown that yp170A and yp170B are encoded by a five gene family (YP1-5) ( Blumenthal et al.. 1984, J. Mol. Biol. 174, 1-18). We have now cloned a developmentally-regulated gene that, based on the timing of expression, size and abundance of the mRNA and finally, hybrid-arreat translation experiments, encodes the 180.000 dalton precursor to yp115 and yp88 . The gene (YP6) was cloned from a C. rary by selecting abundantly expressed sequences that were adult specific but did not hybridize to YP1-5. Genomic clones were selected from John Karn's lambda1059 library. Based on genomic Southern blots the precursor to yp115 and yp88 is encoded by a single-copy gene. It is about 5.5 kb long and hybridizes to a 5.2 kb RNA. YP6 RNA is as abundant in adult hermaphrodites as is the RNA from the YP1-5 gene family but, like YP1-5 RNA, YP6 RNA is not detectable in larvae or adult males. Based on DNA sequence comparison YP6 is distantly related to the YP1- 5 genes suggesting that all of the yolk protein genes evolved from a common ancestor.

Kagawa H et al. (1985) Worm Breeder's Gazette "exson-expression cloning of the unc-15 paramyosin gene and a tropomyosin gene from Caenorhabditis elegans."

Brenner S, Kagawa H, Karn J

[Worm Breeder's Gazette, 1985]

Short genomic clones derived from the C. elegans in gene and a tropomyosin gene were obtained by screening an expression library with specific antibodies. The library was constructed by cloning short Sau3A fragments of genomic DNA into the plasmid vector pUR289, which has a polylinker site at the 3' end of the lacz gene. The identity of the tropomyosin clones was confirmed by sequencing of the cloned fragment. The fragment showed strong homology to rat skeletal alpha-tropomyosin and Drosophila muscle tropomyosin. The sequence of the paramyosin clone showed a hydrophobic amino acid repeat structure, consistent with the known alpha-helical coiled-coil structure of this protein To confirm the identity cf the putative paramyosin clone, Sau3A fragments from a bacteriophage lambda clone containing 14-20 kb of flanking sequence were cloned into lacz expression plasmid vector. Numerous new fragment were recovered which encoded paramyosin epitopes. One of these fragments reacted with a monoclonal antibody to paramyosin which also specifically reacted with a 24 kd CNBr-fragment. Comparison of the N-terminal sequence of the 24 kd CNBr-fragment and the deduced amino acid sequence of the fusion protein showed an exact match of 8 amino acids. Cosmid clones overlapping the paramyosin gene were also isolated. In RNA hybridization experiments, each probe detects a single RNA species of the expected size. The paramyosin mRNA is approximately the same size as the '28s' RNA from nematode Sequencing of the both genes is in progress in Japan.

Goodwin EB et al. (1994) Worm Breeder's Gazette "The laf-1 Gene Functions in the 3'UTR-Mediated Translational Repression of the Sex Determination Gene, tra-2"

Kimble JE, Goodwin EB

[Worm Breeder's Gazette, 1994]

The laf-l Gene Functions in the 3'UTR-mediated Translational Repression of the Sex Determination Gene, tra-2. Elizabeth Goodwin# and Judith Kimble University of Wisconsin, Madison, WI. # Dept. of CMS Biology, Northwestern Univ. Medical School, Chicago Ill.

Hori F et al. (1998) Worm Breeder's Gazette "dpy-20 gene affects the expression of unc-119 gene in the motor neurons, but not in the sensory neurons in C. elegans"

Ali MY, Siddiqui SS, Hori F, Matsuo T

[Worm Breeder's Gazette, 1998]

Mutants in the unc-119 gene are defective in locomotion, feeding behavior and chemosensation (Maduro M, Pilgrim D,1995). The unc-119 gene expresses in many neurons including the cell bodies and processes of several motor neurons in the nerve cord, pharyngeal region, and in the tail lumbar ganglia, pre-anal ganglion and dorso-rectal ganglion, and the male sensory neurons; suggesting that the unc-119 mutants are defective in most of the nervous system. The expression of unc-119::lacZ and GFP shows that the unc-119 gene is expressed shortly before the comma stage in embryogenesis and continues during late embryonic and post-embryonic development (Maduro M, Pilgrim D,1995). In accompanying abstracts in this issue of WBG, we have shown that the dpy-20 may encode a novel transcription factor that affects transcription of various genes in the ventral cord motor neurons (Ali et al., Shibata et al., Ali et al. see abstracts in this WBG issue ) We have found that in the dpy-20(e2017) mutant background the expression of the unc-119::lacZ is reduced significantly in the ventral nerve cord motor neurons, with little or no affect on the staining pattern of the sensory neurons in the head or tail of the animals. In addition we have observed ectopic expression of the unc-119 reporter gene in the vulval region. These results suggest that the dpy-20 is affecting expression of the unc-119 reporter gene in a select class of motor neurons as there is no effect on the expression of unc-119 in the touch neurons and in the embryos. To test the effect of unc-119 mutation on the expression of dpy-20 gene expression, we have done an experiment in reverse, i.e. studied expression of the dpy-20::lacZ in unc-119(e2498) mutant background and found that there is a small effect of unc-119 mutant background on the expression of dpy-20. In the unc-119 mutant background, about 20% animals show reduced staining in the ventral cord motor neurons and also in the hypodermal cells.

Starr TVB et al. (1990) Worm Breeder's Gazette "A CANDIDATE SEQUENCE FOR THE GENE S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE IS LOCATED NEAR THE DPY-14 GENE"

Baillie DL, Starr TVB, Rose AM

[Worm Breeder's Gazette, 1990]

Using the technique of cross-species hybridization to C. briggsae we have initiated a search for coding elements contained within seven cosmids located near dpy-14. Fragments detecting conserved regions between C. elegans and C. briggsae were used to probe Northern blots and screen cDNA libraries. A total of ten conserved fragments detected seven cDNAs. To better align the genetic and physical maps of this region the conserved fragments, cDNA clones, the breakpoint of the deficiency hDf8, and the probes identifying sP1 and hP9 have been positioned on a restriction map of the region. Two cDNA clones, sP1-1 and sP1-3, also detected conserved bands between C. elegans and mammals. Partial cDNA clones of both were sequenced and the sequence data used to screen the EMBL sequence data base. The sP1-1 sequence did not detect any significant matches however, the sP1-3 sequence detected sequence similarities with the gene for S-adenosyl-L-homocysteine hydrolase (AHH) from rat(1) and D. discoideum(2). A comparison of the deduced amino acid sequence indicated that 60% of the amino acids between sP1-3 and AHH are identical; and, if conservative changes are considered the homology between sP1-3 and AHH is greater than 80%. Thus the high degree of similarity between sP1-3 and AHH suggests that the sP1-3 locus encodes the gene S-adenosyl-L-homocysteine hydrolase.