Guo X et al. (1988) Worm Breeder's Gazette "isolation and characterization of basement membrane collagen genes in C elegans."

Kramer JM, Guo X

[Worm Breeder's Gazette, 1988]

Basement membranes(BMs) are believed to be involved in tissue morphogenesis, maintenance of tissue architecture, as well as cell movement and attachment. Although BM components have been studied intensively at the biochemical level, there has been no means to study the functions of these components genetically. We are interested in identifying the genes that encode BM components and in performing molecular and genetic studies of the functions of BM in development. Using fragments of a mouse Type IV (BM) collagen cDNA as probes, we have identified two clones containing BM-like collagen genes (CH#1 and CH#2) from a C. elegans genomic sublibrary of collagen containing phage. The two genomic clones were isolated by screening the sublibrary with a fragment of the mouse Type IV collagen cDNA that does not contain any Gly-X-Y coding sequence and does not cross hybridize to other collagen genes. Under relatively high stringency conditions, CH#1 and CH#2 hybridize more strongly to the Gly-X-Y portion of the mouse Type IV cDNA than do any of the other C. elegans collagen clones. CH#1 and CH#2 have been restriction mapped and both genes span about a 6-7 kb region within the clones. The restriction maps are different from each other indicating that these two genes encode two distinct alpha polypeptide chains of Type IV collagen. Both clones were radioactive labeled and probed to Northern blots. The results show that both genes produce 5.5 kb long transcripts, similar in size to the mouse and human Type IV collagen transcripts, but much larger in size than the C. elegans cuticle collagen transcripts. CH#1 and CH#2 have been placed onto contigs by the MRC group. The genetic location of the CH#1 contig is not known. CH#2 has been mapped to linkage group III, about 100 kb to the right of the lin-12 locus. Several embryonic lethal genes are located in this region. We have examined mutants of these genes briefly. One of these genes, emb- 9, has temperature sensitive mutant phenotypes that could result from defects in BM. Morphologically, emb-9 appears very similar to let-2, a gene previously found to cause BM defects (J. Kramer and J. Priess). Two of the five emb-9 alleles are semidominant, suggestive of a structural protein like a BM collagen. emb-9 is being analyzed to determine if it is the CH#2 BM collagen gene, and this may provide the means to begin a genetic analysis of BM structure and function.

Huang Y-J et al. (1994) Worm Breeder's Gazette "From Ascaris to C. elegans: A Way to Study Gene Structure and Function"

Tobler H, Huang Y-J, Mueller F

[Worm Breeder's Gazette, 1994]

FROM ASCARIS TO C. ELEGANS: A WAY TO STUDY GENE STRUCTURE AND FUNCTION Huang Y-J., Tobler H. and Muller F., Institute of Zoology, University of Pribourg, Perolles, CH-1700 Fribourg, Switzerland

Matthews LR et al. (1997) Worm Breeder's Gazette "The zyg-9 gene encodes a component of the meiotic and mitotic spindles"

Thierry-Mieg D, Kemphues KJ, Matthews LR

[Worm Breeder's Gazette, 1997]

zyg-9 is a maternal effect gene required for cytoplasmic organization and microtubule function in the one-cell embryo. Mutations in zyg-9 result in disorganized meiotic spindles, failure of pronuclear migration, and a mitotic spindle that contains abnormally short astral microtubules. The mitotic spindle forms around the male pronucleus and fails to rotate resulting in an aberrant cleavage division. We have previously reported the cloning and initial molecular characterization of zyg-9. A Blast search revealed similarity between ZYG-9 and the human ch-TOG protein, the S. pombe p93DIS1 protein, and the STU2 protein of S. cerevisiae. ch-TOG shares 48% amino acid identity with a 230 residue domain which is tandemly repeated at the N-terminus of ZYG-9 (51% amino acid identity between repeats). p93DIS1 and STU2 are 32 % and 20 % identical to a region of ~160 AA within the same domain of ZYG-9. The function of this domain is unknown. ch-TOG also exhibits 40% identity with a second domain of ~250 amino acids within ZYG-9. The function of the ch-TOG gene is unknown, although it is over expressed in certain tumor cell lines (1) and its expression is higher in dividing cells than in quiescent cells. The product of the p93DIS1 gene, which is required for sister chromatid separation, localizes to the spindle pole body during mitosis (2). We have generated antibodies against 461 amino acids at the carboxy terminal end of ZYG-9 and shown that purified antiserum recognizes a protein of ~156 kD, the predicted size of ZYG-9, in wild type embryos. This protein is not seen in embryos of three of seven zyg-9 alleles examined. Immunofluorescence microscopy has revealed that ZYG-9 protein localizes to both the meiotic and mitotic spindles. It also localizes to the peripheral region of the poles of the embryo throughout meiosis. At metaphase and anaphase of meiosis I, ZYG-9 colocalizes with tubulin in the meiotic spindle, although its distribution is more diffuse than that of tubulin. Unlike tubulin, ZYG-9 localizes more intensely at the meiotic spindle poles. A similar pattern of localization is observed during meiosis II, although peripheral staining at the poles of the embryo is less pronounced. At the onset of the first mitotic division, ZYG-9 localizes to the newly duplicated centrosomes. Centrosomal localization persists throughout mitosis. Between late prometaphase and early anaphase ZYG-9 also localizes to the kinetochore region. This pattern of localization is observed in all mitotic cells in the developing embryo and in the mitotic cells of the gonad. Localization of ZYG-9 protein to the centrosome may be essential for the formation of the unusually long astral microtubules present in the mitotic spindle of the 1-cell embryo. References: 1.) Charasse, S., Mazel, M., Taviaux, S., Berta, P., Chow, T., and Larroque, C. (1993). Eur. J. Biochem. 243:406-413 2.) Nakaseko Y., Nabeshima K., Kinoshita K. And Yanagida M., (1996) Genes to Cells 1:633-644

Chen WI et al. (1994) Worm Breeder's Gazette "The Interacting Proteins of the C. elegans p21 Ras-Related Rho Subfamily"

Chen WI, Blanc J, Lim L, Yap SF

[Worm Breeder's Gazette, 1994]

We have previously reported the characterization of the C. elegans p21 Ras-related Rho subfamily (WBG 12(5): 75). We have also shown that the nematode CDC42 homologue complements the yeast cdc42 -1temperature sensitive lethal mutation (Chen et al., J. Biol. Chem. 268: 13280-13285, 1993). The human breakpoint cluster region (Bcr) gene product is a member of a group of GTPase-activating proteins (GAP) which act exclusively on this subfamily. A cDNA was isolated (using the Bcr-related region of the genomic clone ZC21 as probe) from C. elegans (Chen et al., J. Biol. Chem. 269: 820-823, 1994). The cDNA clone designated as CeGAP is distributed over a 22 kb genomic region across 3 genomic clones (F42H10 ,C04D8 and ZC21 ) and therefore larger than the Bcr gene defined in the genome sequencing project. The 5 kb insert has an open reading frame of 1438 a. a. which contains a domain with sequence similarity to the COOH-terminal GAP region of Bcr and other known GAPs of the Rho subfamily. It also contains a 'pleckstrin homology' motif present in many signaling proteins including GAPs and nucleotide-exchange factors. However the absence of other domains in human Bcr (kinase and exchange factor) suggests that CeGAP represents a distinct molecule. The Bcr-like domain of CeGAP exhibited activity not only on members of the C. elegans and human Rho subfamily, but surprisingly also on C. elegans Ras protein (let-60 ),human Ras and Rab3 A.CeGAP is therefore the first GAP acting on Ras-related proteins across different subfamilies. Together with the presence of the pleckstrin homology motif, our finding suggests a central and integrative role for CeGAP in a signalling pathway common to Ras and related proteins. Further analyses are underway; these include the isolation of the full length cDNA (as no stop codon is found in the 5' part of the cDNA) and production of antibody. Additionally, the sequence of the nematode rhoGDI (GDP Dissociation Inhibitor) cDNA (cm1 9a7 )encoding 194 a.a. reveals 39% identity compared with the human counterpart (204 a.a.). To assess its activity which requires post-translationally processed substrates, the three nematode p21 Rho proteins (CeRac1 ,CDC42 Ceand CeRhoA) are being expressed in Sf9 insect cells via the baculovirus expression system.

Fields CA et al. (1992) Worm Breeder's Gazette "DEAD and DEAH Box RNA Helicase Genes Identified by EST Sequencing"

Fields CA, McCombie WR

[Worm Breeder's Gazette, 1992]

The DEAD-box family of proteins includes RNA-dependent ATPases involved in processing both structural and messenger RNAs (Linder et al., Nature 337 (1989) 121-122). A number of DEAD-box proteins have been identified as translational initiation factors, including the mouse eIF-4 A1protein (Nielsen et al., NAR 13 (1985) 6867-6880)) and the recently-characterized ME31 Bprotein of Drosophila (de Valoir et al., Proc. Natl. Acad. Sci. USA 88 (1991) 2113-2117). The predicted translation product of EST D18 R(McCombie et al., this issue) is very similar to the C-terminal ends of these proteins, as well as other members of the DEAD-box family, suggesting that D18 Rtags an initiation-factor gene. EST D33 Falso matches these proteins. The DEAH-box family includes putative RNA helicases involved in spliceosome assembly, and hence presumably in splice-site selection (Burgess et al., Cell 60 (1990) 705-717). The maleless gene of Drosophila, which regulates dosage compensation in males by binding multiple sites on the X chromosome, is also a DEAH-box protein (Kuroda et al., Cell 66 (1991) 935-947). Two ESTs with high similarity to yeast PRP16 (Burgess et al.), maleless, and other members of the DEAH-box family have been sequenced. EST H51 Rtags the DEAH-box region of the protein, the putative RNA helicase domain, which is highly conserved between members of the family. EST H57 Rtags a region similar to the C-terminal ends of PRP2 (Chen, J. and Lin. R. Nucl. Acids Res. 18 (1990) 6447) and PRP16 ;the maleless protein is significantly less similar to the PRP proteins in this region than is the C. elegans protein. We do not yet know whether H51 Rand H57 Rtag the same gene. [See Figure 1]

Chen WI et al. (1993) Worm Breeder's Gazette "The p21 ras-related rho Subfamily from Caenorhabditis elegans"

Yap SF, Lim L, Chen WI, Blanc J

[Worm Breeder's Gazette, 1993]

We have chosen the nematode C. elegans as a model system for investigating the signal transduction pathway of the p21 ras-related rho subfamily. Using the coding region of the human rac1 cDNA as probe, a C. elegans mixed stage cDNA library (from Stratgene) was screened under low stringency conditions. Sequence analysis of the positive clones indicated that we have cloned the nematode rac1 ,designated as CErac1 (Chen et al., J. Biol. Chem. 268: 320-324,1993). The 1.6 kb cDNA clone contains a coding region of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. In collaboration with Dr. Coulson et al. at the MRC, UK, the cDNA was mapped to a position on chromosome IV close to cha-1 .The presence of rac1 isoforms is suggested by our genomic rac clone (derived from C. elegans genomic library screening using the same human probe and displaying 85% nucleotide sequence identity compared with CErac1 ),which is mapped to a different location on chromosome IV. The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kb), their expression is developmentally regulated being the highest level at the embryonic stage and decreasing dramatically during development. The glutathione-S-transferase/CErac1 fusion protein (46 kDa) expressed in E. coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity is stimulated by human n-chimaerin, a brain specific GTPase-activating protein for p21 rac. We are currently analyzing other positive cDNA clones derived from the rac1 screening. Preliminary results indicate that we have probably cloned other members of the nematode rho subfamily. In addition, the nematode rho GDI (cm19a 7)and bcr-like (CELZC21) cDNAs have been expressed in E. coli, and their specific activity towards the CErac1 proteins and the other nematode p21s is being determined. The conserved biochemical activity of CErac1 protein suggests that further characterization of these p21 s and their regulatory proteins using genetic analysis, e.g. dominant negative mutant, would be helpful in elucidating not only their role in the signal transduction, but also the biological function of their mammalian homologues.

Harfe BD et al. (1997) Worm Breeder's Gazette "Cetwist-like1, a novel basic helix-loop-helix protein that is active in the M lineage"

Harfe BD, Fire A

[Worm Breeder's Gazette, 1997]

The C. elegans genome project recently revealed the coding region for a novel basic helix-loop-helix (bHLH) factor that is similar to the twist family of transcription factors. In other systems (flies, vertebrates), the twist transcription factor is important in determining different types of muscle fate; twist protein is thought to interact with other muscle specific transcription factors to modulate muscle gene expression. To begin to understand the role of the new C. elegans bHLH gene we have done the following: 1. Identification and sequencing of a cDNA clone from a phage library. This indicates that the gene is expressed and allowed definitive assignment of the intron-exon boundaries. Inside the bHLH domain, the C. elegans gene shares 61% identity to insect twist and 59% identity to mouse and Xenopus twist (Fig.1). This identity is much closer to twist members in other species then to other C. elegans bHLH genes. The twist family is not well conserved outside of the bHLH domain, but all published twist genes have three distinct features. a) Each starts with two Met codons. b) Each has a small conserved domain just 3' of the bHLH domain. c) Each ends just after the 3' conserved domain. The C. elegans gene has none of these features. We are therefore tentatively calling the C. elegans factor Cetwist-like1. 2. Characterization of the regulatory sequences using lacZ and gfp gene fusions and tagging constructs. Both reporter fusions and gfp tagged constructs yielded a similar expression pattern (Fig.2). The Cetwist-like1 promoter is active in the M mesoblast in 3-fold stage embryos and in all M descendants until there are 16 such cells. Promoter activity is then turned off in the 14 M-derived body wall muscle cells but remains on in the two SM's and post-embryonic coelomocytes. Reporter expression can be seen in all the descendants of the SM's until the L4 to adult transition. In addition, reporter activity is seen in the 4 embryonic coelomocytes. Preliminary deletion analysis of the promoter has determined that there are distinct 5' signals for the M lineage and the coelomocytes. Future plans include making a mutation in the Cetwist-like1 gene and determining how this protein interacts with other muscle specific transcription factors like MyoD (Chen et al. Development 120:1631-1641, 1994) and CEH-24 (Fire and Harfe, WBG 13(4): 91, 1994).

Edgley ML et al. (1997) Worm Breeder's Gazette "New Balancers for LG II"

Riddle DL, Edgley ML, Turner CA

[Worm Breeder's Gazette, 1997]

We have been screening for new genetic balancers for the left arm of chromosome II as part of the C. elegans Genetic Toolkit Project, in cooperation with the Rose and Baillie laboratories. We seek to isolate rearrangements that balance to the left of dpy-10, perhaps extending to the end of the chromosome. The existing rearrangement mnC1 balances well from the right end of the chromosome to just left of dpy-10. The semidominant mutation dpy-25(e817) has been used successfully to isolate deficiencies in a relatively small region (Chen et al., Science 256: 240-243, 1992), but we wanted a balancer for generating overlapping deficiencies across the entire left arm. Irradiated dpy-10(e128) + / + unc-104(e1265) males were crossed with unc-85(e1414) rol-1(e91) hermaphrodites. Wild-type F1 cross progeny were isolated and the F2 screened for the absence of Rol-1 non-Unc-85 recombinant animals, indicating suppression of recombination. These screens yielded four balancers (mC4, mC5, mC6 and mC7) in 2048 mutagenized chromosomes, all marked with dpy-10. One of the balancers, mC6, has been found to balance well from rol-1 leftward to let-552, but balancing activity is reduced sharply between let-552 and sqt-2. Initial characterization shows mC6 to be homozygous-viable, and it is probably an intrachromosomal rearrangement. It is stable in crosses and reduces recombination frequency by roughly 98% across the balanced interval. We are using it now to isolate deficiencies in the balanced region. The remaining three balancers do not seem particularly useful to us for isolating deficiencies, since they don't suppress recombination adequately in the region of interest, but two of them are interesting for other reasons. mC7 may be a translocation, as it has relatively small broods and segregates large numbers of dead eggs. Homozygous mC7 animals are segregated at low frequency, and are invariably sterile. We have found that it balances rol-1 quite well (so far), but does not balance unc-85 at all. mC5 suppresses recombination between unc-85 and rol-1, but not nearly as well as mC6, and it alters segregation ratios. Balancer homozygotes account for 31% to 34% of the progeny from an mC5 heterozygote, while animals homozyous for the balanced chromosome, which was never subjected to mutagenesis, account for only 7% to 10%. We see this in both the original isolate (mC5/unc-85 rol-1) and in a constructed stock (mC5/unc-104 rol-1).

Sommer RJ et al. (1995) Worm Breeder's Gazette "Evolution of vulva formation: Part VII: Mutants of Pristionchus with extra or missing Pn.p-ectoblasts."

Sommer RJ, Sternberg PW

[Worm Breeder's Gazette, 1995]

A dramatic reorganization in the ventral cord of Pristionchus pacificus with respect to presumed ancestors involves cell death of P(1-4).p and P(9-11).p. The vulva is formed by P(5-7).p and the remaining ectoblast, P8.p, is not a vulva precursor cell. In a genetic screen for egg laying defective mutants we found mutants with increased or decreased numbers of Pn.p-ectoblasts. Some of the ped (for P-Ectoblast Determination) mutant phenotypes are described below. We have assigned these mutations to separate genes based on complementation analysis (except for ped-2). ped-l(sy312): Some of the posterior Pn.p ectoblasts, P(9-11).p, survive. This phenotype is variable. If the ectoblasts are present, they form vulva-like structures in the posterior body region in addition to the normal central vulva. AC ablation does not influence vulva differentiation of these celIs . ped-2(sy319): The central ectoblasts P(5-8).p are absent, whereas the hypl2 cell is present. We have observed a few embryonic cell deaths in the central ventral cord. However, this analysis is complicated by high embryonic lethality. More than 95% of the eggs are lethal, and most of the remaining larvae die as L1. In the few surviving larvae, the VC neurons (P(5-8).aap in wild-type) die, like the corresponding cells in the anterior and posterior body region. Altogether, the ped-2 phenotype resembles the C. elegans ceh-20 phenotype (Chen & Stern, C. elegans Mtg. 1995, pp20). ped - 6(sy345): P(3,4).p survive and become ectoblasts. This phenotype is highly penetrant. The two additional cells form vulva-like structures with one or two additional anterior invaginations. AC ablations reveal that in ped-6 not only P(3,4).p but also P(5-7).p differentiate in an AC independent manner, indicating that ped-6 is a multivulva mutation, possibly similar to lin-15. ped-5(sy344): P(3,4).p survive as in ped-6, but do not form vulva-like structures. A series of VPC ablation experiments indicate that P(3,4).p are vulva precursor cells. After ablation of P(5,6).p, a normal vulva can be formed by P(3,47).p. Thus, for the anterior region ped-5 reconstitutes the vulva equivalence group seen in C. elegans, indicating a homeotic transformation . ped-4(sy346): P8.p undergoes additional vulva differentiation in appr. 40% of animals. A weaker effect is present on P7.p also. AC ablation show, that both cell can differentiate in an AC independent manner. Thus, both cells of the original ventrolateral pair P7/8L,R are affected. In summary, we can use a genetic approach to identify molecular changes accounting for cellular diversity.

Morita K et al. (1998) Worm Breeder's Gazette "CeBRAM-2 that binds to DAF-1 is a negative regulator of daf-7 TGF-b pathway."

Ueno N, Shibuya H, Shimizu M, Morita K

[Worm Breeder's Gazette, 1998]

TGF-b family of growth factors regulate many aspects of cellular function. The signaling is common in diverse animal species from vertebrates to C. elegans. We have been interested in the signaling pathway of the TGF-b family. We reported about cloning of a full-length cDNA (0.9 kb) of CeBRAM-2, a daf-1 binding protein (abstract, p 250), at the international C. elegans meeting in 1997. Briefly, CeBRAM-2 has a 57% amino acid identity over the carboxy-terminal 60 amino acids of human BRAM2 (BMP receptor associated molecule) identified by yeast two-hybrid system. HA-tagged CeBRAM-2 transiently expressed in COS7 cells was found to bind typeI receptors, both ALK-3 (mammalian) and DAF-1 (C. elegans). CeBRAM-2;;GFP fusion protein was found to be expressed dominantly in amphid and pasmid neurons (e.g. ASI, ASK, ASJ, etc.). This expression pattern was similar to that of daf-1 (C. Gunther and D. Riddle, pers. comm.). Subsequently, the null mutant was created by Tc-1 deletion method. However, phenotype of the CeBRAM2 single mutant was not obvious. We report here the genetic interaction of the CeBRAM-2 null mutant with mutants in daf-7 TGF-b pathway by examining dauer formation of the double mutants. The result suggests that CeBRAM-2 is involved in the daf-7 TGF-b pathway in C elegans as a negative regulator. 20C 23C 25C genotype + CeBRAM2 + CeBRAM2 + CeBRAM2 daf-1 m40 14(137) 2(373) 66(185) 10(348) 100(317) 51(495) m402 61(144) 6(231) 94(220) 24(329) 100(481) 72(750) m213 92(318) 27(245) 95(440) 37(308) 100(614) 80(482) daf-7 e1372 60(210) 4(316) 98(295) 52(261) 100(493) 89(335) daf-11m47 8(154) 5(281) N.D. N.D. 56(1488) 16(2323) daf-14m77 7(247) 3(492) N.D. N.D. 47(1283) 38(1260) TABLE Percent dauer formation of CeBRAM2 and daf-c double mutants at various temperatures .The percentage of animals forming dauers is given, with the total number of animals counted given in parentheses. + indicate the daf-c single mutants, and CeBRAM indicates daf-c;CeBRAM-2 double mutants. These results show that CeBRAM-2 mutant has a week suppressor of DAF-c phenotype. CeBRAM-2 was not suppressed daf-14 SMAD, so CeBRAM-2 is hypostatic to SMAD, and epistatic to daf-7 TGF-b ligand and daf-1 type I receptor. Rescue experiment was done with a CeBRAM-2 genomic fragment, to daf-1 (m40);CeBRAM-2 double mutant. Approximately 80 % of transgenic progeny formed dauers at 25!C, suggesting that suppression of dauer formation in the double mutant was recovered. Together, we hypothesize that CeBRAM-2 functions as a negative regulator by binding TGF-b type I receptors like as FKBP12 (Y., Chen, J., Massague, EMBO J. 16 3866-3876, 1997).