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

Tawe WN et al. (1997) Worm Breeder's Gazette "Paraquat mediates differential gene expression in C. elegans"

Henkle-Duehrsen K, Tawe WN, Eschbach M-L, Walter RD

[Worm Breeder's Gazette, 1997]

In order to identify genes that are differentially expressed as a consequence of stress due to paraquat, we used the differential display technique (1) to compare mRNA expression patterns in Caenorhabditis elegans. A C. elegans mixed-stage worm population, as well as a homogeneous larval population were treated with 100 mM paraquat, parallel to controls. Over 50 mRNA species that are potentially up-regulated in response to paraquat were identified. Sixteen of these candidates were re-amplified, ligated into plasmid vectors and the nucleotide sequences were determined. The induction of four of these expressed sequence tags (ESTs) designated L1, M47, M96 and M132 were confirmed in two independent stress / differential display experiments, as well as by northern blot analysis with RNA from stressed and unstressed worms. The nucleotide sequences of the independently isolated L1 and M47 ESTs were found to be identical. Their corresponding mRNA level increased more than 40-fold in the larval stage, and to a lesser extent in the mixed-stage worm population, in response to paraquat. Induction levels of 3 - 5 fold were observed for the M132 and M96 ESTs, in both larval and mixed populations. All of the isolated ESTs showed homology to portions of the C. elegans cosmids. The L1/M47 EST is derived from a gene encoding one of the putative C. elegans glutathione S-transferases. The paraquat-inducible M132 EST was also identified, and encodes a putative C2H2 - type zinc finger protein, possessing an N-terminal leucine zipper. However, the M96 EST appears to be a novel gene whose product has not yet been identified. Searches of other eukaryotic nucleotide sequence databases did not reveal any significant homologies to known sequences from any organism. Since paraquat is known to generate superoxide radicals in vivo, and the cellular superoxide dismutase (SOD) enzyme complex is responsible for the quenching of the deleterious effects of these radicals, the response of the C. elegans superoxide dismutases (2,3,4) to paraquat was also investigated in this study. Northern blot experiments demonstrated that mRNA steady state levels of the C. elegans manganese type and the copper/zinc type superoxide dismutases increased two-fold in response to paraquat, in the larval population. In contrast, mixed-stage populations did not show any apparent increase in the levels of these SOD mRNAs in response to paraquat. References: 1. Liang, P., Pardee, A.B. (1992). Science 257: 967 - 971 2. Giglio, M-P., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994). Biochem. Mol. Biol. Int. 33 (1): 37-40 3. Giglio, A. M., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994) . Biochem. Mol. Biol. Int. 33 (1): 37-40 4. Larsen, P.L. (1993). Proc. Natl. Acad. Sci. USA, 90: 8905-8909

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

Maduro MF et al. (1996) Worm Breeder's Gazette "eDf2 is not a terminal deletion"

Pilgrim DB, Maduro MF

[Worm Breeder's Gazette, 1996]

The LGIII-derived aberrations eDf2 and eDp6 were obtained concomitantly after acetaldehyde mutagenesis(1). The left-hand eDf2 portion (approximately two-thirds of LGIII) behaves genetically like a terminal deletion, and the remaining portion of LGIII formed the free duplication eDp6, which may be circular (Hunter and Wood, WBG 10#2, p.150). eDf2 complements mutations to the left of unc-119, and eDp6 complements all those to the right. We have previously shown that animals homozygous for eDf2 and eDp6 display the same phenotype as unc-119 mutants, and that eDf2; eDp6 animals transgenic for unc-119 rescuing clones are indistinguishable from wild type, consistent with unc-119 being the only gene affected(2). It is of interest to understand the nature of these aberrations, perhaps as a model for understanding the healing of chromosome breaks in C. elegans. Our previous long-range restriction analysis has shown that there are at least 100 kbp of DNA that have been added beyond the eDf2 breakpoint (WBG 13#2, pp.64-65), suggesting that the healing of the break involved the addition of a large amount of DNA, perhaps from the original right end of LGIII, either as a direct splice or as a result of a recombination event.

McKim KS et al. (1990) Worm Breeder's Gazette "Genetic mapping of spontaneous duplications."

Rose AM, McKim KS

[Worm Breeder's Gazette, 1990]

We have previously shown that duplications spontaneously break and produce new duplications at frequencies ranging between 10+E-4 and 10+E-5 (CSH abstracts 1989 p. 203). We have complementation tested a set of these duplications which break between unc-11 and dpy-14 to mutations in the region. A map of the results is shown in A and B. The spontaneous duplications are indicated by an asterisk. The arrangement of the markers is based on all the duplications except those derived from hDp23. With the exception of him-1 (see accompanying note), there has been no conflicting results. Duplications derived from hDp4 and hDp23 are unique in that they have multiple deletions. The other spontaneous duplications represent terminal deletions. hDp4 was derived from sDp2 by gamma irradiation. hDp78 is a spontaneous derivative of hDp4. hDp78 has three deletions with respect to hDp4, a deletion around unc-57, another around spe-11 and the third in the dpy-14 region. A single deletion event could produce both the unc-57 and dpy-14 deletions if hDp4 was a ring chromosome. The precedence for this conclusion comes from the analysis of eDp6 by Hunter and Wood (Gazette 10(2): 150). To explain the occurrence of three deletions, however, we must superimpose an inversion on the ring chromosome as shown in Figure C. [See Figure 1] hDp23 was a spontaneous derivative of hDp3. The four derivatives of hDp23, hDp26, 27, 30 and hDp59, all have complex structures (Fig. A and B). All of the duplications have a deletion around unc-63 that does not include let-359. Two of the duplications still carry spe-11 ( +) and hDp30 also has a dpy-14 deletion. This complex structure may reflect an unusual structure of hDp23 but unlike hDp4, we have not been able to derive a structure consistent with the results. The mapping data from all of our gamma induced duplications has been consistent. While most of our spontaneous duplications are also consistent in mapping experiments, we stress the importance of using caution when interpreting the mapping data derived from spontaneous duplications (see also accompanying note ). [See Figure 2]

Maduro MF et al. (1994) Worm Breeder's Gazette "The Breakpoints of eDf2 and eDp6 and the Physical Mapping of unc-119"

Pilgrim DB, Maduro MF

[Worm Breeder's Gazette, 1994]

The strain CB1517 is homozygous for the LG III fragments eDf2 and eDp6 and has a phenotype similar to unc-119 (e2498 ),isolated as a spontaneous recessive Unc in an N2 /RW7000background (see WBG 11#4, p.82). Neither eDf2 nor eDp6 complement unc-119 mutations, suggesting that this gene is defective in CB1517 .Our approach toward analyzing the nature of the lesions of eDf2 and eDp6 is based on the hypothesis that the suspected common breakpoint of eDf2 and eDp6 lies within unc-119 .The "gap" is bridged by the YACs 39A1 (see Donna Albertson, Genetics 134: 211-219) and 60D9. By using pulsed-field gel electrophoresis and standard genomic Southerns, we were able to construct a long-range physical map of the region. The size of the overlap of the YACs 39A1 and 60D9 is about 100 kB. Cosmid T27G1 ,which cross-hybridizes to 60D9 and 39A1, picks up a size polymorphism (edP1 )associated with eDf2 ,which suggests that it contains the breakpoint. In order to obtain more clones in the region, we randomly subcloned Sst I fragments of PFG/LMP-purified 39A1 and 60D9. One of these clones, DP#MM008 ,contained DNA which cross-hybridizes only weakly to genomic DNA from CB1517 and which picks up a size polymorphism (edP2 )associated with e2498 .A mut-6 (st702)-inducedphenotypic revertant of e2498 has lost this ~2.4 kB insertion, suggesting that the DP#MM008 clone falls between the breakpoints of eDf2 and eDp6 and contains at least part of unc-119 ,and that e2498 results from the insertion of a transposable element. Using this clone we constructed a short-range restriction map of the region around unc-119 and located the breakpoint of eDf2 and the insertion in e2498 to within 1 kB. We do not know the physical distance between DP#MM008 and T27G1 ,but it cannot be more than 100 kB, since T27G1 contains an end of 40C2. Since the breakpoint of eDf2 lies in DP#MM008 ,and T27G1 also picks up edP1 ,the end of eDf2 may be rearranged with respect to N2 .As well, eDp6 may be circular (see Hunter and Wood WBG 10#2 p.150).

Pilgrim DB (1990) Worm Breeder's Gazette "Unc-119"

Pilgrim DB

[Worm Breeder's Gazette, 1990]

In the course of making N2/B0 recombinants for another reason, two spontaneous mutations popped up on successive days from a N2/RW7000 background. One was a new dpy-20 allele (e2480), the other a strong Unc(e2498). After 10 backcrosses to N2, the phenotype of the Unc remained strong (there is no new Tc1 band on a Southern blot of e2498). It mapped to LGIII, left of dpy-18, right of sma-2, inseparable from vab-7, was uncovered by eDf2, and complemented unc-25, -47, -49, -71 and -81. The phenotype did not change over eDf2. Since this data did not correspond to the position of any known Unc gene, I assigned e2498 to a new gene, unc-119. Since then, I have shown that e2498 is not complemented by eDp6.e2498 is a severe Unc, capable of very little body movement. It is slightly dumpier than wild-type, a coiler, assuming a 'J' shape on the plate. Pharyngeal pumping is normal, and the head seems to be capable of more movement than the rest of the body. e2498 is neither ts nor cs, not Egl, and the phenotype seems equally severe throughout development. e2498 maps very close to the breakpoint of both eDf6 and eDp2. Jonathan Hodgkin reminded me that the original strain carrying eDf2 and eDp6, both of which presumably resulted from the same acetaldehyde mutagenesis event, was isolated as a strong Unc with aberrant segregation (Hodgkin, Genetics 96:649, 1980). Possibly, the chromosomal breakpoint was in or near an Unc gene (generating a ring chromosome [Hunter and Wood WBG 10#2 p150]?). I found that the phenotypes of the two strains (e2498 and eDf2/eDf2;eDp6), were very similar, although eDf2/eDp6 may be slightly dumpier. There is no reason to think that e2498 is a large chromosomal lesion, but it seems surprising that a 'simple' allele of it has not been isolated before. Obviously if this is the lost eDf2/eDp6 Unc, it should be relatively easy to isolate by looking in the region for the chromosomal breakpoints.

Hedgecock EM et al. (1989) Worm Breeder's Gazette "more gonadal cell migration mutants."

Hedgecock EM, Hall DH, Culotti JG, Kowalkowski K

[Worm Breeder's Gazette, 1989]

The complex trajectories of the hermaphrodite distal tip and male linker cells can be analyzed as successive segments of constant direction. These directions are usually along a natural axis of the body wall although an oblique movement of the male linker cell (top right panel) may result from superimposed ventralward and posteriorward migrations. Observations of selective loss-of-function tra-1 mutants and of tra-1 mosaics (C. Hunter and W. Wood, pers. comm.) suggest that the body wall carries only simple, static guidance cues common to both sexes and that complex gonadal cell trajectories are programmed cell autonomously, perhaps by changing cell receptors for these spatial cues. mig-7(X) mutants may disturb the temporal program of gonadal cell migrations. In mig-7 hermaphrodites, the distal tip cells fail to turn dorsalward near L3 lethargus but continue longitudinally along the ventral muscles. In males, the linker cell fails to turn ventralward near L3 lethargus but stops migrating. A possible, though hardly compelling, interpretation is that these cells repeat the navigational program of the previous larval stage, i.e., hermaphrodite distal tip cells continue longitudinally while male distal tip cells attempt to turn dorsalward. Interestingly, occasional seam cells in both existing mig-7 alleles divide near the L4 molt. This phenotype is characteristic of lin-14 gain-of-function mutants that repeat aspects of early larval stages ( Ambros and Horvitz, 1984). We are currently testing whether the mig-7 mutants are, in fact, novel lin- 14 alleles that repeat some L3 events during the L4 stage. In mig-8(X) mutants (not shown), the hermaphrodites distal tip cell trajectory resembles the mig-7 trajectory while the male linker cell trajectory is normal. Distal tip cells in dpy-24(I) hermaphrodites usually turn dorsalward precociously and then continue centrifugally along the dorsal muscles. Often these cells reverse direction near the L3 molt suggesting that longitudinal movements may follow a normal temporal program while circumferential movements are misregulated. Male linker cell trajectories appear normal. As discovered by others, the linker cell migrations in him-4(X) males, and in a new mutant mig(rh155), are abnormal while the distal tip cells in hermaphrodites appear unaffected. In summary, several genes affect the trajectories of either the hermaphrodite distal tip cells, the male linker cell, or both. Some genes, e.g., unc-5 and unc-40, appear to affect directional cues while other genes may affect the temporal program. Double mutants could confirm some of these interpretations and suggest explanations for more exotic cell trajectories. [See Figure 1]

Benian GM et al. (1986) Worm Breeder's Gazette "a portion of unc-22 is homologous to protein kinases."

Moerman DG, Waterston RH, Mori I, Benian GM

[Worm Breeder's Gazette, 1986]

Using a probe from the 3' end of the unc-22 region, a partial cDNA for unc-22 was isolated from Barbara Meyer's lambda gt10 cDNA library. The insert is a 1.6 kb Eco RI fragment which lacks a poly A tail. By comparing this cDNA to an unc-22 genomic clone, we have determined that the cDNA lies approx. 1.5-2.0 kb upstream of the probable 3' end of the unc-22 14 kb mRNA. Both strands of this cDNA have been sequenced and an open reading frame in the direction of transcription with the biased codon usage described by Blumenthal et al. (WBG 8(1) :36) has been found. To our surprise, a search of the protein sequence data bank revealed homology to protein kinases. This portion of unc-22 is roughly 30% homologous to a stretch of about 260 amino acids comprising the catalytic domains of 3 known serine-threonine protein kinases and 10 tyrosine kinases of oncogenes (Hunter and Cooper, 1985, Ann. Rev. Biochem. 54:897). A portion of this region is shown in figure 1a. Most residues (18/24) conserved in all protein kinases are found in unc-22 and these are marked with arrows. Among these is the ala pro glu trio, likely to be important for catalysis. unc-22 also shows strong homology to the presumed ATP binding domain, which is found at the amino terminal portion of the 260 residue region (see figure 1b). This includes a lysine (marked with 2 arrows), which in the v-src protein and bovine cAMP dependent protein kinase can bind an ATP analog resulting in loss of kinase activity. In unc-22, 16 residues to the amino terminal side of this lysine is the sequence gly X gly X X gly (marked with arrows in figure lb). This glycine block is found in an analogous position in all known protein kinases and has also been observed in a number of other nucleotide binding proteins. In addition, unc-22 sequence has been analyzed from three Tc1 insertion sites and a 1.1 kb restriction fragment. A total of 4.3 kb, distributed over an 8 kb region, has been accumulated (see figure 2). We have found seven examples of a 91 residue segment. This repeat is found at the amino acid but not the DNA level. For 3 of these repeats, homology extends further, although less strongly, for a total of about 180 residues. Spacing between repeat units varies from 9 to 73 amino acids. Interestingly, 2 of these repeats bracket the putative kinase. Within the most highly conserved first 91 residues of the repeat, the same 29 residues are found in nearly the same position in at least 4 of the 7 repeats and 7 out of 29 of these residues are the same in all the repeats. Eight of the 29 conserved residues are proline. This proline richness (about 9% overall) seems to rule out alpha helical structure. No sequence homologies to known proteins were found.