Meneghini MD et al. (1997) International C. elegans Meeting "Morphogenesis defects in or34 embryos"

Bowerman BA, Thorpe CJ, Meneghini MD

[International C. elegans Meeting, 1997]

We have identified a maternal effect embryonic lethal mutation called or34. or34 embryos have a highly penetrant morphogenesis defect. These embryos produce mishapen clumps of well differentiated tissues and fail to elongate. One specific defect of or34 mutant embryos is the improper orientation of the mitotic spindle apparatus in an 8-cell stage blastomere called ABar. The ABar spindle orientation is of particular interest to us because a similar defect is caused by mutations in the mom genes which encode Wnt family signal transduction components. While the most obvious defect in the mom mutants is their failure to induce endoderm, this induction appears to occur normally in or34 mutant embryos. We are interested in the possibility that or34 is a weak allele of a new mom gene or defines a new gene required for a subset of processes under control of the Wnt pathway in the early embryo. or34 maps to the right arm of chromosome I. Complementation tests show that or34 is not allelic to mom-4 or mom-5 which also map to the right arm of chromosome I. We are currently pursuing a more detailed genetic and phenotypic analysis of or34.

Perry MD et al. (1995) International C. elegans Meeting "ENIGMA: DECIPHERING MASCULINE MESSAGES"

Wilson E, Extavour C, Perry MD, Rajwans N

[International C. elegans Meeting, 1995]

The her-1 gene on LG V is required for normal male development in X0 animals. Our hypothesis, based on data from genetic and molecular experiments, suggests one of the X0-specific products of this locus is secreted and functions cell-non-autonomously to specify male fates. However, this model does not encompass the locus smaller, more abundant, male-specific transcript comprised of the two P2 promoter-proximal exons. We are investigating whether this other her-1 transcript has a biological function in C. elegans. Phenotypically, mutations that abrogate exclusively the larger her-1 transcript appear indistinguishable from a null allele disrupting both transcripts. In contrast to the masculinizing mRNA, the conceptual translation product from the smaller transcript lacks an obvious secretion signal. To establish if the SL1-trans-spliced polyadenylated RNA is translated we are using anti-her-1 antisera to detect the predicted 10 kDa protein. In parallel, we will assay epitope-tagged variants of this putative protein in transgenic worms. Finally, demonstrating translation of the ORF in vivo will make it feasible to screen nematode cDNA expression libraries for potential interacting proteins.

Perry MD et al. (1991) International C. elegans Meeting "DECIPHERING HER-1'S SEXUAL MESSAGES"

Robertson B, Wood WB, Li WQ, Perry MD, Hageman JM, Ippensen DC, van Auken KM, Trent C

[International C. elegans Meeting, 1991]

The her-l gene, which is required for normal male sex determination, has been cloned using Tcl transposon tagging and shown to have two XO- specific transcripts (1). We are analyzing its functions by determining the structure of the gene and its products. We have sequenced approximately 8 kb of genomic DNA including the her-l gene as well as several cDNAs corresponding to the two transcripts. The first open reading frame in the rare 1.2 kb transcript is predicted to encode a 20 kD polypeptide whose hydrophobic amino terminus could be a secretion signal sequence. The relatively abundant 0.8 kb transcript is predicted to encode the Cterminal (approximately) half of the same polypeptide, with no signal sequence. RNAse protection mapping experiments of the region confirm our earlier evidence that the 1.2 kb and 0.8 kb transcripts include 4 and 2 exons, respectively. DNA- mediated transformation to rescue her-l (lf) mutations has been recalcitrant, and we are now testing smaller and smaller cosmid fragments in hope of removing noxious sequences. To define cisacting sequences in the her-l locus, we have fused different lengths of genomic DNA upstream of her-l to Andy Fire's lacZ constructs and injected them with a rol-6 plasmid. We see poor staining in the resulting Fl male rollers, suggesting that the 2.5 kb of DNA exarnined so far may be insufficient for expression. In an oblique attempt to identify essential elements of the her-l locus, we have used coding region probes in low stringency hybridizations to genomic DNAs and libraries from the male/female species C remanei and WS9-6 and have detected highly conserved fragments. Lack of definitive signals from parallel experiments on the hermaphroditic species C. briggsae suggests that even lower stringencies may be necessary. We are also continuing efforts to obtain anti-her-l antisera using bacterial fusion proteins.

Yandell MD et al. (1995) International C. elegans Meeting "EXPRESSION OF THE C. ELEGANS TGFb HOMOLOG CEG-1"

Wood WB, Yandell MD

[International C. elegans Meeting, 1995]

We have used degenerate PCR to clone a C. elegans homolog of the dpp/BMP2,4 subclass from the TGFb superfamily; we have located it on the left arm of LGV and provisionally called it ceg-1 (for C. elegans growth factor) (WBG 13 (4):65). Sequencing of genomic and cDNA clones has shown that the ceg-1 gene contains seven introns (previously incorrectly reported as 8) and encodes a 1.7 kb mRNA which is transspliced to SL1. In transgenic animals carrying a construct that includes 4.4kb of ceg-1 upstream sequence fused to lacZ, staining first appears in a quartet of cells at about the 100 cell stage of embryogenesis and by the comma stage is present in two rows of cells along the ventral midline. Pretzels and early L1s show staining in what appear to be neuronal and hypodermal nuclei in the head, the juvenile cells of the ventral nerve cord (VNC) and a single cell in the tail. By late L1 there are additional staining VNC cells, suspected to be the Pn cells which migrate ventrally into the VNC. In males, staining is also present in what may be the preanal equivalence group, the post-cloacal sensilla and the dorsal-rectal ganglion. Over-expression of ceg-1 in transgenic animals carrying a ceg-1-cDNA construct driven by a heat-shock promoter show a variety of phenotypes which are consistent with the ceg-1-lacZ expression pattern. Early heat shocks cause embryos to arrest at the comma stage, while later heat shocks produce severely constipated L1 larvae with marked hypodermal defects and notched heads. In progress are further identification of expressing cells, examination of ceg-1 overexpression effects on the male tail, and analysis of ceg-1 expression in Hox gene mutant backgrounds.

Perry MD et al. (1997) International C. elegans Meeting "DETECTION OF A HER-1-BINDING PROTEIN IN C. ELEGANS EXTRACTS"

Rajwans N, Perry MD

[International C. elegans Meeting, 1997]

The genetic pathway regulating C. elegans sex determination has a cell-nonautonomous step. We are testing an hypothesis that a small male-specific HER-1 protein is the secreted signalling ligand for this cell interaction. To initiate characterization of this novel cysteine-rich protein we generated a transgenic strain that overproduces HER-1 from an hsp-16 promoter. Following a masculinizing heat-shock, these hermaphrodites synthesize three new proteins, 16, 17, and 18 kDa, that cross-react with affinity-purified rat anti-HER-1 antibodies (processed HER-1 is expected to migrate at 18 kDa). In parallel, we engineered baculovirus vectors to express HER-1 in insect tissue culture cells. These cells secrete relatively high amounts of HER-1 into the culture medium (~ 2 ug/mL). This recombinant protein migrates slightly slower in SDS-PAGE gels, suggesting it may be post-translationally modified (e.g., N-linked glycosylation). We are now using standard fractionation techniques to purify these HER-1 proteins. A variety of arguments suggest that the 170 kDa transmembrane protein encoded by tra-2 is likely to be a receptor for her-1's signal. We used partially purified HER-1 to probe far western blots of N2 XX hermaphrodite protein extracts and detected an interaction with a ~170 kDa protein. We are exploring several approaches to identify this HER-1-binding protein.

Yandell MD et al. (1991) International C. elegans Meeting "PSORALEN CAUSES SMALL UNC-22 DELETIONS AT A HIGH FREQUENCY"

Wood WB, Edgar LG, Yandell MD

[International C. elegans Meeting, 1991]

We have analysed 23 trimethylpsoralen (TMP)-induced alleles of unc- 22 by Southern blot for RFLPs, using the probes DM17, DM18, DM20, DM22, SL13, and SL14 (many thanks to Carol Trent and Guy Benian!), which cover the entire coding region and about 11 kb of 5' upstream sequence. Eighteen of these mutations were obtained in a single mutagenesis, screening for both homozygotes and heterozygotes (mutation rate of lx10-4). In this mutagenesis, 15 strains were homozygous-viable, and 8 were originally homozygous-lethal. Two of these, however, were homozygous-viable after backcrossing; a third was successfully balanced over sDf22, and 5 were lost before analysis. Of the mutants analysed (the 18 remaining strains and an additional 4 homozygous- viable mutations isolated in previous TMP screens), 9 of the homozygous-viable mutants and the balanced homozygous-lethal mutant show visible polymorphisms within the unc-22 locus. All but one, which remains-ambiguous, were deletions ranging from 0.15 to 10 kb. The average deletion size was 2.2 kb; if the single 10-kb deletion is not included the average size was only 0.94 kb. Therefore, among mutations analyzed from the inclusive screen for both homozygotes and heterozygotes, 44% (8/18) were deletions of less than 10 kb in size. These data suggest that TMP produces small deletions at a higher frequency than any other mutagen in current use, which should be useful in generating mutations causing complete loss of function and likely to be associated with DNA polymorphisms.

Yandell MD et al. (1997) International C. elegans Meeting "SOME STATISTICS ON THE RAPID EVOLUTION OF C. elegans PROTEIN SEQUENCES"

Korf I, Yandell MD, Waterston RH

[International C. elegans Meeting, 1997]

One way to measure the relative rate at which a gene is evolving in two different organisms is to perform a relative rates test. This test uses the relative similarities of a group of orthologus sequences from three or more organisms to determine the relative amount of change in the sequences during the time elapsed since they last shared a common ancestor. C. elegans genes, for example, are often less similar to their orthologs in yeast, than are their counterparts from Drosophila and humans. Similar trends in the conservation of rRNA sequences have lead to the wide-spread belief that C. elegans belongs to a rapidly evolving lineage. There is as yet, however, little information available on how this excess divergence is distributed among different C. elegans proteins. Are all C. elegans proteins more or less equally divergent, or are some C. elegans proteins actually more conserved than their counterparts in other, more slowly evolving, animals. In an attempt to get at this and some related questions, we are examining the relative conservation of several gene families for which there exist orthologs, as determined by bootstrap analysis or synteny, in C. elegans, in a second triploblastic animal, and in a third, more distantly related, organism such as hydra or yeast. These data will then be broken up by gene class and function in the hope that such comparisons will reveal some interesting trends in the rates of evolution of different gene families in different organisms. Finally, EST-sequence data from the nematodes C. elegans, C. briggsae and P. pacificus will also be subjected to a similar analysis in order to examine the variation in the rate of sequence evolution within the nematodes.

Yandell MD et al. (1993) International C. elegans Meeting "Back through the eye of the needle: TGF-b's and reverse genetics."

Perry MD, Yandell MD, Edgar LG, Wood WB

[International C. elegans Meeting, 1993]

Meneghini MD et al. (1995) International C. elegans Meeting "or50: a dominant, extragenic supressor of the maternal C. elegans gene skn-1?"

Martin PR, Meneghini MD, Bowerman BA

[International C. elegans Meeting, 1995]

The product of the maternal skn-1 gene is required for specifying the identity of one 4-cell stage blastomere called EMS. In embryos that do not receive skn-1 (+) gene product, EMS instead develops more like its sister P2, ultimately resulting in dead embryos. In a genetic screen for dominant suppressors of a weak but non- conditional allele of skn-1, zu129, we identified one animal out of about 100,000 in which an apparently homozygous zu129 mother produces all viable embryos. Preliminary studies suggest that the suppressor, or50, is extragenic but on the same chromosome as skn-1 (LGIV). Experiments are underway to determine if the suppressor is allele specific and if it has a phenotype in a skn-1 (+) background. We are also sequencing the skn-1 allele in the suppressed strain to verify that the original mutation (a stop codon) is still the only skn-1 mutation present.

Perry MD et al. (1993) International C. elegans Meeting "her-1(tss) alleles in the P1 promoter: a novel mechanism of temperature sensitivity."

Chamblin C, Trent C, Wood WB, Robertson B, Perry MD

[International C. elegans Meeting, 1993]