Chen W et al. (1995) Worm Breeder's Gazette "The Caenorhabditis elegans Small GTP-Binding Protein RhoA Is Enriched In The Nerve Ring And Sensory Neurons During LarvalDevelopment"

Chen W, Lim L

[Worm Breeder's Gazette, 1995]

The Caenorhabditis elegans Small GTP-Binding Protein RhoA Is Enriched In The Nerve Ring And Sensory Neurons During Larval Development

Savage C et al. (1997) Worm Breeder's Gazette "THE SMALL SCREEN GETS SMALLER."

Savage C, Padgett RW, Krishna S, Cohen S

[Worm Breeder's Gazette, 1997]

We are interested in identifying new components of the TGFb pathway. Analysis of the sma-2, sma-3, and sma-4 genes revealed that they are Smads, which appear to be the primary TGFb signal transducers. They share mutant phenotypes with a subset of phenotypes exhibited by mutations in daf-4. Interestingly, daf-4 is involved in sending two signals, the dauer signal, and the Small/Mab signal. Each of these pathways utilizes a unique set of Smads. Our primary approach has been a series of genetic screens for mutations that are similar to mutations in daf-4, a TGF type II receptor. Since four genes in the Small/Mab pathway mutate to a small body size, we have hypothesized that other genes in the pathway might mutate to similar phenotypes. We have screened 17,000 genomes for additional Small mutants. Most mutants are now mapped and complemented. In this screen, we expected to find a ligand and a type I receptor for the Small/Mab pathway, since these components were not identified when the search began. Three alleles of sma-6 have been obtained in this screen, which we previously identified as a new type I receptor in this pathway. We have also generated an allele of dbl-1, a ligand for the Small/Mab pathway (see abstracts by Y. Suzuki and B. Wood). Of the remaining Sma mutants, there are five loci which are of continued interest, because of their classical Sma phenotype and the presence of multiple alleles. Two non-allelic loci from this group also have fusions of tail rays, in patterns similar to mutations of other small loci. The genes corresponding to these loci are being cloned and further characterized.

Politz SM et al. (1983) Worm Breeder's Gazette "preliminary identification of cuticle collagen precursors in vivo."

Politz SM, Politz JC

[Worm Breeder's Gazette, 1983]

As discussed at the meeting, we've been looking for small collagens in vivo because we believe that the collagens isolated from cuticles ( apparent MWs from 60 to over 200 kd) are cross-linked aggregates of numerous smaller primary gene products varying from about 35 to 50 kd in apparent MW. The collagen genes found by Jim Dramer and Joe Cox are small, the vast majority of worm RNAs that hybridize to a chick collagen cDNA probe are small and collagenase sensitive in vitro translation products obtained from RNA isolated from molting worms are small. Using a Western blotting technique, we have now found small collagens present in worms molting from L4s to adults. In the Western blotting technique that proved useful for us, proteins are electrophoretically separated on an SDS gel, electroblotted to diazotized (DPT) paper and reacted with antibody in situ. Bound antibody is then detected using [125I]-labelled protein A. We screened the sonication supernatant, SDS soluble, and SDS- ME soluble (cuticle) fractions from worms isolated either at the peak of the L4 to adult lethargus or as very young adults. The antiserum used was directed against the soluble fraction of the adult cuticle. This serum contains antibodies that bind to all the adult cuticle collagens (as well as the L4 cuticle collagens) and also cross-reacts to some extent with vertebrate collagen, probably type IV. Small collagens ( identified by their collagenase sensitivity) were most abundant in the sonication supernatant but were also detectable in SDS fractions. These collagens can be concentrated further in a 12,000 X g pellet of the sonication supernatant. We don't understand this yet. The small collagens identified have several properties consistent with their being cuticle precursors. Firstly, they have molecular weights similar to collagens translated in vitro from RNA isolated from molting worms and could be coded for on genes approximately the size of those found by Kramer and Cox. Secondly, they bind to antibody present in an antiserum made to soluble adult cuticle; this cuticle fraction contains the characterized cuticle collagens. Thirdly, and most suggestively, the complement of small collagens present changes during the L4 to adult molt in a way very similar to the collagens translated in vitro from RNA isolated at similar times during this molt. At lethargus peak, small collagens of about 46 to 52 kd predominate, but at the end of the molt, the major small collagens are about 36 and 39 kd in size. This mimics the size difference seen between the collagenase sensitive translation products of RNA isolated near the beginning and end of the molt. We are now further characterizing the small collagens present during the L4 to adult molt to determine if they are, in fact, probable cuticle precursors. If we are looking at primary cuticle gene products, our hope is to begin to screen the precursor population present in morphological cuticle mutants to identify those with possible structural gene mutations.

Maduzia LL et al. (1998) Worm Breeder's Gazette "What Makes My Worms So Small?"

Cohen S, Padgett RW, Maduzia LL

[Worm Breeder's Gazette, 1998]

In C. elegans, two well described TGFb signaling mechanisms, the Sma/Mab and the dauer pathways, have been described. Both pathways share a common type II serine-threonine kinase receptor, DAF-4, which transmits each TGFb signal by associating with different type I receptors, SMA-6 or DAF-1. These distinct receptor complexes can then transduce signals sent by the ligand to the downstream effectors, the Smads, specific for that particular pathway. Mutations in any one of the components of the Sma/Mab pathway leads to small body size, male tail ray abnormalities and crumpled spicules. Previously, we have identified several novel mutants from a screen done in our lab based on small body size. This analysis reveals five new loci - sma-9, sma-10, sma-11, sma-12 and sma-13. All of them are phenotypically small, similar to sma-2, -3, -4, -6 and daf-4 mutants, and complement these existing TGFb signaling components. We are focusing on sma-10 because male tail fusion patterns are similar to those seen in other small mutants. Interestingly, sma-9 has a similar mutant phenotype (C. Savage-Dunn, personal communication). These two genes may represent general TGFb signaling components or regulators. We are currently in the process of cloning sma-10 to identify its role in TGFb signaling.

Adams A (1999) Worm Breeder's Gazette "Philadelphia's Northeast High School Worm Project experiment on the space shuttle Discovery"

Adams A

[Worm Breeder's Gazette, 1999]

We recently had a chance to do something only a handful of people have ever done: send an experiment up into space. We sent up an 'experimental' group of dauer larvae on the space shuttle Discovery's ten-day mission. ITA of Exton, PA, granted us a very small space on the shuttle. So we decided on the relatively small C. elegans. We figured since John Glenn was going up into space to see the effects on him, we'd do a similar study. We wanted to see if the conditions of space had any effect on the C. elegans normal 18-24 day life span. With every science experiment there is a control group and an experimental group. The control group of about 1000 worms stayed in Philadelphia at Northeast High School the other 1000 worms were blasted off into orbit October 29, 1998.

Krishna S et al. (1996) Worm Breeder's Gazette "TGF-B Superfamily Signaling in C. elegans"

Krishna S, Padgett RW

[Worm Breeder's Gazette, 1996]

Previous work examining the transduction of TGF-B superfamily signal at the level of the membrane has resulted in the formulation of a general model by which this may occur. Transmembrane receptors which interact specifically with TGF-B superfamily ligands have been isolated and have been demonstrated to be required for proper transduction in several organisms. A subset of these receptors contain a serine-threonine kinase domain, and can be categorized into two classes, type I and type II, which differ considerably with each other in their non-kinase regions. It is thought that the mature protease-cleaved dimeric ligand interacts with appropriate type II receptors followed by the formation of an oligomeric complex with specified type I receptors. This complex then conceivably transduces the appropriate signal to downstream molecules, eventually resulting in intracellular changes and/or altered expression patterns of specific gene targets. In the worm, the dauer and small pathways are examples of mechanisms controlled by TGF-B like ligands. In the dauering pathway, the products of daf-l, daf-4 are receptors and serve to transduce the signal. Additionally, the dwarfins comprise a group of genes which encode products that have been implicated in the TGF-B transduction pathway in the nematode (Savage et al., PNAS 1996; Savage et al., WBG 1995) and in Drosophila (Sekelsky et al., Genetics 139: 1347-1358, 1995) . In the worm, the sma-2, sma-3, and sma-4 genes are dwarfins that have been characterized by our group. The mutant phenotypes of these genes are a subset of those exhibited by daf-4. However, the sma phenotypes are not observed in other dauer mutants. Thus, daf-4 intersects the dauering and small pathways. Since daf-l does not exhibit the small phenotype, we hypothesized that there should be another type I receptor transducing a TGF-B-like signal to the sma gene products, that when mutated, results in small animals. Using molecular tools, we had identified tre-1 as a novel type I receptor in C. elegans. tre-1 demonstrates a large degree of homology with other type I receptors, particularly the thickveins gene in Drosophila. Searches of extant mutations in the region of tre-1 indicated that sma-6 was nearby. Given that four genes mutate to a small phenotype, other genes in the pathway may also yield the same phenotype. Based on these data, the possibility was raised that sma-6 may encode tre-1. Germline transformation experiments confirmed this hypothesis, whereby a 9kb genomic fragment containing the tre-1 gene and flanking regions extending into, but not encompassing, adjacent predicted transcripts was sufficient to rescue sma-6 mutants and revert F1 transformed progeny to wild-type length. We are currently continuing to characterize the expression of tre-1 and analyze interactions with other components of the small pathway. In addition, we have isolated superfamily ligands in the worm, including tig-1, tig-2, and tig-3 (Krishna, WBG, 1995). We are seeking mutations in the respective regions, and we are continuing to characterize these genes. These results help us to define the specificity of TGF-B signaling by demonstrating that different type I receptors are able to elicit varying biological responses. Furthermore, we have completed a large genetic screen in order to identify other mutations that yield small animals. Our results with sma-6 validate this screen, and we hope to isolate other novel components of the pathway. Based on these results, we are also examining sma-5 and sma-8 for potential participation in the signaling pathway.

Savage C et al. (1996) Worm Breeder's Gazette "Genetic Screens to Elucidate the TGF-b Signaling Pathway"

Savage C, Cao G, Cohen S, Padgett RW, Collins D, Krishna S, Patel S

[Worm Breeder's Gazette, 1996]

We have previously described our analysis of a TGF-b pathway regulating body size in C. elegans. This pathway includes the TGF-b type II receptor DAF-4, the type I receptor SMA-6, and the homologous downstream components SMA-2, SMA-3, and SMA-4. We continue to be interested in identifying novel components of this signaling pathway. To this end, we have been conducting a series of genetic screens. Here we present an update on two of these screens. I. The Small Screen At least four TGF-b signaling components have been identified by their Small phenotypes: the downstream components encoded by sma-2, sma-3, and sma-4, and the type I receptor gene sma-6. To identify other components of the pathway, it seemed reasonable to begin by isolating Small mutants, since this type of screen had not been done systematically before. We therefore screened F2 progeny of EMS-mutagenized N2 worms, and isolated 85 Small mutants from ~17,000 mutagenized genomes. We are characterizing these mutations by first doing a complementation test with the triple mutant sma-4 sma-3 sma-2. Alleles that complement this chromosome are being mapped to chromosomes by STS mapping, using described Tc1 polymorphisms. Then, these alleles are being placed into complementation groups. Fine structure mapping, analysis of male tail phenotypes, and eventually molecular characterization will follow. Alleles that fail to complement known genes include: 2 alleles of sma-1, 38 alleles of sma-2, sma-3, or sma-4, and 1 allele of sma-6. An additional 20 alleles have been mapped to chromosomes and are being placed into complementation groups. II. Suppressors of sma-6 Mutations in sma-6 presumably result in a loss of signaling activity of the type I receptor in the pathway and hence yield the small phenotype. The CB1482 sma-6 strain was screened for suppressors in the pathway, which were identified by wild-type or long phenotypes. The reasoning behind the screen was derived from two possibilities. First, components downstream of the receptor in the pathway, when constitutively active, may be able to circumvent the requirement for full sma-6 receptor signaling potential and thereby restore transduction. In addition, negative regulators of the pathway, when mutant, may revert small animals via higher signal throughput. It is reasonable to argue that mutations which augment the level of signaling in the pathway would be easier to generate against a weak mutant background. Therefore, the CB1482 (obtained from the CGC) strain was used because it is a relatively mild sma-6 mutant biochemically and phenotypically. However, a practical consideration to this approach is the increased difficulty in the identification of suppressed animals. We screened through approximately 19k genomes and obtained nine suppressors, which we are currently mapping and characterizing. Additionally, we are performing the screen with a stronger allele of sma-6.

Han M (1992) Worm Breeder's Gazette "Searching for ras-like Genes in C. elegans"

Han M

[Worm Breeder's Gazette, 1992]

How many ras proteins are there in C. elegans? This has been a common question asked to us since the let-60 gene was shown to encode a ras protein. A high degree of identity in overall structure and a substantial functional overlap may be required for a ras-like small GTPase to be called ras. In worm, loss-of-function in let-60 ras abolished its roles in vulval cell fate specification as well as in early larval growth. It is thus unlikely that C. elegans has another ras protein which has the redundant functions of let-60 ras. Similar words can be said about the Drosophila ras-1 gene. A few years ago, three Drosophila "ras " genes were identified, but ras-2 and ras-3 differ in overall structure from ras-1 and mammalian H-ras significantly and may only be called ras-like small GTPase. The Drosophila ras-3 has been renamed rap-1 . Some years ago, ras homologs in C. elegans were searched for with a mammalian ras gene as probe. Two candidates were identified and named ras-1 and ras-1 (marked somewhere in the physical map), even though they are not very homologous to ras. To investigate the roles of other ras-like small GTPase during C. elegans development, we started to search for let-60 ras homologs. Since ras-like proteins are conserved in their GTP/GDP- binding domain and the so called "effector domain", we used degenerate oligonucleotides homologous to these regions to PCR probe the genomic DNA. Although all ras coding region are similar in size, their intron sizes are expected to be different. Our initial screen detected three bands which differ in size from let-60 .The DNA fragments within the two primers were cloned. The partial sequence of two of the clones are shown below. Both genes are highly homologous to let-60 ras in the two small regions. The genomic DNAs containing the fragments have been isolated from a phage library from Bill Wood's laboratory. We have not been able to isolate cDNAs for the two genes after screening nearly a million plaques out of the Barstead cDNA library. The RNA of these two genes (if they are real genes), could be far less abundant than that of let-60 .We will continue to characterize these two candidate genes and hope to study the functions of ras-like small GTPases by molecular genetic methods including constructing dominant mutations in vitro.

Reinhart BJ et al. (1991) Worm Breeder's Gazette "Further LacZ expression vectors"

Ruvkun GB, Reinhart BJ

[Worm Breeder's Gazette, 1991]

Andy Fire and colleagues (Gene 1990, Vol. 93, 189-198) developed a set of LacZ expression vectors containing a small synthetic intron, an SV40 nuclear localization signal fused to LacZ, and a 3' untranslated region derived from unc-54. For exon fusions the vectors pPD21.28, pPD22.04 and pPD22.11 provide a polylinker in all three reading frames. However, in pPD22.11 the sites upstream of the XbaI site cannot be used due to a stop codon in the XbaI site. To make some of these sites useful for cloning, pPD21.28 was digested with SalI, filled in and religated. In the resulting vector, pPD21.28deltaS, the HindIII, SphI and PstI sites can now be used in the third reading frame. [See Figure 1] In C. elegans, many exons are relatively small (~100bp), thus it happens frequently that convenient restriction sites are close to the 5' end of the exon. Fusions of these sites to the polylinker would create small exons just upstream of the synthetic intron that might not get spliced properly. To circumvent that problem, the intron cassette was deleted by digesting with PpuMI and delegating to yield the vectors pPD21.28deltaI, pPD22.04deltaI, pPD22.11deltaI and pPD21. 28deltaSdeltaI. Occasionally, convenient restriction sites cannot be found in the coding region, but are present in the intron. To make the unique BstBI site in the intron more useful for such constructs, a synthetic oligonucleotide was inserted that provides unique sites. Depending on the orientation of the oligonucleotide insertion the vectors are called LA or LB: pPD21.28LA, pPD21.28LB, pPD22.11LA, and pPD22.11LB. However, we currently do not have data that show that fusion introns get spliced properly in these vectors. [See Figure 2]

Avery L (1995) Worm Breeder's Gazette "Effects of Conus venoms on the pharynx"

Avery L

[Worm Breeder's Gazette, 1995]

Cone snails are predatory snails. They harpoon their prey, which may be small fish, marine worms, or other snails, then inject a paralyzing venom through the hollow harpoon (Olivera et al, Science 249: 257). The venom is a complex mixture of tiny peptide toxins, different in each species. Several of these toxins have been shown to act on cell-surface molecules important for nervous system function such as the N-type Ca++ channel or the voltage-gated Na+ channel (Gray et al, Ann Rev Biochem 57: 665).