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."

Blumenthal TE, Spieth J

[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.

Strome S et al. (1983) Worm Breeder's Gazette "roles of cytoskeletal elements in generation of asymmetry and segregation of P granules."

Strome S, Wood WB

[Worm Breeder's Gazette, 1983]

Between the time of fertilization and first cleavage of C. elegans zygotes, several directed movements of cellular components, which reflect the asymmetry of the zygote, occur. The zygote undergoes contractions of the anterior membrane and pseudocleavage. The egg and sperm pronuclei migrate from opposite poles of the zygote, meet in the posterior hemisphere, and then move to the center. The first mitotic spindle forms in the center of the zygote, but becomes asymmetric with respect to its position along the anterior-posterior axis and the behavior and morphology of its asters. The segregation of P granules to the posterior pole of the zygote, which occurs concurrently with pseudocleavage and pronuclear migration, provides one of the most striking manifestations of zygote asymmetry. In order to determine whether specific cytoskeletal fiber systems are involved in P-granule segregation and other early movements and whether these early events are interdependent, I have analyzed mutant embryos and wild-type embryos treated with microtubule (MT) and microfilament (MF) inhibitors. Analysis of zyg-9(b244) embryos (described in a previous newsletter) and embryos treated with low levels of MT inhibitors (which partially phenocopy the b244 defect) demonstrate that P-granule segregation is not mediated by the mitotic spindle; even though spindle orientation is altered in mutant and treated embryos, P granules nevertheless coalesce and become localized in the posterior pole. Treatment of zygotes with levels of MT inhibitors (colcemid, vinblastine, or griseofulvin) that prevent mitosis does not prevent proper P-granule segregation or pseudocleavage but does inhibit pronuclear migration. Conversely, treatment of embryos with MF inhibitors (cytochalasin D or B) inhibits P-granule segregation and pseudocleavage without preventing pronuclear migration. In addition, the resulting spindle does not become asymmetric in position or in aster behavior in cytochalasin treated zygotes. The results suggest that P-granule segregation does not require either the spindle or cytoplasmic MTs, but that this process as well as generation of other asymmetries do require cytoskeletal functions that depend on MFs. Although MT-mediated events (pronuclear migration) and MF-mediated events (pseudo-cleavage, P-granule segregation) are both normally initiated at fertilization, these two classes of processes can be uncoupled by drugs or mutation and therefore appear to be independent. Details of these experiments will appear in Cell 35, pp. 15-25. In immunoprecipitation experiments designed to identify the molecular components of P granules, one of the anti-P-granule monoclonal antibodies (K76) precipitates a 37,000-dalton polypeptide and another anti-P-granule antibody (L416) precipitates an 83,000- dalton polypeptide from [35S]-worm homogenates. In gel blot experiments both of these antibodies bind to a ~37,000-dalton polypeptide in worm and egg homogenates. However, the K76 antibody also binds a higher and lower molecular weight species as well. Further investigation of the polypeptides recognized by these antibodies, as well as two other anti-P-granule antibodies, is under way.

Ward S et al. (1982) Worm Breeder's Gazette "preparation of antibodies to sperm-specific proteins."

Ward S, Burke DJ

[Worm Breeder's Gazette, 1982]

Specific antibodies can be used both to localize proteins in cells and to identify genes coding for these proteins either by immunoprecipitation of hybrid related in vitro translations or by screening of expression libraries. We have prepared antibodies to three sperm-specific proteins by using spots cut out of 2-D gels for immunization of rabbits. We first identified those proteins that were likely to be sperm-specific by running 2-D gels of [35S]-labeled sperm together with excess cold total hermaphrodite protein and vice-versa. We then compared [35S]-labeled in vitro translated male poly A+ RNA peptides with sperm proteins. Seven major spots were found that appeared to be both sperm specific and present in male poly A+ RNA translations. Five of these spots were cut out of Coomassie blue stained 2-D gels that had been loaded with 3 x 10+E7 cells (480 g total protein). Spots from two gels were combined, broken up by passage through a 20 gauge needle, emulsified with Freund's complete adjuvant and injected subcutaneously into rabbits. At 1 month intervals, rabbits were boosted with the same antigen in incomplete Freunds. Each injection was about 70 g of the major sperm protein ( MSP) and 10 g or less of the other proteins. The sera were tested by indirect immunofluorescence staining and immunoprecipitation. Three out of 5 rabbits were positive. Anti MSP stained cells at a dilution of 1:16,000; anti 'spot 10', a 13,000 dalton sperm protein, stained at 1:16,000; anti 'spot 12', a 15,000 dalton protein, stained at 1:800. All three anti sera immunoprecipitated the injected antigen. All three stained sperm and spermatocytes and no other worm tissue the staining of spermatozoa was predominately in the pseudopod. These results show that spots off 2-D gels can provide sufficient antigen for preparing high titer antisera. The preparative gels can be stored at -70 C after soaking in 50% glycerol so additional spots can be cut out for subsequent immunizations. Technical note: For immunofluorescent staining of sperm and testes, I find that fixation with formaldehyde gives much better morphological preservation and clearer localizations than does fixation in organic solvents. I use 4% formaldehyde in PBS. The formaldehyde must be freshly prepared just before use from paraformaldehyde by heating to 90 C then treating with NaOH until the solution clears and then neutralizing with HCl. Dissected gonads are fixed on BSA or polylysine subbed slides for 20 minutes under a coverslip then the coverslip is removed after by freezing on dry ice. Unreacted aldehydes are blocked by 10 mg/ml glycine in PBS and then cells are permeabilized with 0.5% Triton X-100 for 5 minutes, rinsed, and stained as desired.

Barstead RJ et al. (1988) Worm Breeder's Gazette "update on the deb-1 gene."

Barstead RJ, Waterston RH

[Worm Breeder's Gazette, 1988]

The deb-1 gene encodes a 107,000 dalton peptide found at the base of the muscle dense-bodies. Some time ago we obtained a clone for this gene from an expression library. We have almost completed the sequencing of 12 kb around the original cloned fragment. The sequence data reveals some features which are atypical compared to other C. elegans genes. First, its codon usage is not sufficiently biased to allow us to use measures of codon bias as a reliable means to identify the smaller exons. This may be a reflection of the fact that the gene encodes a relatively low abundance product and may not have the same pattern of codon usage as an abundantly expressed gene. In order to identify the exons we have constructed a cDNA library in the vector lambdaZAP and obtained a 2.3 kb cDNA for the deb-1 gene. This accounts for about 60% of the mRNA size. About 80% of this cDNA has been sequenced. With the direct identification of the intron-exon boundaries through the cDNA we were somewhat surprised to find that many of the potential coding regions in the middle of the gene are not in the cDNA; rather, there is a large 1.6 kb intron. This points to another unusual feature of the gene; it has large and frequent introns. With these introns the gene spans 8-9 kb, whereas the length of its mRNA is only 3.8 kb. Finally, in regard to the identification of splice junctions, in every case except one the splice junction sequences are as similar or more similar to the general eukaryotic consensus sequence than to the nematode splice junction consensus sequence. This exceptional case was significant, however, because it was not identified at all using the general consensus, but was identified when compared to the nematode consensus. Aside from the analysis of the deb-1 cDNA, the cDNA library mentioned above is largely uncharacterized. The initial unexpanded library contained 2x10+E6 phage. Approximately 85% of these were white on X-GAL plates. The median insert size is about 1.1 kb. The maximum detected insert is 4.5 kb and the minimum is 0.5 kb. The library can be screened with both DNA and antibody probes. The lambdaZAP system allows for excision of the cDNA as a plasmid clone by coinfecting with an m13 helper phage, thus eliminating the sometimes tedious step of subcloning. Finally, using a probe obtained from Don Moerman for the unc-52 gene we have also recovered unc-52 hybridizing cDNAs.

Thomas JH et al. (1988) Worm Breeder's Gazette "sensory cells for an endogenous C elegans avoidance factor."

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1988]

When a worm encounters certain noxious conditions with its nose it backs up (see WBG 10-1, p. 89 for how we assay this behavior). In the process of testing various chemicals and extracts for activity in the avoidance assay, we found that C. elegans itself makes an avoidance factor which produces a response that is very similar to osmotic avoidance. This avoidance response is not due to any of the known avoidance chemicals. We have killed various cells with a laser to define which sensory neurons are required for the avoidance factor (Afa) response. The source of avoidance factor for the tests is an ultrafiltered crude lysate of C. elegans. When the amphid sheath is killed animals become Afa nonresponsive, suggesting that the amphid sensillum is required for Afa detection. When the amphid sensory cell ASH is killed animals are less sensitive, and when ASH and ADL are killed together animals are nonresponsive to Afa. When ADL is killed alone the animals respond normally. Interestingly, the primary Afa sensor, ASH, is the same cell that is the primary osmotic avoidance sensor ( WBG 10-1). Both ASH and ADL have synaptic wiring (White, et al. 1986. Phil. Trans. Royal Soc. London 314: 1-340) consistent with being avoidance sensory neurons, because they are unique among amphidial neurons in having synaptic output to AVA and AVD (the interneurons that drive backward movement, Chalfie, et al. 1984. J. Neurosci. 5: 956-964). One model for Afa avoidance is that ADL and ASH act by directly activating AVA and AVD, thus causing the avoidance behavior. This model can be tested by killing other cells to which ADL and ASH synapse, and by killing AVA and AVD. The C. elegans avoidance factor is not yet well characterized. It is not excreted into the medium, but is released upon boiling or sonication of worms. It is made at about normal levels by daf-22(m130) animals, which make no dauer pheromone. It is heat stable and passes through a filter with a 10,000 dalton exclusion limit. No avoidance factor is found in E. coli or Drosophila melanogaster larva extracts, but a factor that C. elegans avoids is found in soil nematodes of the genera Pelodera and Pellioditis. A possible function for avoidance factor is to signal the presence of a feeding predator of nematodes. Most such predators eat nematodes by puncturing their cuticle with a stylet or teeth and pumping up the insides. Other nematodes could detect and avoid the active predator by sensing some chemical(s) released from broken worms. A single adult C. elegans exploded on a plate using a glass needle causes a rapid (within one minute) and strong (maximum radius about 1 mm from the victim) avoidance responses in nearby worms.