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Worm Breeder's Gazette,
1994]
lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA
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Worm Breeder's Gazette,
2013]
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Worm Breeder's Gazette,
1976]
Robert Wyman (Department of Biology, Yale University) reports that Theodore Bullock (University of California at San Diego, La Jolla, California) has isolated a 4.1-meter long nematode from a 3.8-meter whale. The specimen is preserved, and Dr. Bullock is searching for a taxonomist interested in taking the beast. Want Ad: Does anyone know a way to cause a large population of males to release their sperm into the medium? CONTEST REMINDER: Don t forget that bottle of champagne from S. Ward and D. Hirsh promised to whoever scores the most progeny from a single hermaphrodite (see Newsletter, Vol. 1, # 2 for details). Deadline for submission of entries to either judge is February 23rd.
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Worm Breeder's Gazette
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Worm Breeder's Gazette,
1994]
C. elegans U2AF65
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Worm Breeder's Gazette,
1984]
We have analyzed the arrangement of histone genes on cloned C. elegans restriction fragments by constructing detailed restriction maps and by DNA sequencing. We have studied three cloned segments, each carrying a cluster of histone genes. The clusters are surrounded by non-histone DNA and hence give no indication as yet of a major histone locus of multiple, linked clusters. Two of the clusters carry a single copy of each of the core histone genes arranged in the order H4, H3, H2A and H2B. The orientation of transcription of the H3 gene is opposite to that of the other three. In the third cluster there are two copies of each of the core histone genes, arranged as a duplication of a cluster, with genes in the order H3, H4, H2B, H2A. The duplicate clusters are very similar at the nucleotide level. We calculate from restriction site differences that the sequences are 96% conserved, indicating either that the duplication is of fairly recent evolutionary origin, or has undergone a recent correction event. So far, we have not been able to locate H1 genes on our clones. Heterologous H1 probes from chick and sea urchin do not cross- hybridize with C. elegans genomic sequences, so we do not as yet know the number or arrangement of H1 genes. Northern hybridization studies using the cloned C. elegans clusters as probes have revealed four discrete transcripts of sizes expected for the four core histone genes. The absence of a larger transcript of a size expected for H1 suggests that genes for H1 are not present in the cloned clusters and hence must lie elsewhere in the genome. Sequencing of the entire H2A and H4 genes as well as most of the H3 gene in one cluster reveals near identity with sea urchin proteins. Like most histone genes, these sequences contain no intervening sequences and no AAUAAA polyadenylation signal. Interestingly, C. elegans histone genes also do not contain the conserved 3' mRNA processing site found in histone genes of human, chicken, Drosophila, sea urchin and several other organisms. We therefore believe that C. elegans must utilize a different sequence or mechanism for generating the 3' ends of mature histone messages.
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Worm Breeder's Gazette,
1983]
We have been applying hybridoma technology to our research by generating monoclonal antibodies (MAb's) against C. elegans sperm. We have produced 10 mAb's in our lab and have obtained 2 others from Susie Strome at Boulder. Seven of these mAb's target antigens present on the cell surface. We have been purifying monoclonal antibodies from ascites fluid by DEAE Affi-Gel blue chromatography (supplied by Bio-Rad). Although yields have been lower than ideal (40-60%), these antibodies are free of all contaminants except mouse transferrin. Membrane lipid flow and directed movement of pseudopod surface components on sperm was previously reported by Roberts and Ward (J. Cell Bio. 92: 113-120). We have evidence that the movement of antigen-antibody complexes may be due to a continual flow of membrane lipid (see Bretscher, 1976, Nature 260: 21-23) from the tip of the pseudopod back toward the cell body. Bretscher's lipid flow model of membrane movement predicts that membrane components which diffuse slowly are displaced by the lipid stream whereas those which diffuse rapidly effectively escape lipid flow. Because the rate of diffusion is related to molecular weight, large complexes (e.g. antibody- crosslinked membrane proteins) would be swept along more rapidly than individual molecules (e.g.- unbound membrane proteins). We have tested this prediction by comparing clearance rates of various sized complexes form the cell pseudopod. On sperm, relatively small antigen- mAb complexes clear from the pseudopod surface slowly (2-3 min) while larger, cross-linked antigen-mAb-2 antibody complexes are cleared more rapidly (30-45 seconds). Using mAb's conjugated directly to colloidal gold particles (CGP), we have obtained evidence that new surface antigens are preferentially inserted at the tips of pseudopodial projections and then move rearward toward the cell body pseudopod junction (Pavalko and Roberts, C. elegans Meeting Abstracts, 1983). This observation predicts that there must be a pool of antigen in the cytoplasm which would be available for insertion into the membrane. Labelling thin sections of spermatozoa with CGP conjugates of mAb's 11, 63, and 56 (each of which bind to the cell surface in indirect immunofluorescence assays) reveals antigen on the surface (plasma membrane, exposed face of fused membranous organelles and MO contents). In addition, these antibodies label the cytoplasmic laminar membranes and the pseudopod cytoplasm. Many of the gold particles in the cytoplasm lie just beneath the plasma membrane suggesting that they are bound to antigens that were destined for insertion onto the surface. Because there are no vesicles in the cytoplasm, the mechanism which transports these antigens is unknown. We will be trying to characterize the antigens targeted by the mAb's and to solve the question of how they are shuttled around the cell.
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Worm Breeder's Gazette,
1994]
An Electronic Discussion Group For C. elegans Researchers
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Worm Breeder's Gazette,
1986]
Comparison of DNA sequences surrounding six C. tone genes reveals a novel conserved sequence located approximately 80 nucleotides upstream of the translational start site. The six genes include all of the genes in one genomic cluster (one of each of the four core histone genes), and two from another, unlinked cluster (one H2A gene and one H4 gene). All of the conserved sequences 5' of each of these genes are presented in the figure below. Starting at the ATG codon and moving upstream, they include a sequence adjacent to the translational initiation site typically found in eukaryotic mRNAs, a conserved sequence surrounding the transcriptional initiation site similar to one found in some sea urchin histone genes, a TATA box, and the novel sequence. With the exception of yeast, upstream consensus sequences in histone genes of other organisms are restricted to the genes for individual classes of histone proteins (i.e., they are H4 gene-specific or H2A gene-specific, etc.). In yeast a conserved sequence of 16 nucleotides activates transcription of histone genes in a cell cycle-dependent manner (Osley, M. H., Gould, J., Kim, 5., Kane, M. and Hereford, L. (1986) Cell 46, 537-544). Like the C. , this 16-mer is found upstream of different classes of histone-encoding genes. In the figure, numbering of nucleotides begins at the first translated nucleotide, and the number of non-matching nucleotides between each conserved region is given. The asterisk indicates the site of transcriptional initiation in two of the genes (+/- 1 nucleotide) determined from Sl mapping experiments. The arrow over the consensus transcriptional initiation sequence indicates the direction of transcription.
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Worm Breeder's Gazette,
1992]
Background. The extracellular matrix (ECM) plays an important role in maintaining the structural integrity and cellular functions of a multicellular organism. ECM components, including collagens, fibronectin, laminin, vitronectin, tenascin, entactin, and proteoglycans, have been identified and characterized in mammalian tissues. However, the structure and organization of these components in the intact matrix remain unclear. The inability to isolate mutants that are defective in a single ECM component in higher organisms impedes this type of analysis in vivo. With the advantage of the well-characterized genetics in C. elegans, we initiated a project to study the organization and structure of ECM using C. elegans as a model system. Our approach was to generate and characterize a panel of monoclonal antibodies against C. elegans ECM to use as markers for structural studies including immunofluorescent microscopy. In the future, we plan to use these monoclonal antibodies in affinity chromatography for the purification of ECM components and to use as probes for isolating cDNA. Experimental Approach. The ECM components were extracted from a mixed population of adult and juvenile C. elegans using the procedure shown in Figure 1. [See Figure 1] BALB/c mice were immunized intraperitoneally with 100 g of ECM extract homogenized in complete Freund's adjuvant followed by three bi-weekly injections of 50 g of ECM extract in incomplete Freund's adjuvant. Spleen cells from immunized mice were fused with myeloma cells and plated at a concentration of 2.5x10 +E5cells/well. Results. Of the 1,200 wells screened.approximately 32% of the hybridoma supernatants tested positive in ELISA against C. elegans ECM extracts. Supernatants from positive wells were tested by immunoblotting against C. elegans ECM extracts and by immunofluorescent microscopy on whole or fragmented C. elegans Protein species ranging from 20,000-180,000 daltons were detected by the hybridoma supernatants in immunoblotting, and supernatant recognition ranged from complex patterns of multiple bands to a single band. Immunofluorescent studies also revealed diverse staining patterns, which included staining between muscle and hypodermis, around muscle bundles, in layers surrounding intestines and gonads, and around the cuticle. Seven of the hybridomas were purified by limiting dilution cloning and injected into mice for ascites tumor production. We would like to make these monoclonal antibodies available to interested investigators in the near future. (This project was part of a course, Biotechnology Laboratory: Molecular Recognition (BSC 352), which was composed of 70% graduate students and 30% undergraduate students, and was supported by the Department of Biological Sciences and the College of Arts and Sciences at Illinois State University. We also would like to thank Robert Barstead for providing worm fragments and helpful discussion.)