Johnson CD et al. (1982) Worm Breeder's Gazette "monoclonal antibodies to Ascaris neurons."

Johnson CD, Stretton AOW

[Worm Breeder's Gazette, 1982]

With the ultimate goal of isolating neuron specific and/or synapse specific labels which can be used to facilitate the identification of neurons and synapses in light micrographs, we have begun to screen monoclonal antibodies prepared against Ascaris nervous tissue. Balb/c mice have been immunized with ventral and dorsal nerve cords ( prepared by pulling the ventral and dorsal hypodermal ridges off of animals which had been injected with collagenase) or with the anteriormost 2mm of the head (also after collagenase treatment to remove muscle cells). Injections were usually homogenates of nerve cords pooled from the anterior 40% of 10-12 adult females (ca. 200 cm of nerve cord) or 40-60 heads . Initial injections were followed by one or more booster injections, after which the mouse's spleen was removed, dissociated into cells, fused with myeloma strain NS-1 by exposure to polyethylene glycol, diluted and aliquoted into ten 96- well plates (ca. 1 fusion product/well). After two weeks growth, aliquots of the culture media were tested for binding to nerve cord or head homogenates by an ELISA using HRP conjugated goat-anti-mouseIgG as the second antibody. All ELISA positive (and some ELISA negative) cultures were examined anatomically for binding to neurons in a whole mount preparation of the anterior 20-30mm of the head (after removal of muscle with collagenase, fixation and extraction with either organic solvents or detergents). This preparation contains sensory neurons, nerve ring and surrounding ganglia and nerve cords including 14-21 commissures of identified motorneurons ) . In the anatomical screens, Rhodamine conjugated goat-anti-mouseIgG was used as the second antibody. Some cultures were analyzed further by ELISA against separated Ascaris tissues and against homogenates of C. elegans. ( J. Culotti and S. Siddiqui have screened some of the cultures against whole mounts of C.e.; they find ca. 20% cross reaction.) Using these procedures, we have recovered 20-30 hybridomas (from three mice) which reproducibly label neurons in the whole mount preparation, although none of these as yet demonstrate the desired specificity to single or small groups of neurons. Current efforts involve streamlining the immunization and screening procedures and examining neuron-binding antibodies for synapse specificity in frozen sections.

Stern BD (1984) Worm Breeder's Gazette "localization of class C acetylcholinesterase by immunocytochemistry."

Stern BD

[Worm Breeder's Gazette, 1984]

I have partially purified class C acetylcholinesterase (AChE) from triton extracts of N2 animals and prepared polyclonal antibodies to the partially purified protein. A rabbit was immunized with a band cut out of an SDS gel, boosted once with another cut out band and then boosted twice more with undenatured samples. I estimate that each injection contained about 10 g of enzyme. Anti-class C antibodies were detected only after the third boost. By ELISA assay the antibodies show no cross-reactivity to forms from the other two known AChE classes, A and B. However, by immulunoblots the antibodies did show cross-reactivity to a number of other C. elegans proteins. Absorption of the antibodies with an extract from ace-3(dc2) animals resulted in the removal of these contaminating antibodies, as shown by immunoblots. (Animals containing the dc2 allele have no detectable class C enzymatic activity and by ELISA have no cross-reactive material, and therefore are apparently null.) I have used the following procedure to determine the localization of class C in whole mounts: 1. Densely settled animals are squashed on 1.0% BSA subbed slides according to the procedure of Albertson et al (WBG 7(1):73). I find that squashing with a cover slip is most convenient. After freezing in liquid nitrogen and prying off the cover slip with a razor blade the slides are passed through methanol, acetone, and a graded series of ethanols to TBS (Tris buffered saline). The following incubations are then performed in a humid chamber, with 0.035 ml reagent placed over the squashed animals. 2. Incubation with 10% normal goat serum (NGS) in TBS for 30 minutes, followed by a brief wash in TBS. 3. Incubation with primary antibody (1:1000) diluted in 1% NGS/TBS overnight, followed by two 10 minute washes in TBS. 4. Incubation with Goat anti-Rabbit IgG antiserum (1:150) in 1% NGS/TBS 30 minutes, followed by a brief wash in TBS. 5. Incubation with PAP reagent (1:100) in 1% NGS/TBS 30 minutes, followed by 2 ten minute washes in TBS. 6. Repeat the two previous steps. 7. Develop the peroxidase reaction with 0.44 mg/ml diaminobenzidine HCl 0.003% H202 in TBS for 5-10 minutes, followed by a brief wash in TBS. 8. Slides are then mounted with 90% glycerin in TBS. The brown reaction product is visible in the nerve ring, in several cell bodies near the nerve ring, and in two cells located near the vulva. In addition to the specific staining seen in these structures there is a diffuse background which is visible. When staining dc2 there is no specific staining, however the background persists. Some non-specific staining is also observed in the pharyngeo-intestinal valve, both in N2 and dc2. The two cell bodies are located under the lateral alae as are their processes which stain and run posteriorly to the level of the anus and anteriorly to the level of the pharyngeo- intestinal valve. I believe that these are the CAN cells. They stain quite well in late stage embryos and L1s, as well as older animals, and are Present in unc-86 animals. As to why the CAN cells should have class C AChE on their surfaces is anyone's guess. While these cells are required for viability as shown by John Sulston, that viability is not dependent upon class C AChE since the ace-3 animals are viable, and in fact show no visible phenotype. In addition to the above mentioned staining I also occasionally see staining in cells which I believe are the PDE cells, based on their location and morphology. Work is in progress to confirm this result as well as to stain animals with altered CAN cells (vab-8).

Prenger JP et al. (1984) Worm Breeder's Gazette "RNA polymerase II from C elegans."

Tullis GE, Prenger JP, Sanford TR, Golomb M

[Worm Breeder's Gazette, 1984]

As a first step in studying the transcriptional machinery of C. elegans, we have purified nematode RNA polymerase II to near homogeneity. Structurally, the enzyme is a typical RNAP II, having two large (205 and 135 kd) and at least eight smaller polypeptides (10- 30 kd). DR 432, an amanitin-resistant strain with a mutation in gene ama-1 (IV), produces an altered RNAP II that is 150 times less sensitive to amanitin than the wildtype enzyme. We have purified RNA polymerase II from DR 432 for structural and functional comparison with wild-type polymerase. DR 432 enzyme is stable during purification, and its specific activity is identical to that of wild- type RNAP II. In a filter-binding assay, DR 432 RNAP II bound [3H]- labelled amanitin with a K at least 100 times higher than that of N2 enzyme. Thus, amanitin resistance in DR 432 is caused by defective binding of the toxin to RNAP II. The nature of the structural alteration of RNAP II in DR 432 is now being studied. Since interesting alleles of the ama-1 locus are expected to encode unstable or inactive polymerase, we have raised antibodies against nematode RNAP II to aid in identifying polymerase polypeptides in crude extracts of worms. We have obtained a high-titer rabbit antiserum against purified RNAP II which detects the 205 kd subunit in Western blots of worm homogenates. Immunofluorescent staining of hamster cells showed that this polyclonal antiserum cross-reacted with RNAP II in mammalian nucleoplasm (nucleoli were not stained, confirming an apparent lack of cross-reactivity with RNAP I). Rabbit anti-RNAP II IgG did not inhibit polymerase activity. Recently, we have obtained hybridomas secreting monoclonal antibodies to nematode RNA polymerases. Of 480 fusions between plasmacytoma cell line 653 and spleen cells from immunized Balb/c mice, about 30 hybridomas were found to make antibody against purified RNAP II by the ELISA test. Other hybridoma lines appear to make antibody against nematode RNAP I; several of these antibodies stain hamster nucleoli. Monoclonal antibodies against RNAP II are being screened for use in functional mapping of wild-type and mutant polymerase subunits.

Swartz SL et al. (1992) Worm Breeder's Gazette "Production and Characterization of Monoclonal Antibodies to C. elegans Extracellular Matrix Components"

Babwah AV, Lu S, Bobell JR, Lee M, Swartz SL, Brumfield RT, Allikian MJ, Mathus TL, Burow CW, Schroen DJ -COMMENT etc.

[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.)

Robertson SM et al. (1992) Worm Breeder's Gazette "Feminizing-Element Binding Protein (FEBP)"

Robertson SM, Meneely PM

[Worm Breeder's Gazette, 1992]

We reported previously that several X-linked elements that feminize triploid males all contain an octmer sequence which serves, in only one orientation, as a single-stranded DNA-binding site for a protein present in worm nuclear extracts (see Abstracts for 1991 Meeting, pg. 354). We have now purified this binding activity (termed FEBP) and are cloning the gene for this protein. Our purification scheme was simplified by the observation that the sequence-specific ssDNA-binding activity survived boiling. Following removal of denatured proteins by centrifugation, the boiled extract was directly loaded onto an affinity column. The column consisted of a single-stranded oligonucleotide (containing the octamer in correct orientation and derived from the first intron of the act-4 gene) attached to an agarose matrix. Following elution at high salt the predominant protein visible on Coomassie-stained SDS gels was approx. 30-32 kd, with several other bands both above and below 30 kd also visible. These protein bands were individually cut from preparative SDS gels, eluted, denatured and renatured. Band shift activity was not associated with the predominant 30 kd protein, however, but instead with a single band at approx. 24 kd. This purified and renatured protein demonstrated single-strand specificity, octamer specificity (as assayed by competitions), and, when UV crosslinked to the single-stranded probe, gave the same band on a SDS gel as previously reported (30-32 kd). It was, nonetheless, still of some interest to us to know what the predominant 30 kd protein was, since until the denaturation/renaturation experiments it seemed a likely candidate itself for FEBP. Both proteins were therefore gel-purified following the affinity column and microsequenced (William Lane, Harvard Microchem). 30-32 kd protein: Based on the sequence of one trypsin peptide an unambiguous identification could be made. It is a member of a small family of DNA-binding proteins called Y-box binding proteins and appears to be identical to a Y-box binding protein identified and cloned by Fire and co-workers (WBG 12 (2), pg. 72). This protein was initially identified by virtue of its binding to an upstream regulatory sequence of the unc-54 gene. Exactly why this protein is binding to our ss-DNA affinity column is unclear, although reports on Y-box binding proteins in several other systems have detected interactions with single-stranded nucleic acids. FEBP: Three different trypsin peptides of 14, 21 and 25 residues respectively have been sequenced. Protein database searches reveal no homologies to known proteins for two of the three peptides; the third peptide shows a small block of homology (7 of 8 or 9 of 14 identities) to the N-terminus region of E. coli SSB, a sequence non-specific single-stranded DNA binding protein. The significance of this result is unclear as this region appears to lie outside of the SSB DNA-binding domain. Genomic PCR as well as RT-PCR cloning using degenerate primers based upon these peptide sequences is underway. In addition, cDNA libraries are being screened with longer degenerate oligonucleotides. Mice have been injected with purified FEBP (as well as, separately, with the 30 kd protein) and preliminary ELISA results with purified antigens indicate immunoreactivity in the tested sera; fusions should follow the next boost or two. Rabbit antisera await either injection with coupled peptides or with cDNA expression products.