Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Lewis JA (1988) Worm Breeder's Gazette "referencing, crossreferencing, and creating reference lists with Microsoft Word."

Lewis JA

[Worm Breeder's Gazette, 1988]

I've written a little primer on how to use Word (version 3.0 or higher) to automatically generate references (reference number or author-year citation) and cross-references (to page number) within a document using a combination of Word's merge function and table of contents generation facility. Also included in the article are directions for using reference information stored in a database to automatically generate a reference list according to a given style using Word's merge function. The utility of all this is likely to be superseded by future versions of Word and commercially available add- on programs. However, if you are interested, drop me a line and I'll send you a copy of my article.

Kusch M (1982) Worm Breeder's Gazette "more about L4 and adult cuticle proteins."

Kusch M

[Worm Breeder's Gazette, 1982]

SDS-PAGE of L4 and adult soluble cuticle proteins revealed that only bands E and H were present in both developmental stages (Cox et al., 1981. Dev. Biol 86: 456-470). Analysis of these proteins by 2D gels shows that 6 spots (corresponding to 3 bands on one-dimensional gels) are identical. One of the spots makes up all of the adult band E and most of the L4 band E and has an approximate pI of 4.5. Four spots compose all of band H and have pI's from 3.15-3.3. The sixth spot has a higher molecular weight than 210K and a pI of about 4. The remaining major polypeptides are different with the L4 cuticle proteins generally more basic by about 0.5-1 pH unit than the adult cuticle proteins. In addition the major L4 cuticle proteins (H is an exception) all appear as single spots on 2D gels, whereas the major adult proteins (except E) appear as multiple spots with small charge differences. A second difference between L4 and adult cuticle proteins appears when native proteins are treated with pepsin. This enzyme digests nonhelical parts of native collagens but leaves helical regions intact. Purified [35S]-labeled cuticles were extracted with - mercaptoethanol in a buffered salt solution to isolate native molecules. The extracted proteins were digested with pepsin in 0.5M acetic acid at 6 C and analyzed by SDS-PAGE and fluorography. The major labeled bands from L4 and adult cuticles (presumably polypeptides derived from collagen triple helices) had different molecular weights. Each stage has one very prominent polypeptide and 3-5 less prominent ones, some of which may have the same molecular weights in the two developmental stages.

Avery L (1995) Worm Breeder's Gazette "C. elegans Internet Information Servers: Status and Statistics"

Avery L

[Worm Breeder's Gazette, 1995]

As many of you know, there is a gopher server at the CGC(elegans.cbs.umn.edu) and a World-Wide-Web (WWW) server in my lab inDallas (http://eatworms.swmed.edu/) devoted to information about Celegans. These servers are heavily used.There are several other C elegans information servers, but they are notincluded here because I don't have their usage information.The table shows that, although many connections come from net-surfers whovisit once and never again (perhaps having expired from boredom), there isa large group of repeat offenders who use the servers over and over. Many, perhaps most, come from C elegans labs. (For instance,wormworld.ucsf.edu and horvitzlab3.mit.edu are pretty obvious.)My point is, if you need to reach the C elegans community, this is a goodway to do it. There is an Announcements page on both gopher and WWW. Inthe 55-day period analyzed it was read 203 times. If you have a jobopening or a conference or anything else you'd like worm people to knowabout, you probably should be advertising there. Announcements can besubmitted by e-mail to leon@eatworms.swmed.edu, and usually appear withina few hours.

Coulson AR et al. (1989) Worm Breeder's Gazette "genome map."

Ameer H, Albertson DG, Sulston JE, Coulson AR, Waterston RH, Fishpool RM

[Worm Breeder's Gazette, 1989]

The map is now widely distributed electronically (see WBG 10(3), 67), but we are once again providing a summary for the gazette in the form of an output from the routine CHPLT. Do note that this is a provisional best guess, and that some linkages may later go away: please enquire if you need to know about the status of particular areas. When you receive cosmid clones, as stabs, please IMMEDIATELY streak them out on selective medium, pick small colonies, and grow 4ml minipreps (protocol from Alan Coulson if needed). For some cosmids, larger preps are liable to yield deleted DNA. Check that cosmid DNA appears full size (runs slower than lambda on agarose gels), then freeze a sample of good cells in 20% glycerol at -70 C. MRC computer account 'ARC' does not exist; Alan and John share account JES. A database node is now open at Seattle: modem number 206-467-2957; operator Phil Meneely. The summary of clone types given on the next page may be helpful when you are deciding which clones to request for your research. To reveal the most suitable clones for microinjection, the buried clones need to be displayed by the routine CONTASS; we will help you to do this if you ask. [See Figures 1- 3]

Liu FZ et al. (1996) Worm Breeder's Gazette "MOLECULAR IDENTIFICATION OF THE CORE-ASSOCIATED PROTEINS IN C. ELEGANS THICK FILAMENTS"

Ortiz I, Epstein HF, Liu FZ, Bauer CC, Cook RG

[Worm Breeder's Gazette, 1996]

C. elegans thick filaments are complex structures consisting of multiple protein molecules. Biochemical and electron microscope studies show that the outer layer of the filaments contain myosins A and B, which are differentially localized in central and polar regions respectively. The underlying layers contain paramyosin, a homolog of myosin rod. The cores contain a second population of paramyosin (distinguishable by 2D IEF/PAGE) associated stoichiometrically with three proteins, designated as P20, P28, and P30 (Deitiker and Epstein, J. Cell Biol., 123, 303-311, 1993). Image processing and structural modeling of electron micrographs show that the cores consist of seven subfilaments (two strands of paramyosin for each) cross-linked by internal protein rings (Epstein et al., J. Struct. Biol., 115, 163-174, 1995). In order to study potential roles of P20, P28, and P30 in the assembly of thick filaments, efforts were made to purify these three proteins. Thick filaments were purified by sedimentation on 19-38% sucrose gradients and the proteins were precipitated from the filament-containing gradient fractions. The proteins were then separated on SDS-PAGE and transferred to PVDF membranes. Protein bands of interest were subjected to endoproteinase Lys-C digestion, and the resulting peptides were separated by HPLC and then microsequenced. Both P28 and P30 are novel proteins, and cosmids (T14G12 and C46G7) containing the genes encoding them have been completely sequenced by the C. Elegans Genome Project. P28 is a 201 amino acid protein that is composed of only beta sheets interspersed with turns as predicted by secondary structure programs. P30 is a 250 amino acid protein that is predicted to have both beta sheets and alpha helices. Each of the three proteins have been mapped to different chromosomes. P20, which has not been completely sequenced, is located on chromosome II near the unc-52 gene,, whereas P28 maps to the X chromosome and P30 to chromosome IV near unc-82. Anti-peptide antibodies have been raised against both P28 and P30. In preliminary experiments the antiserums have been shown to label body-wall muscle A-bands and isolated thick filaments and cores by immunofluorescence and immunoelectron microscopy. Anti-P28 has been affinity purified against its peptide antigen. The resulting antibody labels isolated cores with a paramyosin-like periodicity and appears to react along the entire length of both cores and thick filaments. These results are consistent with the predictions of the structural model of Epstein et al. (1995) for the inner core proteins. Present approaches to characterize the function of P28 include isolation of a strain with a transposon insertion in the P28 gene by R. H. A Plasterk and K. van der Linden and injection of constructs driven by the unc-54 promoter (Okkema et al., Genetics, 135, 385-404, 1993) which produce anti-sense RNA to P28 mRNA. The possible identity of P30 and unc-82 is being tested by cosmid rescue experiments. Both P28 and P30 have been expressed in E. coli as GST fusion proteins for future biochemical experiments.

Takuwa K et al. (1995) Worm Breeder's Gazette "Tissue Specific Expression of Tropomyosins of Caenorhabditis elegans."

Kagawa H, Takuwa K

[Worm Breeder's Gazette, 1995]

Tissue specific expression of tropomyosins of Caenorhabditis elegans Kyoko Takuwa and Hiroaki Kagawa Department of Biology, Faculty of Science, Okayama University, Okayama 700 JAPAN c5191 8@jpnkudpc.bitnet, hkagawa@sc.okayama-u.ac.jp In C. elegans, there are two different types of muscles; the pharyngeal muscles and body wall muscles. We cloned and sequenced the tropomyosin gene, tmy-1 of the worm which encodes more than three isoforms; two of body muscles and one of pharyngeal muscle. Tmy-1 gene expression was controlled by two promoters and alternative splicing. We have already known the place of the tmy-1 gene expression by injecting series of tmy-1 gene-lacZ fusion plasmids into the oocyte. The expression of promoter activity was detected by histochemical procedure of beta- galactosidase activity. The first promoter controlling two isoforms expressed in the body wall, vulva and male tail muscles. The second promoter specifically expressed in the pharyngeal muscles (Imadzu et al. WBG 13#3p56). To know the results of promoter/lacZ expression were consistent with the results of protein levels, we raised antibody against bacterial produced tropomyosin followed by immunostaining. Fusion protein of b-Gal-CeTMIII was isolated from sonicated bacterial extract by ammonium sulfate precipitation and ion exchange column chromatography. Affinity purification of antibody was prepared by the protocol of Macef and Koch (J. Cell Sci. 92, 61-70 1988), using the proteins immobilized on nitrocellulose membrane. Worms processed with 2X Laemmli buffer run on SDS PAGE. Blotted membrane was stained with 0.01percent Ponseau red in 5percent acetic acid and corresponding band was cut for affinity purificaffon of antibody. Purified protein from SDS-PAGE sometimes is not good antigen for raising antibody for the purpose of histochemistry. We should mention the effect of SDS treatment on the difference between "before" and "after" immunization. Affinity purified antibody by using worm protein was better than that by using bacterial produced protein. Affinity purified anti-CeTMIII antibody stained pharyngeal muscle conversely anti CeTMI/CeTMII antibody stained body wall muscles by indirect immunofluorescence microscopy. Affinity purified antibody also detected a low molecular mass of CeTMII (256 amino acids) on Western blot analysis. These results confirm that promoter-lacZ micro injection experiments were really the result of the transcription and translation of the correct gene. In our micro injection experiments on troponin I (Kuroda et al), troponin C (Matsumoto et al) and ryanodine receptor genes (Sakube et al., WBG 13#2p58), these genes expressed in the body wall muscles. This suggest that muscle contraction in the body wall could initiate with calcium signal of thin filament linked; troponins-tropomyosin. This might suggest that pharyngeal muscle contract with thick filament linked; CaMd-myosin light chain pathway. Upstream carrier of calcium in the pharyngeal may be inositol 1,4,5 triphosphate. Yuji Kohara et al and Baylis et al. isolated the cDNA clones of InsP3 which might express in the pharyngeal (WBG 13#4p18, WBG 13#4p68) .

Desai C et al. (1984) Worm Breeder's Gazette "more about HSN-defective mutants."

Horvitz HR, Desai C

[Worm Breeder's Gazette, 1984]

We have isolated 45 C. elegans egg-laying defective mutants that appear to have functionally impaired HSNs (see Newsletter Vol. 8 No. 1). (The HSNs are two motorneurons that innervate the vulval muscles.) These mutants define at least 14 genes, of which we have now mapped 11 (see Map on the next page). In the last Newsletter (Vol. 8 No. 2) we reported that in the wild type the HSNs are stained by anti- serotonin antisera, and we described the phenotypes of some of these HSN-defective mutants as observed after staining with anti-serotonin antisera. We have now examined the appearance of the HSNs in mutants representing all 14 of these genes using indirect immunofluorescence to visualize serotonin and Nomarski optics to visualize cell bodies ( see Table on the next page; an * in the table indicates a mutant allele studied by microscopy). These HSN-defective mutants can be divided into five categories. Mutants in Category A lack HSNs by both criteria; mutations in three of the five genes of this category appear to cause HSNs to undergo programmed cell death, as these mutations are suppressed by mutations in the genes ced-3 or ced-4 (which block the onset of programmed cell deaths; see Ellis et al., this Newsletter). Mutants in Category B appear to generate abnormal HSNs, which can be absent or variably mispositioned and which when present invariably lack serotonin. The mutant egl-46(n1127), which defines Category C, has HSN cell bodies in almost normal positions but lacks serotonin in the HSNs. (This mutant contains serotonin in the other normally serotonin-positive neurons that are visualized after staining with the anti-serotonin anti-sera.) It has not been unambiguously demonstrated that the cells considered to be misplaced HSNs in Categories B and C mutants are indeed HSNs. The mutant egl-43(n997), which defines Category D, appears to be defective in the migration of the HSNs. (HSN migration is a normal aspect of embryonic development.) As seen with Nomarski optics, egl-43 animals lack HSNs in their normal positions; serotonin-staining reveals ectopic serotonin-positive cells variably located along the path of HSN migration (from the postanal region to near the position of the vulva). A double mutant containing egl-43(n997) and the Category A mutation egl-41(n1069) lacks these ectopic serotonin- positive cells, indicating that they are mispositioned HSNs. The misplaced putative HSNs of egl-43 animals have aberrant processes: some run abnormally long distances along the ventral cord into the nerve ring (normal HSNs run from the region of the vulva along the ventral cord into the nerve ring), others are bipolar, and others send a process posteriorly to the tip of the tail or anteriorly via a sublateral route. The putative HSNs of egl-43 animals seem to migrate slightly farther on average at 20 C or 25 C an they do at 15 C. Category E mutants have HSNs that appear normal as visualized with both Nomarski and fluorescence microscopy. [See figure 1]

Creutz CE et al. (1994) Worm Breeder's Gazette "The Annexins of C. elegans: A Novel Splice Varient May Be Subject To Control By Phosphorylation"

Snyder SL, Daigle SN, Creutz CE

[Worm Breeder's Gazette, 1994]

THE ANNEXINS OF C. ELEGANS: A NOVEL SPLICE VARIANT MAY BE SUBJECT TO CONTROL BY PHOSPHORYLATION C.E Creutz. S.E Snyder. and S N. Daiele Dept. of Pharmacology, University of Virginia, Charlottesville, VA 22908 The annexins are calcium-dependent, phospholipid binding proteins that may function in exocytosis, membrane structure and permeation, and signal transduction. In order to establish a system for genetic analysis of their hypothetical functions, we have been characterizing the annexins of the nematode, C. elegans. Annexins were isolated from postmicrosomal supernatants by calcium dependent binding to lipid vesicles. EGTA extracts of these vesicles appear to contain primarily a single protein of mass 32kDa when examined by SDS-PAGE. Peptides derived from this protein were sequenced and verified that the worm protein is an annexin. An antiserum was raised to the worm annexin and was found to react almost exclusively with a 32kDa band in Western blots of worm homogenates. The antiserum was used to isolate cDNAs for the protein from a lambda gtll library (kindly provided by A, Fire). The sequence of the worm annexin is 42Z identical to that of bovine annexin IV, the closest vertebrate homolog. The single annexin gene was physically mapped to the vicinity of dpy-17 in LGIII. One of three cDNA clones isolated contained a 15 base splice insertion encoding five amino acids near the N-terminus. This insertion contained THR and TYR residues that align exactly with phosphorylation sites in mammalian annexins for protein kinase C and the src kinase.

Powell K et al. (1992) Worm Breeder's Gazette "The Electronic Gazette"

Schatz BR, Powell K

[Worm Breeder's Gazette, 1992]

The Worm Breeder's Gazette (WBG) is now available in electronic format as part of the Worm Community System (WCS). See the accompanying article for WCS availability. We have converted the contents of the WBG into electronic form, including all articles from Vol 1 No 1, published in December 1975, to the latest Vol 12 No 1. The intention was to provide a complete archive so that every page was converted, except for material otherwise available electronically, such as the genome map and the bibliography. Rather than scan in images of the pages, the text was character recognized and marked up so that it can be searched using key words and displayed in a uniform (pretty) format. This makes the information more readily available at the cost of losing the historical variation of submitted printed formats. The figures were scanned and resized for readability. Thus, when using WCS, you can retrieve all WBG articles referencing a given phrase and quickly display their complete text with figures. The process for generating the electronic version was as follows. A paper copy of each issue was scanned using a Hewlett-Packard ScanJet Plus with a sheet feeder running the DeskScan software. This was connected to an Apple Macintosh II FX, where the conversion took place. For text pages, the Omnipage software was used to convert the digitalization to a Microsoft Word document. Surprisingly, most pages were recognized accurately with only a few errors. Word was then used to perform spelling correction to catch scanner errors and to mark up the text in pseudo-SGML to specify location of the title, author, and special words such as gene names. The proofreading was done by an undergraduate biology major. For figure pages (or for figures within pages), the DeskScan software was used to generate a TIFF file. The bitmaps were then resized and cleaned up using Adobe Photoshop. After conversion, the text and image files were downloaded to our Sun file server. The display software converts the marked-up text into its own internal format. It displays the title and author in large boldface followed by the rest of the text in pretty format. It is written in C for Unix machines under X-windows. The figures if any are displayed in adjacent windows from the TIFF format. There are currently some 1450 articles; a typical size is 6000 bytes for a text page and 20 kbytes for a figure image. The recommended usage is to obtain WCS and use the functionality therein. If there is sufficient interest, we will also make the raw files available (please note they contain formatting codes). There is also a stand alone Unix program which displays a table of contents for all issues, and allows you to read them. This project was carried out in about six months, using clerical help and undergraduate students. Special thanks are due to Eric Boyer, Lindy Fletcher, and Clay Sales. Thanks are also due to Samuel Ward, who donated his collection of WBGs as well as serving as the test user and to Mark Edgley, who provided missing copies.