Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

Yasuda H et al. (1987) Worm Breeder's Gazette "screening C elegans expression libraries with nerve specific anit-tubulin antibodies."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1987]

C. elegans, like many other multicellular organisms, has multiple tubulin genes. Linda Gremke has shown that there are at least 4 and 5 alpha tubulin genes in the nematode, based on homology to the chicken tubulin gene probes(1). Similarly, it has been shown that purified tubulin from the nematode can be resolved in to at least 10 isotypes on isoelectric focusing, including 2 major alpha, 2 major , and about 6 minor alpha and isotypes(2). We have screened C. elegans cDNA and genomic expression libraries (kindly provided by Bob Barstead and Bob Waterston), with a set of anti-tubulin monoclonal antibodies, which preferentially stain neurons cytochemically(2). In the present screen 44 clones have been identified, which test positive on rescreening. The insert size of these clones range from 0.6 to 1.7 kb. Among these 29 clones are specific to anti- tubulin, and 14 are specific to anti-alpha tubulin antibodies. Our attention has been drawn to the problem of false positives in screening expression libraries with antibodies(e.g. C. Link and W. Wood, WBG Nov. 1986, and Eric Aamodt, personal communication). These workers have found E. coli DNA in the 'positive' clones, as a major artefact. Affinity purification of antibodies with the fusion protein, may not preclude the inclusion of false positives (Eric Aamodt, personal communication) either. Therefore, we plan to subclone the inserts in an appropriate vector, and determine the nucleotide sequence of the inserts to distinguish the true from the false positives. This is possible for the tubulin clones, as the nucleotide sequence of tubulin variants is available, including that of C. elegans tubulin(1). We plan to use this strategy for other genes for which we have collected 'positives' using expression libraries, including N-CAM like antigen, and N- CADHERIN like antigen of C. elegans, as these genes have conserved sequences, and their nucleotide sequence data are available.

Dichoso D et al. (1994) Worm Breeder's Gazette "C. elegans Homologs of MEF-2, Muscle Specific Transcription Factors"

Krause MW, Dichoso D

[Worm Breeder's Gazette, 1994]

MEF-2 ,(formyocyte-enhancer-binding-factor), is a muscle-restricted, sequence-specific transcription factor known to participate in the regulation of many muscle genes. In vertebrates, MEF-2 binding sites have been demonstrated for both terminally expressed gene products (such as muscle creatine kinase) and MyoD Family members (MyoD and myogenin). Recent results suggest that MEF-2 may play a more important role in myogenic cell fate determination than originally thought. Expression of certain isoforms of MEF-2 occurs in early muscle precursors during development in mouse, Xenopus, and Drosophila. In Drosophila, expression of a MEF-2 clone occurs prior to expression of the MyoD Family homolog nautilus and occurs in cells that are precursors for visceral and skeletal muscle cells (Brenda Lilly and Eric Olson). MEF-2 is a member of a larger group of transcription factors that are know as the MADS Family (MCM1, Agamous, Deficiens, and Serum response factor). MADS Family members share a conserved domain spanning about 55 amino acids at the amino terminal end of the protein. The MADS domain is involved in both dimerization and DNA binding of these transcription factors. The conservation of the MADS domain makes these genes good targets for PCR cloning; so we did. We used degenerate oligonucleotides in RT-PCR reactions of worm total RNA. The clones isolated are listed below; they each are related to MEF-2 and demonstrate that MADS box proteins are present in worms. The sequence results suggest as many as five different MEF-2 gene products. We are presently screening a C. elegans cDNA library with the partial sequence of MEF-2 initially obtained and are also attempting to amplify flanking sequences of the same partial sequence of MEF-2 .We want to know when and where these genes are expressed and how their expression is related to myogenesis in the worm.

Ginsburg MA et al. (1994) Worm Breeder's Gazette "dx5 and Various Aspects of Gonadogenesis."

Ginsburg MA, Lambie EJ

[Worm Breeder's Gazette, 1994]

dx5 and various aspects of gonadogenesis. Marc Ginsburg and Eric Lambie. Department of Biological Sciences, Dartmouth College, Hanover NH 03755. One lucky day, we found a W-induced mutation (dx5) that maps to IR and has strikingly obvious gonadogenesis defects. First, the dx5/dx5 progeny of a dx5/+ hermaphrodite often have proximal germline proliferation in one (and sometimes both) of the gonad arms. Despite such an abnormality, all of these animals are fertile. This phenotype is reminiscent of that seen in lin-12(0) mutants, in which the anchor cell(s) induce proximal germline proliferation. However, dx5 animals do not exhibit either the two-anchor-cell phenotype or vulva morphogenesis defects characteristic of lin-12(0) mutants. To determine whether the anchor cell is responsible for inducing proximal proliferation in dx5 animals, we tested the effect of lin-12(n302), which prevents specification of the anchor cell fate. None of 20 Unc progeny of dx5 unc-75(e950)/ + + I; lin-12(n302) III hermaphrodites had proximal proliferation. 19/49 Unc progeny of dx5 unc-75(e950)/+ + controls had proximal proliferation. This strongly suggests that the anchor cell is the culprit; however, we'll have to do some laser ablation experiments to be certain. The second glaringly obvious effect of dx5 is seen among the progeny of dx5/dx5 homozygotes. These animals have very small gonads and are usually vulvaless (presumably due to lack of an anchor cell). More detailed inspection will be necessary to determine exactly which cell divisions have gone awry. We are optimistic that we can make good progress towards determining the normal function of the gene defined by dx5. For one thing, the dx5 mutation is temperature sensitive, which should permit the isolation of additional alleles by clonal noncomplementation screening. Another good thing is that dx5 maps very close to lin-11 (probably to the left); this region is fairly well-covered on the physical map by cosmids and YACs.

Squire MD et al. (1994) Worm Breeder's Gazette "Molecular Cloning and Functional Expression of a C. elegans Nicotinic Acetylcholine Receptor Subunit (acr-2)."

Fleming JT, Barnard EA, Sattelle DB, Baylis HA, Squire MD, Tornoe C

[Worm Breeder's Gazette, 1994]

MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF A C. ELEGANS NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNIT (ACR-Z). Michael Squire, Camilla Torn0e, Howard Baylis, John Fleming1, Eric Barnard2 and David Sattelle. The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. 1 MGH Cancer Centre, Harvard University, 2Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, UK. As part of an ongoing program to study neurotransmiter receptors in C. elegans we set out to isolate nicotinic acetylcholine receptor (nAChR) subunit clones from C. elegans by screening a C. elegans genomic DNA library with a probe derived from the Drosophila ARD gene (a non-a nAChR subunit clone, kindly supplied by Dr I Hermans-Borgmeyer). A number of putative nAChR subunit clones were isolated. The resulting C elegans clones were mapped to 8 loci on the physical map of the genome (Fleming JT, PhD Thesis, University of Cambridge). We extended our studies on one of these loci (represented by the clone JF#WA56) now called acr-2 which is located between svp-7 and unc-6 on the X chromosome. Iev-9 is also located in this part of the chromosome but cosmids carrying acr-2 do not rescue lev-9. A full length acr-2 cDNA has been isolated and sequenced and the genomic structure is being determined. The cDNA encodes a putative non-a subunit of a nicotinic acetylcholine receptor which shows many of the conserved features of vertebrate and invertebrate non-alpha nAChR subunits, for instance the four putative transmembrane domains and the cysteine delimited extracellular loop. Amongst other nAChR subunits acr-2 is most similar to the Drosophila ARD subunit to which it shows 49% identity. In order to investigate the function of the subunit acr-2, cRNA was produced by in vitro transcription and micro-injected into Xenopus oocytes. When expressed alone acr-2 shows no levamisole gated channel activity. Unusually, such activity is observed when acr-2 is co-expressed with another C. elegans non-a subunit (lev-1, Fleming et al, in preparation). Such activity is normally only observed in the presence of an a-subunit. Whether this activity is the result of endogenous a subunit activity or reflects novel behaviour by the C. elegans subunits remains to be determined. When co expressed with a C. elegans a subunit (unc-38, which is itself unable to form functional homo-oligomers) acr-2 contributed to the formation of a functional (a, non-a) channel. In both of these cases the levamisole induced current was inhibited by mecamylamine.

Dang T et al. (1996) Worm Breeder's Gazette "Isolation and Classification of C. elegans Mutants with the Endomitotic Oocytes (Emo) Phenotype"

Le MT, Francis R, Schedl TB, McCarter JP, Dang T

[Worm Breeder's Gazette, 1996]

We are interested in the regulation of meiotic maturation and ovulation. At maturation, the oocyte cell cycle progresses from prophase to metaphase. At ovulation, the oocyte exits the gonad arm. In many species, maturation and ovulation are coupled. In several characterized C. elegans mutants with the Emo phenotype, maturation is not followed by normal ovulation and the mature oocyte is trapped in the gonad arm where it endomitotically replicates its DNA (Emo = endomitotic oocytes in the gonad arm). Genes with mutant alleles where defective ovulation leads to the Emo phenotype include emo-1 (1), lin-3 (2), let-23 (2), mup-2 (3), and ceh-18 (4). Ablation of the somatic sheath or spermatheca can also disrupt ovulation and cause the Emo phenotype (5). Ablation of the proximal myoepithelial sheath eliminates the contractions necessary to expel the oocyte from the arm. Ablation of the narrow distal spermatheca allows the oocyte to 'slip-back' from the spermatheca into the gonad arm following ovulation. We have isolated and mapped additional mutants with the Emo phenotype. Mutants were isolated in screens for recessive steriles and include: Gene, Allele, Chromosome emo-2(oz136), LG III emo-3(oz138), LG IV emo-5(oz148), LG II emo-6(oz154), LG III Emo(oz196), LG III Emo(oz197), LG V Emo(oz198), LG IV Emo(oz199), LG III Emo(oz200) (6), LG IV Some of the new emo mutants, such as oz136, oz154, and oz196, appear similar in their terminal phenotype to the previously characterized ovulation defective mutants with the Emo phenotype. Endomitotic figures are most prominent in the very proximal portion of the gonad. However, in other new emo mutants, such as oz138, oz198, and oz200, endomitotic figures extend further distally. oz138 is also unusual in that its Emo phenotype is suppressed by feminization. In previously characterized mutants with the Emo phenotype, feminization delayed the onset of endomitosis (because it delays maturation and ovulation), but did not suppress the phenotype. Mating into oz138 females with wild type males results in a maternal effect embryonic lethality, but no Emo phenotype. The oz138 Emo phenotype is therefore dependent on oz138 sperm. In oz197, abnormalities are seen in chromosomal morphology in distal oocytes which are not yet endomitotic. This suggests a primary defects in meiotic prophase progression in this mutant rather than a defect in ovulation. In summary, initial characterization suggests at least three phenotypic categories for mutants with the Emo phenotype. These include 1) ovulation defective mutants, 2) sperm defective mutants, and 3) meiotic prophase defective mutants. Further characterization is in progress. 1 Iwasaki et al., (1996). JCB, In press. 2 McCarter et al., (1996). Midwest Worm Meeting Abstract. 3 Myers et al., (1996). JCB, 132:1061-1077. 4 Greenstein, et al., (1994). Genes & Dev., 8:1935-1948. 5 McCarter et al., (1996). In preparation. 6 We thank Eric Lambie for isolating oz200.

McKay JP et al. (1998) Worm Breeder's Gazette "eat-2 encodes a non-alpha nicotinic acetylcholine receptor subunit"

McKay JP, Raizen DM, Avery L

[Worm Breeder's Gazette, 1998]

In the presence of an abundant supply of bacteria, worms eat as much as they can by initiating rapid pharyngeal pumping. Previously we showed that the motorneuron MC is necessary for this rapid pharyngeal pumping (1,2). Genetic and pharmacological evidence suggests that the MC neurotransmitter is acetylcholine (ACh) and that it acts directly on pharyngeal muscle (3). eat-2 is a gene identified in a screen designed to find mutations that eliminate neurotransmission from MC. eat-2 worms are unable to pump rapidly in the presence of food and they are starved. Two results implicate eat-2 in MC neurotransmission. First, electrical recordings from the pharynxes of eat-2 mutants were similar to recordings from MC- worms. Also, when MC was ablated in two possible loss of function alleles, pharyngeal pumping rate did not decrease when compared to the same mutants in which MC was intact (3). Fourteen independent recessive mutations were identified in eat-2 that all caused a starved appearance and reduced pumping rate. There is complex intragenic complementation among the 14 eat-2 alleles as well as allele specific interactions between eat-2 and a semidominant allele of another slow pumping mutant, eat-18 (3). eat-18 is MC- by the same criteria as eat-2. A ! model consistent with these data is that eat-2 and eat-18 interact in a protein complex that is involved in ACh neurotransmission. Mapping experiments placed eat-2 at the end of the right arm of LGII near unc-52, a region of the genome that is currently being sequenced. Eric Jorgenson pointed out to us that a recent sequence update contained a nicotinic ACh receptor subunit in an area consistent with the map position of eat-2. Transformation of eat-2 mutants with a YAC containing the nicotinic receptor subunit rescued the slow pumping and starved phenotypes. To determine if the nicotinic receptor was responsible for rescuing eat-2 we used PCR to amplify 10kb of genomic DNA that included the coding region of the receptor and 4kb of upstream sequence. This 10kb PCR product was sufficient to rescue eat-2, showing that the nicotinic receptor is encoded by eat-2. We also found that the eat-2 allele ad451 contains a missense mutation that changes a glutamate to a tyrosine in the amino-terminal extracellular region of the protein. No specific func! tion is known for this amino acid but it is highly conserved among nicotinic receptors. We were able to rescue MC nuerotransmission in an eat-2 mutant by expressing an eat-2 genomic clone in pharyngeal muscle using the myo-2 promoter. Rescue by this construct shows activity of eat-2 in pharyngeal muscle is sufficient to restore MC neurotransmission. Comparison of eat-2 with other receptor subunits shows it is most similar to vertebrate alpha7 subunits of neuronal nicotinic ACh receptors. However it does not have the vicinal cysteines present near the ACh binding site characteristic of all alpha subunits and is therefore a non-alpha subunit.

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.