Zhou MH et al. (1996) Worm Breeder's Gazette "Liquid Culture of the Worms with a Fermentor"

Zhou MH, Yang H, Walthall WW

[Worm Breeder's Gazette, 1996]

The yield of C. elegans and their food (E.coli) are relatively low in the large liquid cultures growth (Koelle et al.,1994) and the whole growth process is tedious. Here we report the successful growth of C.elegans with a fermentor. The following protocol is based on the protocol by Michael Koelle and Tory Herman (1994) from Internet.

Hengartner MO et al. (1992) Worm Breeder's Gazette "unc-50, a Gene Required for Acetylcholine Receptor Function, Encodes an Unfamiliar Protein"

Horvitz HR, Tsung N, Hengartner MO, Lewis JA

[Worm Breeder's Gazette, 1992]

unc-50, a gene requiired for acetylcholine receptor function, encodes an unfamiliar protein Michael Hengartner, Nancy Tsung, Jim Lewist, and Bob Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA. t Division of Life Sciences, U. of Texas at San Antonio, San Antonio, IX 78249

Parker JA et al. (1999) Worm Breeder's Gazette "C. elegans HIP1 expression is restricted to a few neurons"

Parker JA, Rose AM

[Worm Breeder's Gazette, 1999]

We have been characterizing the expression of ZK370.3, which shares sequence identity with human HIP1 (Huntingtin Interacting Protein) and Sla2p of yeast (Synthetic lethal with Actin Binding protein 1). ZK370.3 spans approximately 4.5 kb of genomic DNA, consisting of 10 exons and codes for a 2.7 kb message. The 923 amino acid product has a molecular weight of approximately 100 kDa, and shares identity with cytoskeletal associated protein talin. To study the expression of C. elegans HIP1, 1000 nucleotides of the genes 5 upstream region, along with the first 30 nucleotides of the coding region, were fused to a GFP fusion vector (pPD95.75, kindly provided by A. Fire). The fusion construct was injected into dpy-5 animals, along with a dpy-5 rescuing clone (pCeh361, kindly provided by C. Thacker). Rescued animals were examined, and fluorescence was detected in the pharynx and tail of the animals. Expression in the pharynx appears to be limited to a set of neurons, possibly M4 and I3. To better understand the function of the C. elegans HIP1 gene product, we conducted a yeast two-hybrid screen in collaboration with the Michael Hayden laboratory at the University of British Columbia. We used a full length C. elegans HIP1 cDNA fused to the bait vector pGBT9 (Clonetech) to screen a library kindly provided by R. Barstead. Two clones were isolated from the screen. The first shows similarity to a type II transcription factor, and the second shows similarity to the Drosophila hedgehog protein. We are currently characterizing the protein interactions.

Blumenthal TE et al. (1994) Worm Breeder's Gazette "C. elegans U2AF 65."

Zorio DAR, Lea K, Blumenthal TE

[Worm Breeder's Gazette, 1994]

C. elegans U2AF65

Yuan JY et al. (1986) Worm Breeder's Gazette "progress in the cloning of ced-3 and ced-4."

Horvitz HR, Yuan JY

[Worm Breeder's Gazette, 1986]

ced-3 and ced-4 are two genes required for the initiation of programmed cell death during the development of C. elegans (Ellis & Horvitz, Cell 44: 817-829 1986). We are trying to clone ced-3 and ced- 4. We have started by walking from a restriction fragment length polymorphism closely linked to ced-3. A 5.1 kb EcoRI Bristol-specific Tc1 band and a 2.7 kb EcoRI Bergerac-specific Tc1 band had been mapped between ced-3 and unc-26 (which are about 0.2% apart) by Hilary Ellis ( C. elegans Newsletter Vol. 8 No. 3). We have identified two cosmids that contain the 5.1 kb Bristol-specific Tc1 band. Alan Coulson and John Sulston have identified two more cosmids that overlap with our cosmids. We will now use these clones to probe genomic blots of the existing ced-3 mutants, including both the seven EMS-induced alleles and the three putative Tc1-induced alleles (see below). We are also using the mut-2 mutator strain TR679 generated by Phil Anderson's laboratory (C. elegans Newsletter Vol. 9 No. l) to isolate spontaneous ced-3 and ced-4 mutants, in the hope of identifying Tc1-induced mutations. Such mutants could be used for the direct cloning of these genes. We have used the fact that ced-3 and ced-4 are recessive suppressors of egl-1 to do complementation screen experiments to identify new ced-3 and ced-4 alleles. egl-1 males were mated with mut-2 hermaphrodites to construct a mut-2; unc-76 strain. Several putative mut-2; unc-76 strains were identified on the basis of expressing an Egl e as well as being weakly Him and giving rise to spontaneous mutants. We have mated ced-3 egl-1 males with mut-2; unc-76 hermaphrodites and looked for rare nonEgl F1 progeny. So far, we have screened about 16,000 F1's and have found three new alleles of ced-3. All three seem to be linked to a recessive lethal mutation. We do not know yet whether these lethals are in ced-3. We also have mated unc- 79 egl-1 males with mut-2; unc- 76 hermaphrodites and looked for rare non-Egl F1 progeny. We have screened about 30,000 F1's and have found one new allele of ced-4. This allele is homozygous viable. We have identified three spontaneous revertants of this new ced-4 allele. We are now mapping the Tcl's in our new ced-3 and ced-4 strains to see if these new ced-3 and ced-4 mutations were generated by Tcl transposition.

Morgan WR (1994) Worm Breeder's Gazette "An Electronic Discussion Group for C. elegans Researchers."

Morgan WR

[Worm Breeder's Gazette, 1994]

An Electronic Discussion Group For C. elegans Researchers

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.

Aamodt EJ et al. (1990) Worm Breeder's Gazette "Transformation of C."

McGhee JD, Aamodt EJ, Chung MA

[Worm Breeder's Gazette, 1990]

To identify the control regions of the C. elegans gut-specific esterase gene (ges-1), we are using a variety of approaches including a comparison of the upstream control regions of ges-1 and the homologous esterase gene from C. briggsae (cloned and sequenced from the Baillie library by Brian Kennedy and Fran Allen). For this approach to be valid, we needed to show that the esterase gene from one species is properly expressed in the other species. It was easy to show that the C. briggsae esterase is expressed in the gut of C. elegans by transforming the C. briggsae gene into a ges-1(0) strain of C. elegans and then staining the transformants for esterase. We did this experiment using both transient transformation and extrachromosomal heritable transformation. To show that the C. elegans properly expressed in C. briggsae, we needed to make a strain of C. briggsae that was transformed with the C. elegans tried the rol-6 dominant mutant gene and followed the transformation procedure of Mello et al. (WBG 11-1:18 and WBG 11-3:12). Roller strains of C. briggsae were easily obtained (the rachis of C. briggsae is even easier to hit than that of C. elegans). When the ges-1 gene was used in a cotransformation with the rol-6 gene all of the esterase expression was confined to the guts of the transformants i.e. the transformants looked exactly like wildtype C. briggsae. We then used isoelectric focusing gels on extracts of the transformed C. briggsae followed by staining of the gel for esterase activity to show that the C. elegans indeed being expressed. Thus, the C. elegans expressed in C. briggsae and it is expressed only in the gut. A deleted construct of ges-1 that contains only 521 base pairs 5' from the translation initiation codon is expressed in the pharynx muscles of C. elegans (E. Aamodt, M. Chung and J. McGhee, manuscript submitted). When this deleted ges-1 gene was transformed into C. briggsae, the transformants also express the esterase gene in their pharynx. In other words, not only is the intact gene properly expressed but the ectopic expression of the deleted ges-1 is identical between the two species. Thus, both ges-1 and the mutant rol-6 genes are properly expressed in C. briggsae. Cotransformation of C. briggsae using the rol-6 marker gene should be useful to others who wish to see if their favorite C. elegans gene works in C. briggsae.

Giebelhaus DH et al. (1990) Worm Breeder's Gazette "Spectrin Genes in C. elegans?"

Clark-Maquire SD, Mains PE, Giebelhaus DH

[Worm Breeder's Gazette, 1990]

Nonerythyroid spectrin (fodrin) and its associated proteins (protein 4.1 and ankyrin) have been implicated in the establishment of specialized membrane-cytoskeletal domains in differentiating cells. Since the spectrin family of proteins is phylogenetically widely distributed (Giebelhaus et al, 1987, Byers et al., 1987), we have begun to investigate the expression and role of possible fodrin-like genes in C. elegans. Southern blots of genomic DNA from C. elegans and C. briggsae were hybridized with a 1.2 kb human alpha-fodrin clone under moderate stringency (Hybridization: 30% formamide, 5xSSC, 48 C. Washes: 2xSSC, 1% SDS, 55 C). Two distinct bands were seen from DNA digested with EcoRI or HindIII from both C. elegans and C. briggsae. We are currently screening genomic libraries with the human alpha-fodrin clone to isolate clones which may contain C. elegans spectrin-like genes.

Kuersten S et al. (1994) Worm Breeder's Gazette "Mono- and Polycistronic pre-mRNA Splicing in a C. elegans Embryo Extract."

Blumenthal TE, Kuersten S

[Worm Breeder's Gazette, 1994]

Mono- and Polycistronic pre-mRNA Splicing in a C. elegans embryo extract