Zarkower D et al. (1994) Worm Breeder's Gazette "mab-3 YAC Rescue"

De Bono M, Hodgkin JA, Zarkower D

[Worm Breeder's Gazette, 1994]

mab-3 YAC rescue David Zarkower, Mario de Bono, and Jonathan Hodgkin MRC Laboratory of Molecular Biology, Cambridge, England

De Stasio E et al. (1994) Worm Breeder's Gazette "Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea."

De Stasio E, Stanislaus D, Lephoto C

[Worm Breeder's Gazette, 1994]

Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911

Plasterk RHA et al. (1994) Worm Breeder's Gazette "Reverse Genetics Using Transposon Tc1: A Progress Report"

Plasterk RHA, de Vroomen M

[Worm Breeder's Gazette, 1994]

Reverse genetics using transposon Tcl: A progress report Ronald H.A. Plasterk, Marianne de Vroomen, the Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121,1066 CX Amsterdam, the Netherlands, fax + 31 20 6172625, phone +31 20 5122081, E-mail RPLAS@NKI.NL

Wiegner O et al. (1996) Worm Breeder's Gazette "Large communication channels in C. elegans embryos"

Bossinger O, Schierenberg E, Wiegner O

[Worm Breeder's Gazette, 1996]

The pattern of cell-cell communication in embryos of C.elegans has beeninvestigated by injecting tracer dyes into individual blastomeres (Bossinger and Schierenberg, 1992). It has been shown that in embryos from the 4-cell stage onward all blastomeres are well coupled for the diffusion of negatively charged Lucifer Yellow (LY; MW 457 Da) while uncharged Rhodamin-coupled dextran (MW 4.000 - 70.000 Da) remains restricted to the injected cell and its descendants. However, in Cephalobus spec. (another soil nematode), LY and Rhodamin-dextran (even at a molecular weight of 70.000 Da) diffuse along discrete pathways from cell to cell (Bossinger and Schierenberg, 1996). We extended our dye-coupling studies in C. elegans using negatively charged dextrans (LY-dextran and FITC-dextran) with molecular weights of 10.000 and 70.000 Da. Suprisingly, we found that in embryos between the 4-and the 24-cell stage all blastomeres are coupled for negatively charged dextrans whereas under all tested conditions uncharged Rhodamin-dextran (MW 10.000 Da) remains in the injected cell and its descendants. The diffusion of the negatively charged dyes appears to occur quickly and freely between all blastomeres. In contrast to Cephalobus we did not observe preferential pathways of dye spread from the injected cell, suggesting that all blastomeres are equally coupled. However at the 24-cell stage, when the primordial germline cell P4 has been generated, diffusion into D and P4 cells becomes retarded.The diffusion block lasts approximately 5 minutes until D joins the somatic compartment and P4 remains uncoupled for at least 20 min. A similar observation has been made for the much smaller LY in C. elegans indicating that it is a diffusion block and not a reduction in channel diameter. In summary, our findings suggest that in the developing C. elegans embryo all somatic blastomeres are coupled by communication channels much larger than conventional gap junctions. These channels seem to be permeable for negatively charged molecules up to a molecular weight of at least 70.000 Da but are impermeable to uncharged dextrans. Moreover, these channels seem to be equally present between all blastomeres of the somatic cells. Presently we can only speculate about the function of these communication pathways. Bossinger, O; Schierenberg, E. (1992) Dev. Biol. 151: 401-409 Bossinger, O; Schierenberg, E. (1996) Dev. Genes Evol. 206: 25-34

Loer CM et al. (1990) Worm Breeder's Gazette "Neurotransmitter Synthesis in Worms: An Aromatic Amino Acid Hydroxylase Gene from C."

Kenyon CJ, Loer CM

[Worm Breeder's Gazette, 1990]

Specification of neurotransmitter phenotype is an important requirement for generating a functional nervous system. A specific neurotransmitter phenotype requires the coordinate expression of biosynthetic enzymes, a degradation or re-uptake system, and perhaps a specific packaging apparatus. The Drosophila dopa decarboxylase gene ( Ddc), the last enzyme required for serotonin (5-HT) and dopamine (DA) synthesis is differentially regulated and spliced in the nervous system and the epidermis, where is required for cuticle synthesis ( Morgan et al., 1986, EMBO J. 5: 3335; Bray et al., 1988, EMBO J. 7:177). A number of neuron-specific enhancers in the 5' region of Ddc have been identified, including one that binds a POU-type homeodomain protein. Mutation in this enhancer region eliminate DA expression in a subset of dopaminergic CNS neurons (Johnson and Hirsh, 1990, Nature 343: 467). The biogenic amine neurotransmitters 5-HT and DA are used by a number of neurons in C. elegans, including the HSN, NSM, and some CPs (5-HT) and ADE, PDE, CEPs, and Ray neurons 5A, 7A, and 9A (DA) ( Sulston et al., 1975, J. Comp. Neurol., 163: 215; Horvitz et al., 1982, Science 216:1012). 5-HT and DA are derived by two enzymatic steps from the aromatic amino acids tryptophan and tyrosine, respectively (see below). In vertebrates and flies, specific enzymes catalyze the first step in the reaction (i.e., tryptophan hydroxylase and tyrosine hydroxylase), whereas the decarboxylation step for both 5- HT and DA is catalyzed by a single enzyme with broader specificity (i. e., dopa decarboxylase). [See Figure 1] As a first step towards analyzing the control of neurotransmitter phenotype, we are seeking to clone genes encoding biosynthetic enzymes for these neurotransmitters. We used the polymerase chain reaction ( PCR) with degenerate oligonucleotides encoding highly conserved regions of aromatic amino acid hydroxylases to amplify a genomic fragment encoding a C. elegans AAA Hydroxylase. Of 5 clones sequenced, 3 were garbage and 2 encoded the AA sequence shown below. As indicated, the sequence included a typical small worm intron with good consensus splice donor and acceptor sequences at an evolutionarily conserved splice site. Typical C. elegans codon usage was also apparent in the coding region sequenced. [See Figure 2] We used this 185 bp fragment (TH1,2) to isolate a lambda genomic clone and also to probe a genomic southern blot. At high stringency, the TH1,2 fragment showed a single band in all lanes except for a SacI digest (TH1,2 contains a SacI site); thus, the gene seems to be found in a single copy in the genome. We are using the lambda genomic clone to isolate cDNAs for sequencing. We plan to use in situ hybridization to define sites of expression of the gene. Since we may not be able to determine what kind of AAA hydroxlase this gene encodes by sequence alone, such information may clearly indicate the probable specificity of the enzyme (e.g., if it is found only in dopaminergic cells, it should be a tyrosine hydroxylase!). We also plan to fuse -gal or another reporter gene into the AAAH gene to use for identifying mutations that alter its expression and to examine expression in certain known mutants. Such a screen should yield genes that are required for the regulation of neurotransmitter synthesis (the cat mutations may be alleles of such genes) as well as genes that determine which neurons produce a specific neurotransmitter.

Russell RL et al. (1977) Worm Breeder's Gazette "a cellular model of wave propagation in C elegans."

Byerly L, Russell RL

[Worm Breeder's Gazette, 1977]

Starting with the published anatomy of the ventral nerve cord (White et al., 1976), we have developed a model for contractile wave propagation in C. elegans. This model attempts to rationalize the known circuitry of the ventral cord with several known features of C . elegans movement, most notably the ability of waves to propagate at different rates and in opposite directions, and the ability of the animal to reverse immediately from an existing waveform. The central features of the model are that the O motor neurons act inhibitorily to keep dorsal and ventral sides in anti-coordination, that both the A and the B motor neurons act excitatorily to facilitate wave propagation, that the A motor neurons are active during backward movement while the B motor neurons are active during forward movement, that the long, 'undifferentiated' portions of the A and B motor neurons adjacent to their neuromuscular outputs are stretch sensors, and that the alpha and interneurons act as 'command fibers' for the A and B motorneurons respectively. This model accounts satisfactorily for several otherwise difficult features of movement, and seems in accord with what is known about sensory inputs that can affect movement; it also makes an interesting suggestion about the lineage division which separates the VA and VB motorneurons. Building on Stretton's (personal communication) report of a commissural 'repeat unit' in Ascaris and Sulston's (1976) lineage study of the C. elegans central cord, we have examined the notion that the motor neurons of C. elegans and Ascaris might be very similar. Under this assumption, we predicted that the commissure repeat unit of Ascaris should contain Ascaris homologs of 1 DA, 1 DE, 1 DD, 2 VD and 2 DAS cells. By initial stimulation near the lateral line and by later use of preparations from which broad muscle strips had been removed to expose the commissures, we stimulated individual commissures or commissure pairs in Acaris and determined the nature of any observed effect on muscles (dorsal or Ventral, excitatory or inhibitory). The results strongly suggest that the DD and VD cells are inhibitory, whereas the DA and DAS cells are strongly excitatory and the DB cells are weakly so. These results are predicted by the model.

Nawrocki LW et al. (1988) Worm Breeder's Gazette "characterisationa and cloning of unc-24."

Thomson JN, Nawrocki LW, Southgate E

[Worm Breeder's Gazette, 1988]

The behavioral phenotype of unc-24 worms is characterized by severely kinked L1 worms that do not wriggle more than one quarter body length in either forward or backward directions, whereas worms from the L2 stage onward toward adults are only slightly kinked in motion and make progress similar to N2 worms. These observations appear to indicate that the unc-24 gene may be affecting the embryonic motoneurones (DAs, DBs and DDs). Reconstruction of three different alleles of unc-24 have not been consistent. The canonical allele (e138) reconstruction shows DA motoneurones to be lacking commissures to the dorsal musculature. A Tc1 allele (e2386) reconstruction contains one DA motoneurone with the same aberration but another with normal morphology. The P32 induced allele (e927) reconstruction presents DA motoneurones with normal morphologies, but the commissures of all D motoneurones (DAs, DBs and DDs) proceed toward the dorsal cord on the opposite side to the wild type condition. To clear up this ambiguity, a reconstruction of an L1 from, the canonical strain is being completed. The unc-24 gene has been isolated. A 5.6kb BamHI Tc1 containing fragment found in an allele (e2386) isolated from the TR679 strain that also shifts to a 4.0kb BamHI fragment in revertants and Bristol strains was cloned into pUC 8. A 1.0kb non-Tc1 containing fragment was isolated from this plasmid by BamHI and EcoRV digestion and was used as a probe to screen the Coulson and Sulston cosmid library. Two true positives were identified that mapped to a contig containing the vit-6 gene on linkage group IV. Fortunately, a larger cosmid in this contig which covered both newly identified cosmids had been used as an in situ hybridization probe by Rita Fishpool and Donna Albertson and showed binding to chromosome IV. The same 1.0kb non-Tc1 fragment was used to isolate 16 lambda clones from the 2a cDNA library of Julie Ahringer. The lambda inserts range in size from 0.6kb to 2.6kb. The largest of these was used as a probe and showed polymorphic band shifts on the genomic Southerns composed of different alleles of unc- 24. This insert is being sequenced.

Schinkmann K et al. (1996) Worm Breeder's Gazette "unc-4 regulates neurotransmitter (FMRF-amide-like)expression in VC motor neurons"

Li C, Miller DM, Nonet ML, Schinkmann K, Emambokus N, Butkiewicz K

[Worm Breeder's Gazette, 1996]

unc-4 encodes a homeodomain protein [1] that determines the pattern of synaptic input to VA motor neurons in the ventral cord [2]. unc-4 mutations lead to an uncoordinated phenotype whereby animals can crawl forward but not backward. Electron microscopic examination of unc-4(e120) revealed that VA motor neurons, which are necessary for backward movement, receive synaptic input normally reserved for their sister cells, the VB motor neurons [2]. Experiments with unc-4-lacZ reporter genes detected expression in all A-type motor neurons (SAB, DA, VA) and demonstrated that unc-4 activity in the VAs in L1/L2 larvae is sufficient to establish connections with the appropriate interneurons [3]. Thus, one unc-4 function is to distinguish the fates of VA and VB sisters cells with respect to presynaptic input.

Zwaal RR et al. (1991) Worm Breeder's Gazette "Tc1 insertions in the gpa- genes"

Plasterk RHA, Anderson P, Rushforth AM, Zwaal RR

[Worm Breeder's Gazette, 1991]

Guanine nucleotide binding proteins (G-proteins) play a role in a variety of transmembrane signal transduction pathways. They consist of 3 subunits, the alpha-, -, and gamma-subunit, of which the alpha- subunit contains the guanine nucleotide binding site, determining the activity or inactivity of the protein. The variability in structure of the alpha-subunits seems to cause the variability in G-protein function. For much of the Galpha-subunits however the specific signal transduction pathway they participate in is not known, nor is the receptor- or effector molecule they interact with. In C. elegans a number of alpha-subunit genes has been discovered. The genomic sequence of novel, possibly C. elegans specific genes, gpa-1, gpa-2 and gpa-3, is known, as well as cDNA sequences of goa-1, gq-1 and gsa-1, genes homologous to resp. mammalian Goalpha, Gqalpha and GSalpha (da Silva and Plasterk, JMB 215, 483-487 (1990); Lochrie et al., Cell Regulations 2, 135-154 (1991); Mendel and Sternberg pers. comm.; Oshima, pers. comm.). To investigate the functions of the different alpha-subunits we are trying to obtain Tc1 knock-out mutants in the C. elegans specific gpa- genes, using the PCR technique to screen populations of worms for Tc1 insertions, followed by sib-selection to isolate the mutant animal (as described by Rushforth et al., WBG 11/5, 65). Via this method, starting with approximately 25,000 animals of the MT3126 strain, a worm was obtained in which a Tc1 element is inserted in the gpa-2 gene between the translation stop codon and the first putative poly-adenylation site. The animal seems to have a wild-type phenotype although RNA levels are not checked yet to see if it really is a knock-out. In the same screen a second Tc1 insertion was found in the gpa-2 gene in an intron and one was found in the gpa-1 gene. Both of these are inheritable and are now being purified. A general conclusion can be that the method of PCR detection and sib- selection of Tc1 insertions seems widely applicable. We found inheritable insertions in both genes we looked at, at a frequency that makes purification of the mutant relatively easy. Elaborating this approach we hope to be able to systematically study the role of different G-proteins in C. elegans development.

Mueller F et al. (1991) Worm Breeder's Gazette "CHROMATIN ELIMINATION IN ASCARIS LUMBRICOIDES TAKES PLACE WITHIN A SHORT, PRECISELY DEFINED CHROMOSOMAL DNA SEGMENT"

Tobler H, Mueller F, Wicky C

[Worm Breeder's Gazette, 1991]

The chromosomes of Ascaris lumbricoides undergo chromatin diminution in those cells destined to form the somatic lineages of the animal. During this process, the heterochromatic ends of the chromosomes break off and eventually degrade in the cytoplasm. The shortened chromosomes are stable, suggesting that elimination is accompanied by a de novo formation of telomeres. In order to test this hypothesis, we decided to analyze the structure of the somatic telomeres of A. lumbricoides. Bal 31 experiments revealed that they carry multiple copies of the tandemly repeated hexamer TTAGGC, a sequence that is similar to those found at the chromosomal ends of vertebrates and some lower eukaryotes. Since we expected the regions flanking the somatic telomeres to be involved in the elimination process, we designed a cloning strategy for their isolation from the genome of A. lumbricoides. One of the positive clones obtained (pTel 1) was analyzed in detail. By sequencing we found pTel 1 to carry 27 perfect repeats of the hexamer TTAGGC, oriented 5' to 3' towards the end of the fragment. Thus, the orientation of the G-rich strand corresponds exactly to the defined orientation of eukaryotic telomeric repeats. The subtelomeric DNA sequences of pTel 1 are unique and therefore specific for a single somatic telomere. The corresponding region within the germ line genome is located at an internal chromosomal site rather than on a telomere. Elimination, however, does not take place merely at the locus cloned in pTel 1, but also at many more sites, all of them being scattered throughout a 3 kb long specific DNA segment within this region. The nucleotide sequence of this DNA segment is very AT-rich, but so far no specific elimination signals could be identified. Comparison of pTel 1 with the corresponding germ line sequences shows that chromosomal fragmentation is followed by the addition of tandem arrays of the telomeric repeating units TTAGGC, which are not present on the germ line chromosome at this site. The interesting question, whether this de novo formation of telomeres is carried out by the action of a telomere terminal transferase activity, is currently investigated in our laboratory.