Maine EM et al. (1994) Worm Breeder's Gazette "Mutations That Enhance glp-1 Identify Genes Required For Various Aspects of Germline Development."

Gelber MB, Smardon AM, Shu P, Qiao L, Maine EM, Lissemore JL

[Worm Breeder's Gazette, 1994]

Mutations that enhance glp-1 identify genes required for various aspects of germline development. Eleanor Maine, Li Qiao, Jim Lissemore-, Pei Shu, Anne Smardon, and Melanie Gelber. Biology Dept., Syracuse University, Syracuse, NY 13244 and Biology Dept., John Carroll University, Cleveland, OH 44118.

Lambie EJ (1994) Worm Breeder's Gazette "Easiest Worm Plate Medium"

Lambie EJ

[Worm Breeder's Gazette, 1994]

In my abstract for the '93 C. elegans meeting, I provided the recipe for Flo Youngren's EZ worm mix. Since then, I have come up with a better formulation, which has less salt (for superior lawn consistency) and does not include yeast extract. These plates have three main advantages over conventional NGM plates: 1 ) Annoying salt crystals do not readily form 2) No stock solutions need to be added after autoclaving, and 3) There is minimal fuss and/or room for error when undergraduate assistants make up the plates. Here is the recipe for MYOB (Modified Youngren's, Only Bacto-peptone). For 100 liters of plates: Combine 55 g Tris-CI (Sigma reagent grade), 24 g Tris-OH (Sigma reagent grade), 310 g Bacto Peptone (Difco), 800 mg Cholesterol and 200 g NaCI. Mix thoroughly and be sure to avoid chunks; this powdered mixture can be stored for many months prior to use. Dissolve 5.9 g in 1 liter of water, add 20 g of agar and autoclave.

Dusenbery DB (1976) Worm Breeder's Gazette "chemotaxis."

Dusenbery DB

[Worm Breeder's Gazette, 1976]

The group of mutants previously described (Genetics 80: 279, 1975) by Sheridan, Russell and myself have been further characterized. Additional complementation tests indicate that RS2 and RS7 belong to the same group as DD79, that DD72 and DD75 complement with all other groups including one another. RS3 and DD76 remain unclassified. Sixteen of these strains have been tested for their chemotactic responses in 11 different tests. The six strains belonging to the complementation group of DD79 have similar patterns of responsiveness. This pattern is a nearly normal response to C02 in pH 6.0 phosphate buffer, to acid, and to pyridine and little if any response to Na+, Cl- , OH-, CO2 in pH 8.8 borate, cAMP, and D-tryptophan. A particularly interesting observation is that three other strains (representing at least two different complementation groups) avoid cyclic AMP instead of being attracted. All but two of the 16 strains had substantial responses to C02 in phosphate and to acid. Most strains were defective in most other tests. Recently I tested two of Don Riddle's dauer-defective mutants (E1378 and E1382) they also demonstrated relatively normal responses to C02 in phosphate and to acid. This observation prompted me to test the Caltech chemotaxis mutants for defective dauer production. Initial results indicate that RS4 and DD71 do not make dauer larvae in normal numbers and that DD73 makes dauers even in the presence of bacteria. Thus it looks like lack of chemotaxis to NaCl and defective dauer production select for much the same kind of mutants. I hope to collect a much different set of mutants by selecting for those defective in avoidance of acid or C02 in phosphate. We have recently discovered that the worm avoids mM concentrations of glucose, fructose, and mannose. Experiments to further define the specificity are in progress. Questions: 1. Does anyone have suggestions as to the possible adaptive significance of the chemotactic responses such as attraction to ions or pyridine and avoidance of D-tryptophan or certain hexoses? 2. Is anyone presently working on the identification of 'natural' chemical stimuli such as those in bacterial cultures?

Zhen M et al. (1997) Worm Breeder's Gazette "A genetic screen for mutations affecting D neuron synapse formation and synaptic plasticity"

Jin YS, Zhen M

[Worm Breeder's Gazette, 1997]

Long-term synaptic plasticity is widely observed in diverse organisms from insects to mammals. It often involves two aspects of concurrent changes in the neurons: the structural change and the change in synaptic strength. In C. elegans, one example of such neuronal plasticity is the synaptic rewiring of the DD motor neurons. DD motor neurons are GABAergic inhibitory neurons born during embryogenesis. In L1 larval stage, DDs receive synaptic inputs from DA and DB motor neurons on the dorsal side and innervate ventral body wall muscles. However in older larvae and adults, DD neurons receive inputs from VA and VB neurons on the ventral side and form NMJs on the dorsal side. The original DD neuron connectivity is replaced by another class of GABAergic neurons, VDs, which are generated by postembryonic cell division (l). Little is known about either the detailed process of the synaptic rewiring of DD neurons or the molecular mechanism underlying such a synaptic polarity switch. We are interested in elucidating the molecular mechanisms regulating the remodeling of DD synapses. We are using a synapse specific GFP marker to visualize the synaptic rewiring (2), and to perform a genetic screen for genes regulating the structural and synaptic changes of DD neurons during the rewiring. The GFP marker, Punc-25-VAMP-GFP, is specifically expressed in the pre-synaptic zones of DD and VD neurons (together called type D neurons). VAMP-GFP chimeric protein is localized to the presynaptic zone of neurons (3). The promoter of the unc-25 gene, which encodes GABA biosynthetic enzyme glutamic acid decarboxylase, is used to specifically activate the expression of VAMP-GFP in DD neurons in L1 stage and DD&VD neurons in L2 to adult stages. In this screen we are recovering mutant animals showing any abnormal GFP expression patterns in the D neurons so that a wide spectrum of genes involved in the synapse formation of the D neurons can be isolated. But the further characterization will be focused on those disrupting the DD neuron synaptic rewiring (most likely to be the ones with little or abnormal GFP expression in the dorsal cord from L2 stage onward). So far we have screened 8,000 haploid genomes using EMS as the mutagen. New alleles of known genes required for axonal growth and synaptic formation, such as unc-5, unc-6, unc-11, unc-30, unc-33, unc-51, unc-76, and unc-104 have been isolated. The new allele (ul9) of unc-104 gene, which encodes the kinesin heavy chain and is essential for vesicle transport, is particularly interesting since the mutant animals show very little abnormality in movement. The GFP expression pattern in ju19 indicates that this mutant is able to make nearly normal synapses in the ventral cord, but fail to make synapses in the dorsal code. This suggests that ju19 may be defective in long-distance vesicle transport. More than 100 lethal/sterile mutants, most of which are defective in postembryonic cell division (thus fail to generate VD neurons) were also isolated from the screen. Intriguingly we have isolated a number of viable mutants with severely disrupted GFP expression pattern in either the DD or VD neurons (or in both), yet only subtle abnormality in locomotion. Because of the subtle visible phenotype of these mutants, they might represent new genes which are essential for the normal D neuron synaptic formation, but fail to be uncovered by previous unc screens. We are in the process of characterizing and mapping these mutations. Ref: 1) White JG, Albertson DG and Anness M. Nature 271: 764-766, 1978 2) Jin YS and Horvitz HR. 1995 Worm Meeting Abstract: 291. 3) Nonet ML WBG 13(5): 40, 1995

van Luenen HGAM et al. (1993) Worm Breeder's Gazette "Mechanism of Tc Transposition: Starting at the End"

Vos JC, van Luenen HGAM, Plasterk RHA

[Worm Breeder's Gazette, 1993]

The transposable element Tc1 is thought to excises via double strand DNA breaks made at the ends of the inverted repeat (Plasterk (1991) EMB0 J. 7,1919-1925). Excised transposons have been detected as extrachromosomal structures (linear or circular; Rose and Snutch (1984) Nature 311, 485-486; Ruan and Emmons (1984) Proc. Natl. Acad. Sci. USA 81, 4018-4022). Excised, linear elements have also been detected for the related transposon Tc3 (Van Luenen et al. (1993) EMBO J. 12, 2513-2520). We have examined how the Tc1 and Tc3 transposons are excised by determination of the exact double strand cleavage sites. Excision of Tc3 is not very frequent and thus the amount of extrachromosomal Tc3 in "natural" mutator strains is limited. Therefore we have made a stable transgenic line containing the Tc3 transposase under the control of an inducible promoter. Upon induction Tc3 transposase is made and this results in excision and transposition of Tc3 elements (Van Luenen et al. (1993) EMBO J. 12, 2513-2520). The level of excision and thus of the amount of extrachromosomal Tc3 elements is high. The 3' end of the excised Tc3 transposon was determined in two independent ways. A restriction site in the inverted repeat located close to the end of the transposon was used to generate a small fragment containing the end of the transposon. This fragment and a size marker were run on a denaturing polyacrylamide gel. The DNA was Southern blotted and probed with a transposon end specific sequence. The length of the Tc3 fragment was compared to the size marker and from that we conclude that the 3' end of the excised element is the G nucleotide (see figure). Alternatively, the extrachromosomal material was amplified by PCR after 3' tailing with terminal transferase and subsequently the PCR product was sequence. The 5' end of the excised Tc3 element was detected using the same Southern blot approach as used for the 3' end. The results indicated that the excised element ends at the G residue (see figure). In other words: the excised Tc3 element has a 2 nucleotide 3' extension. The same cleavage site is used most likely also for Tc1 excision. This has been observed in an in vitro cleavage reaction using recombinant Tc1 transposase. Cleavage was found directly after the G residue. What are the implications of these results for the mechanism of transposition? Since the sequences of Tc1 and Tc3 were determined it was unclear whether the TA dinucleotides flanking the integrated transposon are part of the transposon or result from target site duplication. Our results show that the TA dinucleotides are the result of target site duplication. The element has to integrate using the 3' end, otherwise the last 2 nucleotides of the transposon will be lost. Integration through a nucleophilic attack of the 3' 0H group is also seen for other transposons. The presence of a 3' OH group at Tc3 ends is indirectly demonstrated by the possibility to add nucleotides to them by terminal transferase tailing. Upon excision the cell is left with a double strand break with a 2 nucleotide 3' extension at both sides. The result of the cellular repair process is seen as a footprint left behind after excision of the element. We have sequenced somatic Tc3 footprints after PCR amplification. The footprints generated by Tc1 and Tc3 are very similar. The footprints are most often 4 basepairs in length (a TA basepair and two other basepair: CA or TG) and we conclude that these result from the 2 nucleotides at the 5' ends of the element which are not co-excised.