Klein RD et al. (1989) Worm Breeder's Gazette "notes on galvanotaxis."

Kim SK, Klein RD, Meyer BJ

[Worm Breeder's Gazette, 1989]

As a joke, Stuart and I decided to put worms in an electric field. We did this by placing worms on an agarose mini-gel (0.3% in 1X M9 salts) with the running buffer just touching the sides of the gel. Interestingly, when we turned the power on, the worms rapidly moved toward the cathode. As a result of these observations, we have been following up this behavior in order to determine if it can tell us anything about worm biology. Galvanotaxis shows two distinct phases. The first occurs at relatively low voltage (~0.07 V/mm in M9 salts) and consists of animals moving somewhat haphazardly toward the cathode in a manner similar to that seen in chemotaxis assays. As the voltage increases, the response gets stronger until a new type of behavior is seen. At approximately 0.4 V/mm (in M9 salts) the worms move toward the cathode in a much more directed manner. They very quickly decide on the direction in which they are going and, once decided, rarely stop or change directions. Although the worms move in a straight line at the higher voltage, they do not move directly toward the cathode. Rather, worms initially placed in a single spot will move out of that spot in two streams, each of which makes an approximately 45 angle with a line between the initial spot and the cathode. An animal traveling along one of these angles will generally (but not always) have its ventral side closer to the cathode. Occasionally a worm will flip onto its other side causing it to change its angle of movement such that it now moves at an angle that is flipped relative to its starting angle (i.e. it makes a V-shaped pattern). The exact angle that a group of worms adopts is somewhat variable and appears to depend on the agarose concentration. Adult and L4 hermaphrodites as well as males show the strongest response. However, male worms tend to get distracted and make slower progress toward the cathode. L3 and L2 hermaphrodites show weaker responses in that younger animals spend a greater proportion of their time moving in a direction other than toward the cathode. L1 animals show little if any response to an electric field. Finally, dauer larvae tend to galvanotax fairly strongly over short periods of time. However, they often can be seen going in a direction other than toward the cathode. In order to determine if galvanotaxis might be a tractable genetic behavior, we have checked (and are continuing to check) existing mutations for the ability to galvanotax. These include a large number of unc, daf, che, lin, mec, and osm mutations. To date only three mutations appear to be defective (to varying extents) in galvanotaxis; lin-32(u282), 33), and n1754, a mutation isolated by L. Bloom as defective in thermal avoidance and also found to be chemotaxis deficient. All three of these mutations are quite pleiotropic in their defects and clearly are not specific for galvanotaxis. One possible explanation for this behavior could be that the worms are responding to a heat or salt gradient set up over time by the electric field. We do not believe this to be true for two reasons: (1) the response starts as soon as the power is turned on and stops immediately after the power is turned off, and (2) two mutations, n1937 and n1938, isolated by C. Bargmann as being completely chemotaxis deficient, are wild type for galvanotaxis. Therefore, it is likely that at least some components of galvanotaxis are distinct from chemotaxis. In order to find mutations that are specific for galvanotaxis, we have begun a mutant screen for animals that do not respond to an electric field. To date we have screened approximately 3000 haploid genomes without finding any galvanotaxis mutations. In order to try to increase the number of worms we can screen as well as make the assay easier to set up, we have adapted the assay to 9 cm tissue culture plates.

Charnay Y et al. (1980) Worm Breeder's Gazette "characterization of two sub populations in C elegans, Bergerac by bacteria in the digestive tractus."

Charnay Y, Brun J

[Worm Breeder's Gazette, 1980]

In our laboratory C. elegans, Bergerac strain,has been routinely cultivated in xenic conditions on nutrient agar since several years. Different populations were maintained separately. The intestinal tractus of the worm was studied cytologically by the 'frottis' technique followed by Feulgen reaction. We have distinguished two populations categories. Results are homogeneous in each of them what ever the larval and adult stages considered and are similar after 100 generations. - First category (majority of populations) - The intestine nuclei alone are Feulgen positive, intestine lumen is clear. - Second category - Feulgen positive material is found throughout the intestine lumen with the same intensity from the very anterior region to the rectum. This Feulgen positive material when observed under Nomarski differential interference contrast optics in the light microscope appears to be constituted by many thousand particles. Their size is just larger than resolution power allowed by microscope. These Feulgen positive particles are neither revealed in Nematodes cultivated in axenic conditions, nor when the hydrolysis is omitted during Feulgen reaction. All these results allowed us to consider that the positive Feulgen material originates in the staining of bacterial chromosomes. So, one or more bacteria strains would be able to colonize the intestine lumen of this Bergerac sub population and to be tolerated by the worm even in large amounts. This result is interesting in the perspective of better knowledge of Nematode-bacteria relations. Which and how much bacteria species are implicated in the nutritional requirements? Are these bacteria symbiot or not?

Cox J et al. (1979) Worm Breeder's Gazette "more on roller mutants."

Edgar RS, Cox J

[Worm Breeder's Gazette, 1979]

We have now completed a study of some 90 roller mutants. The accompanying table indicates the genes which can mutate to give a roller phenotype. Several new genes have been identified and several mapping errors were found. e187, a widely used roller allele of dpy-2, is the canonical allele of another closely linked gene rol-6. The E8 E26 strain usually used for complementation testing at the dpy-2 locus is in fact a triple mutant containing a non-visible allele of the rol- 6 gene, which is where the confusion among these mutants arose. unc- 76 is closer to the cluster on chromosome V than previously indicated. It is within 0.3 map units of rol-4, which is located 2.9 + .8 map units to the right of unc-42. The new position of unc-76 was checked by mapping against dpy-11.We have found that the genes fall into five different classes with regard to phenotypic characteristics: left roller (LR), right roller (RR), left squat (LS), right squat (RS), and left dumpy roller (LDR). Mutants of genes in these groups show remarkably distinctive phenotypic differences in terms of roller handedness, time of onset of phenotype, presence or absence of a dumpy phenotype, and effects on cuticle morphology. All roller alleles of these genes possess a helically twisted cuticle. Some genes contain nonroller alleles and the cuticles of these mutants are found to be straight. Other types of cuticle aberrations are listed in the table. The most bizarre phenotypes are manifest by the squats which are rollers as heterozygotes but straight non-rollers as homozygotes. The right squat homozygotes roll only during the L3 and dauer stages and not as L4's or adults except when the L4 or adult arises from the dauer. This latter finding indicates that adults derived from L3's and dauers are not equivalent, at least in some respects. Dominant or semi-dominant mutations have been detected in five genes: sqt-1, sqt-3, positions of these genes are indicated below. [See Figure 1] [See Figure 2] [See Figure 3]

Shen MM (1986) Worm Breeder's Gazette "lineage alterations in mab-3 mutants."

Shen MM

[Worm Breeder's Gazette, 1986]

Mutations in the gene mab-3 cause a variety of male-specific defects. mab-3 males lack most of their rays, possess a greatly diminished fan, and produce large quantities of yolk proteins (see 1985 CSH C. elegans Meeting Abstracts, p. 129), while mab-3 hermaphrodites appear wild-type. An examination of terminal phenotypes of mab-3 males suggested possible defects in the lateral hypodermal lineages (and perhaps in the B lineage), but probably not in the germline (WBG 9 #1: 87) or ventral hypodermis. All of these structures appear normal in mab-3 hermaphrodites. I have followed the lineages of the lateral hypodermal blast cells V5, V6, and T from mid-L2 to late L4 in mab-3(e1240) males. (e1240 is a candidate for a null mutation, as its terminal phenotype seems identical to that of e1240/Df, using deficiencies that span the mab-3 locus. The lineage alterations in the mab-3 male lateral hypodermis appear to be confined to the nine ray precursor (Rn) sublineages that generate nine ray cell groups (R1 descends from VS, R2-6 from V6, and R7-9 from T). The mutant lineage that mab-3 ray precursors can follow is depicted in the figure below, along with the wild-type ray precursor lineage. In mab-3(e1240) males, R1-4 generally adopt the mutant lineage, while R7-9 usually remain wild-type, and RS and R6 often follow an intermediate lineage. These results are consistent with the terminal phenotype. The anterior-posterior distribution of mutant lineages may reflect a gradation in the requirement for mab-3 gene product; this distribution is also observed in the terminal phenotypes of two weaker mab-3 alleles, e1921 and e2093. I have begun to examine the effect of mab-3(e1240) in combination with mutations that create ectopic ray cell groups. For example, males of lin-22(n372) (isolated by Bill Fixsen; see WBG 7 #2: 40) form an ectopic postdeirid and a pair of ectopic rays from each of V1-V4. mab-3;lin-22 males produce ectopic postdeirids, but instead of producing ectopic rays, they generate alae (based on inspection of late L4 animals). Thus, it appears that mab-3 mutations are capable of creating a transformation from ray to seam cell fates. This is reasonably consistent with the mab-3(e1240) terminal phenotype, in which patches of abnormal alae-like structures are seen in the tail region, where normally no alae are found. [See Figure 1]

Ray C et al. (1995) Worm Breeder's Gazette "Evidence for Proteolytic Processing of a Cuticle Collagen in a Plant-Parasitic Nematode"

Hussey RS, Ray C

[Worm Breeder's Gazette, 1995]

Collagen is the major structural component of nematode cuticles. In Caenorhabditis elegans, the N-terminal regions of cuticle collagen proteins contain the highly conserved motif, R-X-X-R, which has been predicted to contain a potential subtilisin-like proteolytic processing site required for cleavage of the collagen proteins and normal cuticle collagen function (Kramer, 1994, FASEB 8:329-336). Cuticles of adult females of the plant-parasitic nematode Meloidogyne incognita contain a 76 kDa collagen which comprises over 50% of the B- mercaptoethanol-soluble cuticular proteins (Reddigari et al., 1986, J. Nematol. 18:294-302). We determined the N-terminal amino acid sequence of this major collagen and found it to be identical to the predicted amino acid sequence, starting at amino acid number 66, of the M. incognita Lemmi 5 cDNA clone (Van der Eycken et al., 1994, Gene 151:237-242) (Fig.1). A putative subtilisin-like protease recognition site was found immediately upstream of the region of amino acid homology between LEMMI 5 and the N-terminal sequence of the 76 kDa collagen (Fig.1). Our data support previous speculation about the existence of this novel method of collagen maturation and provide further evidence that this mechanism has been conserved during nematode evolution. In addition to protein processing, the expression of the Lemmi 5 gene was transcriptionally regulated: Lemmi 5-specific transcripts were present in adult females but not in eggs or second-stage juveniles. Also, Lemmi 5 analogs were present only in four Meloidogyne species, but not in C. elegans, Heterodera glycines, or tomato. .. PREDICTED LEMMI5 AMINO ACID SEQUENCE .. 1M A T L V V M P Q L Y S Q I N D L N L R V R D G V Q A .. F R V N T D S A W N D L M E L Q V A V T P Q S K P R S * N P F Q S L YR Q K RS L P D Y C I C Q P L E I N79 S L P D Y C I C Q P L E I N ............................................................ CONFIRMED N-TERMINAL AMINO ACID SEQUENCE .............................................................. Figure 1. The partial predicted amino acid sequence (residues 1-79) of the Lemmi 5 collagen gene is shown in roman letters. The putative subtilisin-like protease recognition sequence, R-Q-K-R, is boxed and the first amino acid after the proteolytic cleavage site is indicated by an *. The empirically determined N-terminal sequence of the 76 kDa from adult female M. incognita is italicized.

Politz SM (1984) Worm Breeder's Gazette "stage-specific surface antigens and antibodies."

Politz SM

[Worm Breeder's Gazette, 1984]

I am developing stage-specific cuticle antibodies to help identify genes controlling the stage transitions in C. elegans. I want to use these antibodies to select heterochronic mutants expressing stage- specific cuticle antigens at alternative times in development compared to wild-type. Rabbit anti-cuticle antibodies elicited by immunization with whole cuticles or solubilized cuticle collagens bind to the surface of live worms, making it fluoresce uniformly when fluorescein conjugated (FITC) second antibody is applied. Surface immunofluorescence was at first visualized in the compound microscope. Recently, I have developed an arrangement for viewing immunofluorescing worms on agarose plates under a low power dissecting microscope adapted for illumination by an FITC excitation source. This allows me to pick individual live fluorescing worms by the normal method. The basic strategy for using stage-specific antibodies to select heterochronic mutants is to bind antibody to worm populations under conditions of synchrony or genetic background where only mutants expressing stage-specific antigens heterochronically will fluoresce, enabling them to be recognized amongst thousands of nonfluorescent wild-type worms. Approaches involving adult-specific antibodies are in progress; characterization of adult-specific antibodies and surface antigens is described below. For antibody characterization, I devised a quantitative radioimmunoassay to measure surface binding of antibodies. Aliquots containing about 500 worms are incubated with antiserum, washed, incubated with [125I]-labeled Staph. aureus protein A, washed, and counted for total bound radioactivity. Binding is saturable by worms, antiserum, or protein A, indicating stoichiometric reaction. Pre-immune serum gives minimal binding. This assay has been used to study stage-specificity of antibodies and antigens. Rabbit anti-adult cuticle serum cross-reacts with the surface of live L4's. I have made the serum adult-specific by adsorption; after adsorption with saturating numbers of live L4's, serum no longer binds detectably to L4's, but binding to adults is reduced by only 50%. As expected, this adult-specific serum does not bind to adults of strain CB0912(lin-4), a retarded cell-lineage mutant that never develops adult cuticle. After a serum made to L4 cuticles was adsorbed with saturating numbers of adults, no binding to either L4's or adults was observed. These results indicate the existence of at least two distinct classes of surface antigens; i.e., those common to both L4 and adult cuticles, and those restricted to the adult cuticle (adult-specific surface antigens).

Schriefer LA et al. (1990) Worm Breeder's Gazette "PCR and the muscle tropomyosin gene in C."

Schriefer LA, Waterston RH

[Worm Breeder's Gazette, 1990]

As part of our ongoing effort to identify genes for known muscle proteins, we have used an approach that starts with the purified protein and works backwards to the gene. To test this approach and gain experience with it we selected tropomyosin (Tm) a relatively abundant, easily purified, actin associated protein. A candidate Tm clone had previously been identified by Hiro Kagawa, but it was not known whether this gene produced the major muscle form or another form of Tm. Tropomyosin from total worm homogenate was brought to near 100% purity by salt extractions, ethanol and low pH precipitations, and FPLC column chromatography. The N-terminal was blocked so this material was treated with CNBr, and the fragments fractionated on a Laemmli acrylamide gel. The peptides were then transferred to an Immobilon-P membrane and Coomassie stained. Cleavage was partial with about 10 bands present ranging in molecular weight from 39kd to 15kd. 5 major bands were excised and given to the Washington U. Protein Chemistry Lab. to sequence. These yielded protein sequence data on two separate regions of Tm. Both regions had high homology to rabbit alpha skeletal muscle Tm and are presented below: [See Figure 1] Two degenerate oligonucleotides were made to the arrowed regions in the upper fragment. These oligos were used to amplify the predicted 80 bp PCR band from a cDNA library and genomic DNA. This PCR fragment was gel purified and then cloned into M13 and sequenced. This sequence yielded the expected protein sequence and is presented below: [See Figure 1] The 80 bp PCR fragment was hexamer labelled and used to probe the YAC library. The positive clones Y38D6 and Y68B7 map to the left of unc-54. This position and amino acid sequence agreed with the results independently obtained by Hiro Kagawa (this issue and personal communication) and indicates that Hiro has cloned and sequenced the major muscle tropomyosin gene. Deficiencies that map to the left of unc-54 were obtained from the CGC stock center and these were used as a substrate for PCR amplification. The arrested homozygous deficiency animals were digested with proteinase K and amplified using the oligos to Tm. Our results indicate eDf10, elete Tm. Tentative data indicates that eDf15 does not delete Tm while eDf16 does. The genes lev-11 and let-205 are known to be in this region. This project has shown the power of micro-sequencing of immobilized proteins, PCR amplification, the C. elegans YAC library, and the C. elegans genetic map with its deficiencies. These tools can lead the C. elegans researcher from their favorite protein to localize its gene.

Byerly L et al. (1978) Worm Breeder's Gazette "voltage-clamp studies of the electrical currents of the Ascaris pharyngeal muscle."

Masuda M, Byerly L

[Worm Breeder's Gazette, 1978]

It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.

Waterston RH (1986) Worm Breeder's Gazette "reversed field gel electrophoresis of C elegans DNA."

Waterston RH

[Worm Breeder's Gazette, 1986]

Schwartz and Cantor (Cell 37, 67-75 (1984) and Carle and Olson (NAR 12, 5647-5664, 1984) have devised an orthogonal field agarose gel system that allows the separation of large DNA molecules (>2000 kb). A recent modification of the method by Carle et al. (Science 232, 65- 68, 1986) allows these separations to be obtained using a conventional gel box by periodically reversing the field. Control of the switch times through a microcomputer is convenient and allows the switch time to be varied at will during the run. This is important as the switch time controls the range of sizes that can be separated in a given run. Chris Bond of the MRC electronics laboratory designed and built an extremely convenient switch box employing Mosfett power bridges, which have an excellent response time and a long life time. Details of the set up I'm using are available to anyone interested (see also Carle and Olson's Science paper). To make high molecular weight (chromosomal?) DNA from C. elegans, I am currently using pure populations of L1's as starting material. These are embedded in 0.5% agarose in 0.125 M EDTA 0.125 M Tris pH9 and then lysed by overlayering with 1% sarcosyl, 1 mg/ml proteinase K and 7% -ME as described by Carle and Olson (1985) for yeast. Without restriction of the lysed worms very little ethidium stained material enters the gel under a variety of conditions with the exception of some low molecular weight material, assumed to be degraded RNA, and 2 or 3 ethidium stained bands with a mobility corresponding to about 100 kb but of unknown origin. After digestion with any of a series of restriction enzymes large amounts of DNA enter the gel. Two enzymes, NotI and SfiI which both have 8 bp recognition sequences composed entirely of G:C pairs yield a smear extending from about 100 kb to more than 1000 kb. A few distinct bands are visible against the background smear but it is impossible to directly estimate the number of fragments present. From parallel experiments using digests of random cosmid clones, NotI sites are estimated to occur every 600-700 kb on average, predicting be less than 150 bands. SfiI sites are slightly more frequent. After BglI digest, a 6 bp recognition site enzyme that does not cut within the ribosomal repeat sequence (Ellis et al. NAR 14, 2345-2364, 1986), most DNA appears to be about 50-150 kb although bands are visible at 300 and 350-400 kb and a single distinct band is present with a mobility between that of yeast chromosomes XI (~700 kb) and X. This band hybridizes strongly and specifically with an rDNA probe provided by R. Fishpool, and places an upper limit of 100 rDNA of 7.2 kb repeats. Current efforts are being directed toward using this methodology to characterize genome rearrangements in the sma-1 region of LGV. I am also trying to identify the free duplication fragments mnDp30 and eDp6 in unrestricted DNA preps. The methodology may provide a level of resolution intermediate between that of cytogenetics and the cosmid mapping.

Kennedy BP et al. (1987) Worm Breeder's Gazette "fairly large scale oocyte preparation."

McGhee JD, Ito K, Kennedy BP

[Worm Breeder's Gazette, 1987]

1. Preparation of fer-1 dauer larvae A temperature sensitive sperm defective mutant, fer-1 (hc-1) is grown at 20 C on 150mm NGM plates with 10 ml of whole egg homogenate ( 50 ml H20/egg) dried on top. Leave the plates for 10-14 days (or even longer - but the number of dauer begin to decrease) by which time most worms on plate are dauers. Wash worms from plate with M9 or H2O and preclean either by multiple washing or by filtration through a 44 micron Nytex screen. The worms are further cleaned by flotation in 35% w/v sucrose or if only dauers are required by 1st treating with 1% SDS for 30 minutes and then sucrose flotation. 2. Adult fer-1For each 150 mm NGM plate, add 10 ml whole egg homogenate plus 1-3 g of E. coli and dry onto plate in sterile hood. Once the plate is dry or semi-dry, add 0.7 ml of packed fer-1 dauers and incubate at 25.5 C for about 2 days. This is the non-permissive temperature and necessary for a synchronous shift out of dauer hood. After 2 days, worms are ready to harvest; by this time greater than 95% should be young adults full of oocytes. Worms are cleaned by 35% sucrose flotation. The yield from 10-12 150mm plates is about 60-80 ml packed worms. 3. Preparation of oocytes The clean worms are given a final wash in egg salts buffer (esb=110 mM NaCl, 4 mM KCl, 5 mM CaCl2, 5 mM MgCl2 and 5 mM Hepes pH 7.2). Resuspend each 10 ml of packed worms in a 50 ml tube with 40 ml of esb and sonicate on ice. We have a Braun Sonic 2000 with micro and medium probes. If micro probe is used, the settings are 60-80 watts for 30- 40 sec. and if the medium probe is used, the settings are 40 watts for 30-40 sec. You can test your probe by sonicating worms at various power settings and analyzing the sonicate - too high destroys oocytes - too low just tickles worms. After sonication, the worms are allowed to settle and the supernatant removed and stored on ice. 10ml of esb are added to the worms and mixed, the worms are allowed to settle again and the supernatant saved. The settled worms are again resuspended in esb to 40ml and the sonication and settling procedure repeated 4-6 times. The supernatants from all the sonications are centrifuged at 500 xg for 5 minutes. The pellet is taken up in 10 ml of cold esb and transferred to a clear 15ml polystyrene tube; the supernatant is discarded. By this time, the oocytes are at sufficient concentration that they aggregate and settle faster than the contaminating worms. Let the suspension settle and remove the supernatant. Repeat this procedure at least 20 times (nobody said this was elegant) or until no more worms can be seen. You may have to break up the oocyte aggregate a few times to get rid of all the worms. The yield from about 60ml packed adult fer-1 is about 1ml oocytes. 4. Purity check of oocytes Presence of worms or developing embryos can be detected under polarized light for their gut granules. Usually less than a few percent of the preparation shows gut granules.