Fitzhugh N et al. (1995) Worm Breeder's Gazette "Nematode Gymnastics: Do They Run and Tumble?"

Thomas JH, Fitzhugh N

[Worm Breeder's Gazette, 1995]

Nematode Gymnastics: Do They Run and Tumble? Nicol Fitzhugh and James H. Thomas Department of Genetics, University of Washington, Seattle, WA 98195 In an effort to elucidate the behavioral mechanism governing the chemotaxis response in C. elegans, we performed and analyzed the results from a series of single-animal chemotaxis assays. We hypothesized that the movement toward attractants by C. elegans might include a component analogous to the "run and tumble" phenomenon that occurs in bacteria in response to chemical attractants. This response is characterized by the suppression of tumbles (reorientation movements), in favor of runs (continuous movement in one direction), when a bacterium is traveling up an attractive chemical gradient. C. elegans has movements that are somewhat analogous: continuous forward movement (runs) occasionally interrupted by bouts of backing and reorienting (tumbles). Based on our hypothesis, we predicted that the length of each nematode run would increase if the animal were traveling up an attractive gradient, and that the frequency of tumbles would increase if the animal were traveling down an attractive gradient. In order to assay chemotaxis behavior, we followed an experimental protocol similar to that devised by Bargmann et al. I, except that we directly recorded movement of one animal in an assay. We performed two types of assays: those with 7 microL of isoamyl alcohol as attractant, and those without attractant. We used keystrokes interpreted by a computer program to record such qualities as direction of movement, times and lengths of tumble bouts and runs, speed of movement, etc. After running a series of both types of assays, all data were compiled, and the run lengths and tumble frequencies that occurred in the absence of attractant and in the presence of attractant when moving up or down the attractive gradient were compared. Contrary to our hypothesis, our data indicate that run length does not seem to be affected by the presence or absence of attractant or orientation of movement on the gradient. Additionally, preliminary evidence suggests that the animals' initial choice of direction following reorientation was not strongly biased by the gradient of attractant. We conclude that the chemotaxis response to this odorant by C. elegans does not include a significant component analogous to the run and tumble response in bacteria. We infer that another type of orientation mechanism must control the response to attractant. We posit that this alternate mechanism is one that allows the animal to adjust its direction of movement while running based on information about the gradient. Bargmann, C. I., Hartweig, E.. and Horvitz, H. R., Cell 74: 515-527, 1993

Loer CM (1992) Worm Breeder's Gazette "C. elegans Spectrin/Fodrin-Like Clone Free to a Good Home"

Loer CM

[Worm Breeder's Gazette, 1992]

In the course of cloning and sequencing PCR-generated bands while looking for something else, I got a fragment that when translated and run through a homology search ("blast") pulls out a sequence that is repeated in spectrin and fodrin from several different organisms. Apparently, there are similar repeats found in alpha-actinins and dystrophin (Davison and Critchley, Cell 52:159,1988). If anyone out there would like to have the clone, it's yours for the asking. [see Figure 1]

Schauer IE et al. (1990) Worm Breeder's Gazette "Update on Early Embryonic Transcription"

Schauer IE, Wood WB

[Worm Breeder's Gazette, 1990]

In previous gazette abstracts we have reported the use of a run-on transcription reaction to quantitate and characterize transcription in pregastrulation C. elegans embryos and to identify clones of genes that are preferentially expressed during this time. We report here on further experiments addressing the validity of the in vitro assay and characterizing the early embryonic genes identified so far. The presence of 0.5% sarkosyl or 1 mg/ml heparin, inhibitors of polymerase II initiation, in the embryonic extracts and run-on transcription reactions enhances the activity by about 50-100%. The transcripts produced in the presence and absence of these initiation inhibitors have about the same size range and proportion of sequences homologous to the 3' and 5' ends of several specific genes. Thus the enhancement of activity represents a slight increase in elongation rate (as opposed to inhibition of reinitiation accompanied by a large increase in elongation rate). We conclude that little or no reinitiation occurs in the in vitro reaction. Since initiation therefore occurs in vivo, the in vitro reaction is, at that level, regulated in vivo. We have previously demonstrated correct regulation in the in vitro reaction for several temporally regulated genes (C. elegans meeting abstracts, 1989) and now also for the sex-specific gene, her-1. Radiolabeled run-on transcripts produced in extracts of him-8 embryos hybridize strongly to cloned her-1 DNA while those produced in extracts of N2 embryos do not. This result strongly suggests that the sex-specificity of her-1 transcript accumulation (C. Trent, WBG 10,3) is controlled at the level of transcription. These observations also strengthen the argument that the patterns of transcription observed in vitro do reflect in vivo regulation. We have also continued to screen for early embryonic genes using run- on transcripts to probe an early embryonic cDNA library. Several new clones, including new isolates of two of the genes found earlier, have brought the total number of identified early embryonic genes to fourteen. Hybridization to YAC grids has placed several of the new genes on the physical map. Unfortunately, and against all odds, all of them so far fall into genetically unmapped contigs. Of the fourteen total genes identified three are genetically mapped, two to LGX and one to LGIV, eight fall into genetically unmapped contigs, and three have not yet been mapped. Sequence analysis of cDNA clones of five genes has yielded open reading frames but no significant homologies. We have also extended our RNA gel blot analysis of the early embryonic genes to include RNA preparations from glp-1(q231ts) and fem-1(hc17ts) adults, both grown at the nonpermissive temperature, as well as the early embryonic, late embryonic, and L1 RNAs used previously. None of the early embryonic transcripts are present in the glp-1 (soma only) RNA, but all appear to be present at very low levels in the fem-1 (soma and germline) RNA. Levels of these transcripts increase about 20- to 50-fold in early embryonic RNA. In contrast, levels of several predominantly maternal transcripts increase only about 5- to 10-fold, a consequence of packaging of transcripts into oocytes and of a low level of continuing embryonic expression. The RNA gel blot and run-on transcription results together indicate that there is a small number of genes whose expression is limited to a period of time beginning just prior to fertilization and ending sometime early in embryogenesis. We are continuing to pursue various approaches to determining their functions in the early embryo.

Fields CA et al. (1989) Worm Breeder's Gazette "gm2 automated DNA sequence analysis system available. C Fields, C Soderlund."

Soderlund CA, Fields CA

[Worm Breeder's Gazette, 1989]

A prototype automated DNA sequence analysis system, gm2 is available to laboratories interested in serving as external test sites. gm2 consists of a set of pattern recognition and statistical analysis modules, together with a geometric modeling system. It accepts as input a DNA sequence, consensus matrices for locating splice sites, translational start sites, and polyadenylation sites, match-quality cutoff values for consensus searches, and base frequency and codon usage standards for coding regions and introns. It produces as output schematic models of the possible genes contained in the sequence that show the locations of the coding sequences, introns, and control signals; it also produces translations of each of the gene models into amino acid sequences. The current version of gm2 generates all possible models of the gene content of a sequence that are consistent with the input parameters. It is capable of analyzing sequences containing partial genes or multiple genes as well as sequences containing a single complete gene. gm2 is implemented entirely in C. It employs a simple, prompt- driven user interface; it can also accept input from a file. It prints output to ASCII files. gm2 can be run from a conventional text- only terminal. The system has been tested on Sun 3 and Sun 4 workstations. It will run on a Sun 3/50 with 4 Mb memory; a larger memory improves performance. The system has been tested on a number of C. elegans sequences in the 10 kb size range, and on composite sequences of up to 40 kb. Complete and correct models of multi-exon genes, e.g. myo-2 and unc-15, can be generated on a Sun 3 in run times ranging from less than 1 minute to roughly 30 min, depending on the search parameters used. Runs on the Sun 4 are approximately four times faster. gm2 is available to university or other nonprofit laboratories, under the condition that they do not redistribute the software. Users of the software will be asked to supply us with results of running gm2, descriptions of problems that are encountered and suggestions for improvements. Source code and documentation for gm2, and for ...

Jagan Srinivasan et al. (2002) Worm Breeder's Gazette "Towards a Pristionchus genome map III - SNP searches and analyses in Pristionchus pacificus"

Gunther Raddatz, N Ram Kumar, Ralf J Sommer, Hanh Witte, Waltraud Sinz, Stephan C Schuster, Jagan Srinivasan, Alexandra Brand

[Worm Breeder's Gazette, 2002]

The availability of polymorphic strains of P. pacificus and a genomic BAC library of Pristionchus pacificus with large insert sizes encouraged us to use a BAC end based strategy to achieve our goal of constructing genetic and physical maps of the satellite model system Pristionchus pacificus . We looked for polymorphisms in the sequenced BAC ends using the SSCP ( single stranded conformation polymorphism) technique. The BAC end sequences were obtained from our Genome Centre at the institute and primers were designed to amplify 180-250 bp amplicons within these BAC end sequences. We used an inhouse program Prime Array 3.0 to design the primers. These primers were used to amplify both California and Washington DNA's and the resulting PCR products were run on an agarose gel to check for the presence or absence of bands. Later they were run on an SSCP gel to check for mobility differences. We found a total 131 SNP's . BAC ends that showed mobility differerences were then rechecked by sequencing the Washington PCR product and compared to the already available California sequence. Sequence comparison was done on a commercially available program Sequencher3.0. We found that the percentage of insertions and deletions in Pristionchus pacificus was more than in C.elegans (data from Wicks et al ) However the single base pair substitutions were lesser than in C.elegans . This result indicates that insertion and deletion events are more common in Pristionchus pacificus than single base pair substitution events.

Schauer IE et al. (1987) Worm Breeder's Gazette "transcription in early embryos of C elegans."

Schauer IE, Wood WB

[Worm Breeder's Gazette, 1987]

We are characterizing early transcription in embryos of C. elegans. While general embryonic transcription appears to begin at about the 100-cell stage, midway through gastrulation (Hecht et al.), there is evidence for some transcription as early as the four-cell stage (L Edgar, personal communication). We have used run-on transcription in extracts of staged N2 embryos and gel blot analysis of separated RNAs from adults, oocytes, L1's and staged embryos to analyze the pattern of embryonic transcription and to look for genes that are preferentially expressed during early embryogenesis. To obtain embryos, L1 hermaphrodites are synchronized by hatching overnight in M9 without food. The starved L1s are then plated at 10-20,000/plate on NGM plates and kept well fed for 34 days at 16-20 C. The worms are observed as they reach adulthood. 'Early'' embryos are harvested by hypochlorite treatment when the most mature worms contain 4-6 eggs. The result is a low yield of embryos of which 90-95% are pregastrular, with <30 cells. 'late' embryos are obtained by hypochlorite treatment of heavily gravid adults followed by 2 hr of aeration of the embryos in M9. Of these embryos, 95-99% have >30 cells; usually about half the embryos are premorphogenesis and half are in the process of morphogenesis. Extracts prepared from early and late embryos are able to incorporate [32P]-UTP into run-on transcripts. In vitro transcription is 100% rifampicin-resistant and ~80% amanitin-sensitive, indicating that most of the incorporation results from eukaryotic pol II activity. The transcripts include sequences homologous to the actin gene act-1, but not to the vitellogenin gene vit-5, consistent with normal developmental regulation. Labeled run-on transcripts from early and late embryonic extracts were used to probe duplicate filter lifts from an N2 genomic library ( prepared by C. Link). With the reservations that there may be artifacts in in vitro transcription and that some later embryos are present in the early embryo preparations, the following observations suggest that substantial transcription occurs prior to the 30-cell stage. 1) Early extracts incorporate labeled nucleotide at about 50% of the rate seen in late extracts, on a per nucleus basis. 2) When library filters are probed, early run-on transcripts hybridize as strongly as late run-on transcripts to 25-50% of the plaques. 3) RNA gel blot analysis of actin gene expression indicates that act-1 and act-3, but interestingly not act-4, are already being expressed in early embryos. When duplicate library filters are probed with early and late running transcripts, about 0.02% of the plaques hybridize more strongly to early transcripts. Several such phage have been isolated and further characterized by gel blot analysis of RNAs from the following developmental stages: adults with oocytes (temperature sensitive fer-1 adults raised at the non-permissive temperature), early embryos, late embryos, and L1's. Nine independently isolated phage clones have been found to recognize transcripts that are present primarily in early embryos. Three of these transcripts were recognized by more than one of the clones, suggesting that the number of primarily early transcripts may not be large. Six different transcripts were recognized by the nine clones. These six transcripts fall-roughly into the following three classes: 1) Low abundance in adults with oocytes, high in early embryos, low in late embryos (one transcript). 2) Absent from adults with oocytes, high in early embryos, barely detectable in late embryos (one transcript). 3) Absent in adults with oocytes, high in early embryos, 2- to 5-fold lower in late embryos (four transcripts). None of the transcripts persist as far as early L1. All six are small, ranging from 0.7 to 2 kb. We are currently looking for cDNA clones that correspond to these transcripts and planning to use the cDNA's for in situ hybridization and sequencing. Five of the six sequences have been placed in contigs (J. Sulston and A. Coulson, personal communication), but none of the relevant contigs are mapped or marked. Plans to begin genetic analysis must, therefore, await further developments.

Soderlund CA et al. (1990) Worm Breeder's Gazette "gm Sequence Analysis System Update"

Soderlund CA, Fields CA, Shanmugam P

[Worm Breeder's Gazette, 1990]

A number of improvements have been made in the gm automated DNA sequence analysis system: (1) The ratio of AT-containing to CG-containing dinucleotides has been added as a test for introns. This works better than AT frequency alone in C. elegans.(2) A branch site consensus sequence or an enhanced dinucleotide ratio can be required as an additional test on introns. (3) Predicted amino-acid sequence files are generated in a format appropriate for input to the Dana-Farber motif-identification program plsearch. (4) A graphic interface based on X-windows, version 11 is available as an option. (5) The complete analysis algorithm is significantly faster than the previous version. (6) A greedy model evaluation algorithm is available as an option. This algorithm generates the longest, non-overlapping models that cover a sequence and is much faster than the complete analysis algorithm. The program has been tested on Sun3, Sun4 and VAX machines running Unix (Ultrix on the VAX). Results for a series of tests run on a Sun 4/60 are shown in the table. [See Figure 1] gm can be run remotely on our machine, using the Internet. To do this, telnet to haywire.nmsu.edu, and logon as gm_guest with password gmuser. Read the README file for information on running gm. We are also distributing gm as C source code to nonprofit laboratories, either via anonymous ftp to haywire.nmsu.edu, or on tape. If you would like to receive gm on tape, send a 1/4' cartridge or 1/2' reel tape to: Chris Fields, Box 30001/3CRL, New Mexico State University, Las Cruces, New Mexico 88003-0001, USA; Telephone (505) 646-2848.

Laufer JS et al. (1982) Worm Breeder's Gazette "no 5-methylcytosine in Ce DNA."

Laufer JS, Powers S, Wood WB

[Worm Breeder's Gazette, 1982]

Methylation of DNA has been implicated in gene regulation. About 4% of the cytosine residues are methylated in mammalian DNAs, and some specific changes in methylation are correlated with changes in gene activity (e.g., McGee & Ginder, Nature 280, 419, 1979). On the other hand, it was recently reported that Drosophila DNA contains no 5- methylcytosine (News & Views, Nature 290, 363, 1981). We undertook to determine whether C. elegans DNA contains methylated bases by reversed-phase high pressure liquid chromatography of L1 DNA hydrolyzed to free bases with formic acid. We detected no 5- methylcytosine or N5-methyl adenine. By comparison with hydrolyzed calf thymus DNA run on the same column, we can place an upper limit for 5-methylcytosine in C. elegans DNA at about 0.01% of total nucleotides, corresponding to <0.04% of cytosine residues.

Honda BM (1984) Worm Breeder's Gazette "transcription of pol III genes in C elegans cell-free extracts."

Honda BM

[Worm Breeder's Gazette, 1984]

We've recently prepared crude C. e extracts which support the transcription of pol III genes. S100 fractions were initially prepared from ncl-1(e1865) (generous gift of Ed Hedgecock) as it was possibly RNase deficient, but more recently N2 extracts have also shown transcription activity. There are still some problems with variability from preparation to preparation; general assay conditions ( optimal salt, temp., etc.) are similar to those described for yeast systems. When a single C. elegans 5S DNA repeat in pBR322 is used as a template, a 120N transcript is produced. The product hasn't yet been fingerprinted, but transcription of 5S DNA minus termination signal ( run of T's) yields the expected (185N) transcript (terminated at a run of T's in psR322), suggesting initiation of 5S transcription at the correct place upstream. Transcription of a Xenopus tRNAmet gene shows products with sizes characteristic of primary transcript and processed product(s)--processing may be aberrant, as product sizes look different from those obtained in a Xenopus oocyte extract. It's interesting that C. elegans extracts will not efficiently transcribe Xenopus 5S DNA; similarly, Xenopus extracts are very inefficient in transcribing heterologous worm 5S DNA. This is perhaps not surprising given the fairly large sequence differences between the internal promoter regions of the two genes. However, the addition of C. elegans protein fractions 'complements' Xenopus extracts and allows efficient transcription of worm 5S DNA. This might provide a good assay to purify a worm 5S specific protein without the problems associated with the instability of fractionated components (this would be analogous to the first scheme used to purify TFIIIA from Xenopus oocytes). We're also looking indirectly for TFIIIA-like or tRNA- specific fractions, and a pol II system to look at more 'interesting' ( ?) genes.

Skimming J et al. (1984) Worm Breeder's Gazette "growing a lot of (ugh! smelly!) worms."

Skimming J, Lewig JA, McLafferty SA

[Worm Breeder's Gazette, 1984]

In our efforts to make monoclonals against the levamisole receptor, we've needed a passel of worms. Which means growing a helluva alot o' bacteria. 250 liter fermentors do not seem to abound in these parts and so we're unable to emulate the Johnson-Rand gang (1.5 kg of worms from a 250 L fermentor). We've turned to making the best of our Lab- Line S.M.S. fermentor (see an earlier article by S. Ward and E. Hogan, WBG 5 2, p.10). Using pure oxygen and a slightly modified version of the SLBH medium of Sadler, Miwa, Maas and Smith, Laboratory Practices 23, p.642, 1974, we've been able to grow 260 gm of E. coli per 3.3 liter of medium in a 7 hour fermentation run with very good reproducibility. A simplified version of the method described by Sadler et al. works just fine. We use all El Cheapo chemicals from Sigma for our SLBH medium. If you follow this method, be very careful with the oxygen. As you should know, it makes things highly combustible. Apart from the cost of the regulator and a tank rental charge, 244 cubic feet of oxygen cost us only $6.40 and it lasts 4 or 5 runs, giving us an average of 80 gm more a run than the 180 gm yields described by Ward using air. [see Figure 1] Use of the Sigma chemicals leads to a precipitate in the SLBH medium but otherwise things work just fine. To start things off, a 300 ml overnight culture is grown at 37 C in SLBH. We use E. coli strain NA22 whose genotype seems to be lost in the Dark Ages but the genotype does include a ts lethal so careful temperature control is important. The next morning the S.M.S. high density fermentor containing 3 liters of the complete SLBH medium is inoculated with the overnight culture and run at 320 rpm with an initial O2 flow rate of 1-2 liter per minute at 1 or 2 psi. Unlike the method of Sadler et al., all the glycerol needed is present from the start. Also, to set up a run, we autoclave the growth chamber and media separately (partially necessitated by the small size of autoclaves in Missouri). The growth chamber bottle is autoclaved lying on its side in a sling made from Tom Sanford's car seat belts. Another useful invention of Tom's is that the exiting gas from the fermentor is directed down a drain and up the sewer side of the trap. Such an exhaust tube goes a long way to making you more socially acceptable in your building. After 3 hours into the fermentor run, the O2 flow rate is turned up to 4-5 liters per minute and left at this rate for the remainder of the run. Around 4 hours, a slow trickle of cooling water is turned on to cool the hot little growing bugs. One advantage of the S.M.S. fermentor is the bacteria produced are in such a small volume that they can easily be collected in about 10 sterile 500 ml centrifuge bottles. Getting worms from the bacteria. We've found Nalgene transparent polycarbonate carboys to be excellent worm growth chambers. As advertised, they are lightweight and durable but somewhat more expensive than glass (~$40 from VWR). Due to our puny autoclave size, we've had to cut most of the necks off our carboys and create makeshift caps out of the lids of Sigma kilogram bottles. Two holes are cut in a lid as entrance and exit ports for 3/16 inch by 1/16 inch by 5/16 inch Tygon tubing to carry air into and out of a carboy. A third 3/4 inch diameter hole is cut as a sampling port and plugged with a #3 silicone rubber stopper. To keep the water level up, sterile water is also added through this port during worm growth (we don't think pre-humidifying air is good for the 'sterility' of worm cultures). The Tygon tube bringing air into the carboy is run to the bottom of the carboy where it is held weighted by stainless steel nuts loosely held onto the tubing by inserting a flanged plastic tubing adapter into the end of the tubing. The exit tubing begins just inside the makeshift lid and both inlet and exit tubing are glued to the lid by epoxy resin. Air coming in through the inlet tube is filtered through sterile cotton packed in a gas drying tube. Carboys are autoclaved filled with 16 liters of the modified S medium described by Ward (op cit). E. coli are added suspended in S medium to give a final concentration of 28 gm/liter of bugs and a final volume of 18 liters in the carboy. Before inoculating with worms, a carboy is vigorously bubbled for 24 hours. Aeration should be violent enough to form a standing wave 1 to 2 inches high. We have otherwise proven a few times too many that the worms will die of anoxia. The inoculum consists of 600 ml of a clearing culture of worms grown at a similar bacterial concentration in a 2800 ml Fernbach flask by rotary shaking. With such an inoculum, the carboy clears in about 4 days and becomes mostly dauers about a week after that, yielding 100 to 130 gm of dauers from ~500 gm of E. coli. Carboys are sat in an ice-water filled sink for 2 hours and then allowed to continue settling overnight at 4 C. After siphoning off the liquid, settled worms are cleaned by sedimentation through 14% w/w sucrose and flotation on 30% w/w sucrose in 250 ml centrifuge bottles spun at 2000 rpm in a Sorvall GSA head at 4 C. Sucrose is employed because on this scale, Ficoll 400 would be prohibitively expensive. For regular stages of worm, 35% w/w sucrose probably could be substituted in the flotation step with some loss of worms to the bottom of the bottle. A yield of over 200 gm of worms per carboy should be obtainable if the worms are not allowed to go to dauers. As for fermentor runs, directing the exhaust air from worm carboys down a drain helps make the air in the lab breathable. Mixing some air in with the pure oxygen from fermentor runs is probably also a good idea to avoid accumulating an oxygen atmosphere in your local branch of the sewer.