Hogan JD et al. (1996) Worm Breeder's Gazette "Mapping the C. elegans Src homologue, src-1."

Collins JJ, Hogan JD

[Worm Breeder's Gazette, 1996]

The oncogene src encodes a tyrosine kinase implicated in signal transduction pathways that control cell growth and development. When activated by mutation, src can induce neoplastic transformation. Despite intensive study for many years in a variety of organisms (almost exclusively vertebrates), a good understanding of src function still eludes us. We are trying to knock out src-1, a C. elegans homologue of vertebrate c-src, in order to characterize the role of src-1 in C. elegans development. Such knowledge will contribute to the understanding of src function in all organisms. We have isolated a src-1::Tc5 mutant using the PCR / sib-selection screen of Rushforth and Anderson (Zhang et al, C. elegans Meetings Abstracts - 1995). In this mutant, Tc5 resides within the fifth intron of src-1. The presence of Tc5 does not confer a visible phenotype because it is spliced out with the intron leading to normal mRNA production. Efforts to isolate a deletion caused by Tc5 excision are in progress. src-1 resides on a small "orphan" contig that has not been placed on the physical map. The mutant we isolated provides the opportunity to map the gene via a PCR based approach using the src-1::Tc5 allele as a molecular marker. Using a primer within Tc5 and a primer in exon 6, we are able to specifically amplify a 1.2 kb product only from individuals heterozygous or homozygous for src-1::Tc5. We crossed strain TW399 [src-1 (cj290::Tc5)] by strains containing markers near the center of each linkage group: LGI dpy-5, LGII dpy-10, LGIII unc-36, LGIV unc-22, and LGV dpy-11. Individual F2 animals homozygous for the marker allele were picked and screened by PCR for src-1::Tc5. In each case, approximately 75% were positive indicating that src-1 was not linked. We established that src-1 is not X-linked by following its inheritance through two generations of XO males. We continued our mapping efforts by crossing strain TW399 by strains containing markers located on either arm of each linkage group: I L bli-3, I R unc-54, II L sqt-2, II R unc-52, III L dpy-1, III R bli-5, IV L dpy-9, IV R dpy-4, V L unc-34, and V R unc-51. In each case but one, approximately 75% of individual F2 animals homozygous for the marker allele were positive for src-1::Tc5. The one exception was F2 animals homozygous for bli-3, a marker on the left arm of linkage group one. Only 5% (3/60) were positive for src-1::Tc5. Control reactions with the same DNA confirmed that the negative reactions were in fact due to the absence of src-1::Tc5. Hence we conclude that src-1 is located near bli-3 on the left arm of linkage group I.

Roberg-Perez KJ et al. (1999) Worm Breeder's Gazette "WormPD -- a Comprehensive Proteome Database for C. elegans."

Lew-Smith JE, Hogan JD, Fancher AM, Roberg-Perez KJ, Lengieza C, Davis BP, Cusick ME, Tillberg M, Lingner CA

[Worm Breeder's Gazette, 1999]

DeFelice LJ et al. (1993) Worm Breeder's Gazette "Preliminary Biophysical Characterization of Ion Channels in C. elegans Sperm Membranes"

Machaca KA, DeFelice LJ, L'Hernault SW

[Worm Breeder's Gazette, 1993]

Biochemical and pharmacological studies have revealed that ion transport across the C. elegans sperm plasma membrane probably accompanies formation of spermatozoa (Nelson and Ward, Cell 19: 457-464, 1980; Ward, Hogan and Nelson, Dev. Biol. 98: 70-79, 1983). We have been examining these phenomena by patch clamping techniques. Spermatids were prepared from him-5 (e1490)males in Sperm Medium (SM; see Nelson and Ward, ibid.) and the patch pipette was filled with SM (for cell attached recordings) or various other solutions (for cell detached recordings). The small size (~5-6 microns for spermatids) of this cell required narrow tipped pipettes, which were fire polished so that the tips had an internal diameter of about 1 micron and had a resistance of 4-10 megaohm. Utilization of these narrow tipped pipettes allowed formation of high resistance (greater than 20 gigaohm) membrane seals to patch pipettes on both cell attached and broken cell detached (inside out) preparations, as measured on a List EPC-7 ,bandlimited at 1000 Hz by standard techniques (Wellis et al., Biophys. J. 57: 41-48, 1990). Cell attached patches, which have an area of about 5 microns (2) (by capacitive measurement), do not seem to affect cell viability for at least 30 minutes and can be obtained routinely on either spermatids or spermatocytes. Distinct levels of current appear at each applied voltage, indicating at least four types of ion channels in spermatid plasma membrane. In figure 1, the lowest conductance level observed is about 15 pS and the highest is 95 pS. These currents reverse between 10 and 15 mV negative to rest (arbitrarily set to 0 mV). A 200 pS ion channel is also occasionally observed (not shown in Fig. 1). These results indicate that it is feasible to study C. elegans sperm differentiation by patch clamping. Our first investigations will involve patch clamping spermatids and inducing their differentiation into spermatozoa while monitoring ion channel activities.

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

Lewig JA, McLafferty SA, Skimming J

[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.