Dorman S et al. (1997) Worm Breeder's Gazette "Optimal Buffer and Storage Conditons for ENU"

Dorman S, De Stasio E

[Worm Breeder's Gazette, 1997]

N-ethyl-N-nitrosourea, ENU, is a likely mutagen for genetic studies of C. elegans. We are working to determine optimal buffer and storage conditions in which ENU toxicity remains low with mutagenicity similar to that of EMS. Wild type N2 C. elegans were used in tests of ENU stock and storage conditions. 50 mM ENU stock solutions were prepared in one of four solutions: M9 buffer, 10 mM acetic acid, 10 mM DMSO, and 10 mM Tris pH 6.5. All ENU solutions were diluted to 0.5 or 1.0 mM in M9 buffer and used immediately after dilution. Mutagenesis was for four hours at room temperature. The parent generation of worms did not show any noticeable difference in either lethality or complete sterility, regardless of ENU buffer conditions used. We therefore used Po brood size following mutagenesis as a measure of ENU toxicity. The data show that immediate use of 1.0 mM ENU diluted from a fresh ENU/acetic acid stock provides the highest average brood size at 143 F1 per mutagenized hermaphrodite. This is approximately a two-fold reduction in normal brood size. Results obtained using ENU stock both initially prepared and diluted in M9 are similar, 124 F1/hermaphrodite. Use of ENU stocks prepared in DMSO or Tris appears to be much more toxic as average brood sizes are twenty-fold lower than normal. In an effort to reduce worker exposure to powdered ENU, we undertook experiments to determine whether ENU solutions could be stored without increasing toxicity. Experiments with M9, acetic acid or DMSO as initial solvents indicate that, over time, ENU breaks down to a molecule more toxic to worms. Average brood size drops, sometimes severely, when ENU stock solutions are kept at -20C for as little as two days. ENU stock solutions prepared in Tris were not included in this portion of the study as brood sizes were already remarkably low after immediate use. M9 buffer and DMSO solutions had the largest decreases in brood size. The average brood size resulting from a 1.0 mM ENU solution diluted from an M9 stock dropped from 124, when used immediately, to 39, when mutagenesis was performed after storage for only 2 days at -20C. Use of acetic acid as the initial solvent provided the most suitable environment for storing ENU. After immediate use, the average brood size was 143; after two days of storage at -20C the average brood size had dropped only to 119, a 17% reduction. After eight days of storage, average brood size was further reduced to 34 F1/hermaphrodite. Thus, acetic acid is the optimal solvent for storage of ENU stock solutions for short periods of time only. We previously found that ENU prepared in M9 buffer induces mutations at the same frequency as does EMS. We are currently testing the reversion frequency induced by ENU made from 10 mM acetic acid stocks. In addition, we plan to try reducing the amount of time worms are exposed to ENU.

Hodgkin JA (1986) Worm Breeder's Gazette "observations on strains with high Tc1 copy number."

Hodgkin JA

[Worm Breeder's Gazette, 1986]

In order to identify Tc1 polymorphisms close to the sex-determining gene tra-1 III, congenic strains carrying the interval vab-7 - dpy-18 ( markers that flank tra-1) from several different high copy number strains have been constructed. The first of these, using the Bergerac strain N62, revealed several polymorphisms mapping between tra-1 and dpy-18 (see WBG 8-1: 45). However, neither of the strains DH424 and TR403 appear to contain any extra Tc1 sites in the vab-7 - dpy-18 interval. Also, unique probes derived from the two of the N62 polymorphisms were used to show that the N62 insertion sites are vacant in another Bergerac strain, RW7000. Thus, different high copy number strains may have markedly different Tc1 patterns in a given interval. Carol Trent reported at the 1985 C. elegans Meeting that a strain carrying Sma-1(e30), derived from N2 by Sydney Brenner, had considerably more than thirty copies of Tc1 (the normal N2 number). The Cambridge working stock of Sma-1(e30) (designated CB4000) has been grown continuously at 15 C for more than fifteen years, by a succession of workers at the MRC. DNA from this stock was compared with DNA from the oldest available stock of e30 (designated CB30, formerly E30), which has been stored frozen since approximately 1969. CB30, which is the ancestor of CB4000, has a Tc1 pattern (EcoRI digest) identical to N2, but CB4000 has many more copies of Tc1 (more than 100, i.e. a smear resembling that seen with DH424 or TR403). The CB4000 strain differs from CB30 in other ways: it grows poorly and has a slightly different small phenotype, males are unable to mate, the strain is slightly Him, and it has a mutator activity (twitchers and other mutants are occasionally segregated). Therefore it seems that a Tc1 mobilization event took place at some point during the maintenance of e30 as a 15 C stock, before 1975 but after the original stock, CB30, had been frozen. So, who was Prince Charming, and how did he wake up Sleeping Beauty?

Meneely PM et al. (1982) Worm Breeder's Gazette "embryonic lethal amber mutations on linkage group II."

Meneely PM, Wood WB, Pesavento P

[Worm Breeder's Gazette, 1982]

In the last newsletter, we described a selection of lethal amber mutations linked to dpy-10, using sup-7. We have revised this procedure slightly to take advantage of the balancer chromosome C1. Our current procedure is to mutagenize unc-4(e120); ) hermaphrodites and mate them with sup-7 males. Neither marker is suppressed by sup-7. From the F1 progeny, we pick wild-type animals and score their F2 progeny. Broods that have fertile Uncs and Dpys, but no fertile Unc Dpys are candidates for an amber lethal linked to unc-4. We have been outcrossing such candidates with C1/unc-4 males to establish stocks of genotype C/unc-4 am, which segregate am am mutant homozygotes. We also have been establishing stocks of genotype C1/unc-4 am; sup-7/sup-7. This is readily distinguished from the former stock not only by the suppressible lethal, but also by the suppression of unc-52(e669), one of the markers on the C1 balancer. Thus far, we have isolated more than thirty candidates, and have outcrossed about half. From these, two separate amber mutants have been maintained in both suppressed and nonsuppressed stocks. The balancer makes stock maintenance and mutant complementation much easier. We hope to identify amber mutations of genes known only by temperature-sensitive alleles, as well as new essential genes.

Diana McCulloch et al. (2001) Worm Breeder's Gazette "N2 (ancestral) is a mutant with reduced fertility and longevity"

David Gems, Diana McCulloch

[Worm Breeder's Gazette, 2001]

Genetic variation among different laboratory lines of N2 affects fertility and lifespan. A previous study found that median lifespans of different lines ranged from 12.0+/-0.8 to 17.0+/-0.6 days (20 o C)(1). To attempt to establish which of these best resembles the original N2 isolate, the lifespans of F1 hybrids between N2 variants, and those of seven C. elegans wild isolates, were examined (1). The results suggested that the N2 male stock currently distributed by the CGC (here designated CGCM), is most similar to the original N2 isolate. This strain is the longest-lived N2 variant. An alternative approach to establish which variant best resembles wild type is to examine an N2 stock frozen at an early date. One such stock is N2 (ancestral), which is only 6 generations removed from a stock frozen in the Brenner lab in 1968. We compared the fertility and lifespan of N2 (ancestral) with 3 N2 variant lines: CGCM, CGCH (slightly short-lived) and JW (very short-lived). Our expectation was that it would most closely resemble CGCM. Table 1: Longevity and fertility of N2 (ancestral) and other N2 lines Strain Median lifespan (days) Maximum lifespan (days) N* Mean brood size N* N2 CGCM 20.5 +/- 1.0 24.4 +/- 0.7 5 (127) 292 +/-11 1 (18) N2 CGCH 18.3 +/- 0.8 21.9 +/- 0.7 7 (136) 270 +/- 9 1 (18) N2 JW 13.4 +/- 1.0 23.1 +/- 1.0 8 (194) 213 +/- 9 1 (15) N2 (ancestral) 13.6 +/- 0.7 21.2 +/- 1.0 10 (243) 260 +/- 7 1 (19) N2 CGCM/N2 (ancestral) 19.7 +/- 0.3 25.7 +/- 0.7 3 (53) ND - N2 JW/N2 (ancestral) 15.0 +/- 3.0 22.0 +/- 0.0 2 (46) ND - +/- Standard error. *Number of trials (number of animals scored). To our surprise, the lifespan of N2 (ancestral) was significantly shorter than CGCM and CGCH (median 20 o C lifespan, p < 0.001 in each case)(Student's t test) (Table 1), yet indistinguishable from JW ( p > 0.1). Furthermore, its fertility was significantly lower than that of CGCM ( p < 0.02) but not CGCH ( p > 0.1), but higher than that of JW ( p < 0.001). Does this mean that the longer-lived line CGCM is a mutant variant after all? We favour an alternative explanation: that as early as 1968 there existed a number of genetic variants of N2 in the Brenner lab (Cambridge), and N2 (ancestral) is a short-lived one. In 1975, when Don Riddle moved from Cambridge to Missouri, he took with him CGCH, CGCM and JW (obtained from John White), and froze them upon arrival. These three strains have different lifespans (1). Thus, by 1975 a number of variants certainly did exist. One possibility is that N2 (ancestral) and JW are related. To test this, the lifespans of F1 hybrid hermaphrodites issuing from crosses between CGCM, JW and N2 (ancestral) were examined. The lifespan of CGCM/N2 (ancestral) was indistinguishable from that of CGCM, and significantly longer than that of N2 (ancestral) ( p < 0.001). A reduction in median lifespan was seen in JW/N2 (ancestral) hybrids relative to CGCM/N2 (ancestral) hybrids, but due to the large standard error this did not reach significance. However, the maximum lifespan of JW/N2 (ancestral) was significantly reduced ( p < 0.02). These preliminary results support the view that N2 (ancestral) and JW are related, short-lived mutant variants, and that the mutations concerned are recessive. N2 (ancestral) thus appears to be another short-lived N2 variant, related to JW. We therefore advise that N2 (ancestral) not be used as wild type; instead, we recommend CGCM. (1) D. Gems & D.L. Riddle (2000) Defining wild-type life span in Caenorhabditis elegans. J. Gerontol . 55A: B215-B219.

Carta LR et al. (1992) Worm Breeder's Gazette "Call for Worms"

Edgley ML, Thomas WK, Carta LR, Fitch DHA

[Worm Breeder's Gazette, 1992]

In response to the growing interest in worms related to C. elegans for comparative studies, we are coordinating an effort to collect a comprehensive array of cryopreservable species belonging to the order Rhabditida. This collection would be maintained at the Caenorhabditis Genetics Center, would be freely available to all interested scientists, and would provide an excellent resource for worm breeders who are interested in applying a broader phylogenetic viewpoint to comparative biological investigations. Of course, an important advantage of a universally accepted canonical set of living type species is that species identifications can be tested biologically through cross-mating experiments. The utility of such a collection has already been demonstrated in the Drosophila system a remarkable collection of species from around the world is maintained, for example, at the Bowling Green Stock Center in Ohio. We request that interested parties please send their wild isolates to David Fitch, Lynn Carta or Kelley Thomas, along with the following data: the date, source and method of isolation, any ecological information concerning the isolate, pertinent literature references, the names and addresses of the collector, the depositor and the taxonomist(1), and any specifics about stock maintenance. Other data about the species should also be included, such as measurements(2) and male tail characteristics. Scale illustrations and any anatomical, developmental, cytogenetic or molecular data are greatly appreciated. If the isolate is hermaphroditic, males should also be provided, since most of the morphological characters used in species identifications are associated with males. Males may occur spontaneously or can be induced by heat-shocking L4 or young adult hermaphrodites (usually, but not always, at 30 C for 6 hours). Males obtained in this way can be mated to hermaphrodites to maintain a stock containing males. Kelley will provide a molecular "identification tag". David and Lynn will determine if the species has been previously identified in the literature and serve as liaisons to nematologists with taxonomic expertise to help verify the species identification. We will then deposit the species with the CGC. Eventually, we hope to make all of the information associated with each species in the collection available in a database. (See the abstract by Fitch et al. in this issue for the latest information on current CGC species depositions). So hesitate not to share your pet species with us! We think that the effort in building a phylogenetically broad and comprehensive live collection of Rhabditida will be more than compensated by the valuable opportunities it will provide for developing novel approaches to many areas of nematode research.

Hodgkin JA (1984) Worm Breeder's Gazette "a selection for suppressors of tra-3."

Hodgkin JA

[Worm Breeder's Gazette, 1984]

The mutations fem-2(b245) (a ts feminizing mutation) and tra-3(e1767) (a weak masculinizing mutation) are mutually suppressive at 25 C, so that the double mutant is an abnormal intersexual animal, occasionally self-fertile. A double mutant strain CB3688 was persuaded to grow at 25 C as a self-fertile stock. Initially the strain was barely viable even at 25 C, but after some generations it improved to the point where it could be mutagenized. On shift-down from 25 C to 20 C or 15 C, the suppressive effect of b245 is removed and the Tra-3 phenotype ( an abnormal sterile male) is expressed, unless another suppressor has been induced. Since CB3688 grows as a self-fertile stock, dominant or recessive or even maternal effect suppressors can be selected, depending on how many generations elapse between mutagenesis and shift down. Strong suppression may lead to a female phenotype; fortunately there are usually stray fertile XO males in the CB3688 population after shift-down, which can fertilize the females. Thus, both male/female and hermaphrodite revertant strains can be recovered. This simple and powerful technique has yielded a large number of suppressors. So far I have characterized 20 of these: tra-1(dom) 8 alleles fem-1 2 alleles fem-2 3 alleles (i.e., double mutants with b245 in cis) fem-3 7 alleles The last gene is a new sex-determining gene on LGIV (see map below), with effects similar to those of fem-1. Although the null phenotypes of fem-2 and fem-3 are not yet certain, it is likely that the three fem genes are similar, because they have the following properties in

Herman RK (1978) Worm Breeder's Gazette "stockkeeping, mapkeeping, and genetic clearing house-keeping."

Herman RK

[Worm Breeder's Gazette, 1978]

With lots of help from the Medical Research Council, especially Marilyn Dew, we now have a core collection of C. elegans E stocks, and we (Claire, Kari & I) hereby offer to supply requested strains. At John Sulston s request, I have also agreed to handle two other functions: maintaining the genetic map and administering the genetic clearing house. At present, we have no special support for this work. As many of you know, Don Murphy at NIA is exploring the possibility of supporting a stock center, which could be here or somewhere else. At any rate, I think it makes sense to have the mapkeeping and clearing house operations affiliated with the stock collection, and on the advice of John Sulston and Bob Horvitz, I have defined what, with your support and help I propose to do, for the time being at least: 1. Stockkeeping: Keep and supply requests for at least one mutant per mapped gene or know where in North America the mutants can be obtained. 2. Mapkeeping: (a) Solicit new data (actual counts, complementation results, etc.) on both old and new genes, and update the official map records as such data become available. (b) Submit new data for publication in the Newsletter. These data would include, besides linkage results, interesting properties of new or old genes. 3. Genetic clearing house-keeping: (a) Register new lab symbols, which, in accordance with Bob Horvitz s nomenclature paper, are one or two letters, used as non-ized upper case prefixes for strain names and ized lower case prefixes for mutation names. (b) Register new gene names and list them in the Newsletter. The laboratory assigned to a gene name is then responsible for issuing gene numbers. I hereby solicit data, comments, suggestions, etc. What follows is a list of E stocks available, some new map data, and a few clearing house assignments. Please note: We have taken a cue from the E. coli stock center and have not attempted to confirm the gentoypes of all stocks. We certainly have checked all the more obvious phenotypes, such as for unc and dpy mutants, and we ve been careful in handling heterozygotes, but we have not confirmed some of the other mutants, such as cat, che and flu. If you request any of the latter class, it will be useful if you inform us, say upon acknowledging receipt of the stocks, how they checked out, and we can record that information. In the list that follows, if no mutation name is given for a homozygous single mutant, it can be derived from the strain name, (i.e. , strain E769 has the following genotype: bli-1(e769)).

Bakaev VV et al. (1998) Worm Breeder's Gazette "An Attempt to Slow Aging in C. elegans. 10. The Slight Positive Effect of C. elegans Larvae Extract"

Bakaev VV, Ivlev-Duntau AP

[Worm Breeder's Gazette, 1998]

C. elegans larvae (1-2 days old) were collected from separate stock, frozen and after 12 hours or more time, thawed, centrifuged and mixed with S medium containing E. coli (1:1), this mixture was used as a medium for worms in experimental group. The medium for the control group was prepared by mixing S medium containing E. coli with S medium without E. coli (1:1). Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and with or without larvae extract) during 12 hours, then they were transferred in next wells everyday (one worm in one well). Dilutions of larvae extract (1:10, 1:100 and 1:1000) were applied on third day. This investigation was carried out in temperature +24C and in the darkness. The obtained results are presented in the following table.

Fodor A (1980) Worm Breeder's Gazette "Letter Addressed to B. Edgar Regarding New Data on Bal-X-1"

Fodor A

[Worm Breeder's Gazette, 1980]

Dear Dr. Edgar: 28.11.1979. Probably you have got our two papers for News Letters (dealing with Precocene in Nematodes and Bal-X-chromosome, respectively. I should like to complete the Bal-X-1 paper with some recent data arriving from Szeged today. The genotype of hermaphrodites of the stock: dpy-8( e1321) * unc-3(e151) over * lon-2(e678) *.The distribution of the self- progeny was the followings: [See Figure 1] The occurrence of males having one of the X chromosomes alternatively among P1 progeny indicate the the presence of Bal-X-1 chromosome causes some anomaly in meiotic pairing of the homologs.The reason of this could be i.) The presence of a dominant him mutation ( homozygous lethal) nearby to the lon 2 marker; ii.) chromosome aberration, probably translocation. Considering,that we could not manage to ride of this dominant effect by several outcrosses ( recombination) we prefer the second hypothesis to test. Best regards: Andras Fodor (BRC, Szeged)

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.