Kenyon CJ et al. (1994) Worm Breeder's Gazette "A polyray Suppressor"

Kenyon CJ, Maloof JN

[Worm Breeder's Gazette, 1994]

A polyray-l suppressor Julin Maloof and Cynthia Kenyon, Dept of Biochemistry, U.C.S.F, San Francisco, CA 94143 The polyray-l (pry-l) gene is required to keep homeotic selector (HOM-C) genes repressed where their expression has not been initiated (see accompanying abstract and WBG 12(5):39). pry-l mutant worms have numerous homeotic transformations and are very sick, so we were intrigued by a plate of pry-l mutant worms which appeared much healthier than normal. I outcrossed the strain and found F2s with the full pry-l mutant phenotype, suggesting that the "healthy" strain contained both pry-l and an extragenic suppressor. Does the suppressor have a phenotype on its own? Since pry-l causes homeotic transformations due to ectopic HOM-C expression, it seemed possible that the suppressor would cause transformations in the opposite direction, similar to those seen in HOM-C loss of function mutants. Indeed some F2s from the outcross had descendants of the QL neuroblast in the anterior of the worms, a phenotype also caused by loss-of function mutations in the HOM-C gene mab-5. We are calling this newly identified gene spy-l for suppressor of polyray-l. spy-l seems to suppress all of the pry-l mutant-phenotypes: pry-l; spy-l double mutant worms do not have extra rays, are no longer Muv, are not Unc, and have anteriorly placed QR descendants. spy-l could suppress pry-l either by preventing ectopic HOM-C expression, or by preventing ectopically expressed HOM-C genes from having any effect. mab-5-lacZ expression was examined and found to be normal in the p1y-l; spy-l double, so wild-type spy-l is required for the ectopic HOM-C expression seen in pry-l mutant animals. The fact that there is a QL migration defect in worms mutant for spy-l alone suggests that spy-l may normally be required to turn on mab-5 in QL. We compared expression of mab-5-lacZ in wild- type and spy-l mutant worms at 3.5-5 hours and at 8-10 hours after hatching. At 3.5-5 hours 100% (n=20) of the mutant and 95% (n=20) of the wild-type worms had expression in QL. At 8- 10 hours 0% (n=30) of the mutant worms has expression of mab- 5 in the descendants of QL, but 87% (n=30) of the wild-type worms showed expression. It appears that spy-l is not required to initiate mab-5 expression in QL, but that it is required to maintain expression once it is initiated. It is possible that spy-l is required to maintain the ON state of a number of HOM-C genes, in a manner analogous to the brahma and trithorax genes of Drosophila. spy-l is located on X, in a region well covered by cosmids. Hopefully a molecular analysis of spy-l along with the isolation and study of more spy-l alleles will enable us to determine how similar the C. elegans and Drosophila maintenance systems are, and how spy-l and other maintenance genes are used to maintain and specify pattern.

Grant BD et al. (1994) Worm Breeder's Gazette "sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein."

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1994]

sel-l, a negative regulator of lin-12 and glp-l activity, encodes a novel extracellular protein. Barth Grant and Iva Greenwald HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 31. No positive effect of glycerol"

Lyudmila M Bakaeva, Vladimir V Bakaev

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of glycerol in water solutions on the nematode life span. In this experiment glycerol was used in following dilutions: 100 g/L, 10 g/L, 1 g/L, 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without glycerol) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without glycerol in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with glycerol in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table.

Yandell MD et al. (1994) Worm Breeder's Gazette "ceg-1 is Very Similar to decapentaplegic."

Wood WB, Yandell MD

[Worm Breeder's Gazette, 1994]

CEG-I IS VERY SIMILAR TO DECAPENTAPLEGIC Mark Yandell and Bill Wood Department of MCD Biology, University of Colorado, Boulder We have cloned a TGF-beta homolog which we have provisionally called ceg-l for C. elegans growth factor-l. ceg-l is a member of the dpp/bmp2-4 subclassl of TGF-beta proteins (see PILEUP diagram) and is very similar to the Drosophila gene decapentaplegic (dpp) and the mouse gene GDF-5, the product of the brachypodism locus, alleles of which cause defects in limb morphologyv.2 We previously reported (WBG 12(5): 82) that ceg-l maps to chromosome V just to the left of the cluster. We have now mapped ceg-l to the genetic map by PCR from single dead embryos homozygous for either the deficiency sDf30 or the deficiency sDf20 and find that it maps to the cluster of lethals near dpy-l l which lie under both deficiencies. Developmental Northern blots show that ceg-l encodes a moderately rare transcript that is present in late embryos and L1 larvae. Sequencing of genomic and cDNA clones reveal that the ceg-l gene has eight introns and encodes a 1.7kb message which is trans-spliced to SL1. We are now preparing to inject heat-shock and LacZ contructs. We are also collaborating with Ronald Plasterk to obtain a Tc1 insertion in ceg-l. Kingsley, D. M., Genes and Development 8: 133-146, 1994. 2 Storm, E. E., Huynh, T. V., Copland, N. G., Jenkins, N. A., Kingsley, D. M., and Lee, S. Nature 368: 639-643. 1994.

Hong K et al. (1992) Worm Breeder's Gazette "A Method for Direct DNA Sequence Analysis of PCR Amplified DNAThat Works Very Well"

Driscoll MA, Hong K

[Worm Breeder's Gazette, 1992]

We have begun DNA sequence analysis of 50 recessive mec-4 mutations. By combining aspects of a few different protocols we have come up with a method for sequencing of PCR products that is reliable and gives high quality data. We hope that this protocol may be of use to others involved in sequencing projects! A. Generation of the template DNA for sequence analysis 1. Amplify genomic DNA in standard PCR reaction. We use 2 g genomic DNA and approximately .5 g each primer (our primers average 21 bp in length) in 100 l reaction volume. 2 Run an agarose gel to assay the concentration of amplified DNA. Ideally, the concentration of DNA should be at least 1 g in 10 l. If the DNA concentration is very low, either ethanol precipitate to concentrate it for the next step or reamplify. Also if large visible amounts of primers remain, it is good to remove them as outlined in Step 4 below. 3 Amplify one strand of DNA. In separate experiments we have used either a new primer internal to the original boundaries of the PCR product or we reused on of the original primers for the single stranded amplification. Both approaches work quite well. Usually we use 10 l of the DNA generated in Step 1 above (About 1 g if possible) and about 200 ng of primer in 100 l volume. 4. Purify the ss amplified DNA away from unincorporated primers by passing over Miniprep Spun Columns form Pharmacia. Alternatively, make homeade columns with Sephacryl S-400. 5. Check DNA concentration on an agarose gel. For certain success, about 2 g of single-stranded DNA is used in each sequencing reaction. If the DNA concentration is too low, DNA can be ethanol precipitated to concentrate the sample or a second single-stranded amplification step is suggested. The amount of DNA template is very important. B. Denaturation of the template and annealing of sequencing primer 1. Add 8 l of template DNA and 1 l of 1 M NaOH to a 1.5 ml microcentrifuge tube; incubate at room temperature for 10 minutes 2. Add 1 l of 1 M HCl to neutralize the solution and then immediately add 2 l (about 200 ng) of sequencing primer (from the complementary strand, of course) and 2 l of sequencing buffer (as in Sequenase kit from US Biochemical). Note: perform this step on each tube individually, ie., if you are doing 10 reactions do not add HCl to each tube and then go back and add primers. Neutralize the first tube and add the appropriate primer and buffer and then start the next tube. This is important. 3. Incubate at 37 C for 15 minutes to allow the primer to anneal to template DNA C. Sequencing reactions Reactions are according to instructions in the USB Sequenase kit with a few changes: Labelling reaction: -14 l annealed DNA (the entire reaction prepared above) -1 l DMSO (added fresh) -1 l DTT. 0.1 M -2 l diluted dITP mix (1:5 dilution) -0.5 l 35S -2 l diluted sequenase* *When diluting Sequenase for reactions the dilution mix is made as follows: enzyme dilution buffer, 6.5 l pyrophosphatase, 0.5 l Sequenase, 1 l as usual, do this just before starting reactions Termination reaction: Although the starting volume is slightly larger than in the USB protocol, still add 4 l of labelled DNA into 2.5 l of each dITP for termination reactions, add 4 l stop solution. Notes: 1. We can read at least 400 bp for each reaction. 2. This procedure works very well for sequencing double stranded plasmid DNA.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 32. No positive effect of peroxide"

Vladimir V Bakaev, Lyudmila M Bakaeva

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of hydrogen peroxide in water solutions on the nematode life span. In this experiment hydrogen peroxide was used in following dilutions: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without hydrogen peroxide) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without hydrogen peroxide in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with hydrogen peroxide in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Conclusion: If hydrogen peroxide solution was applied to C. elegans, it was not able to increase their mean longevity significantly in comparison with control.

Mains PE (1994) Worm Breeder's Gazette "Interactions Between mei-1 and the unc-116 Kinesin"

Mains PE

[Worm Breeder's Gazette, 1994]

Interactions Between mei-l and the unc-116 Kinesin Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada

Bennett KL (1993) Worm Breeder's Gazette "An Old-Fashioned Protocol for in situ Hybridization to Nematode Embryos"

Bennett KL

[Worm Breeder's Gazette, 1993]

SAMPLE PREPARATION-Egg Preps The Bennett laboratory uses the same protocol Ascaris or Caenorhabditis, although we currently have a lot more experience with Ascaris. We use the hypochlorite solution (40% Chlorox, 50% 1 N NaOH. 10% water) for gravid C. elegans adults or synchronous cultures of Ascaris eggs. When adults have dissolved and eggs have been stripped of their proteinaceous coat, rinse 3-4 times in M9 (or water). Transfer egg pellet to 1.5ml eppendorf tube. Add equal volume of 6:3:1 (methanol:acetic acid:chloroform) to permeabilize vitelline membrane. Invert. Immediately dilute in M9 or water and spin down eggs. Rinse several times. Fix in 4% paraformaldehyde in M9 or PBS (in DEPC water) on ice for 15 min. Rinse 2-3 times. A few embryos should be checked with DAPI at this point to assure they are intact and permeabilized. Mix fixed egg pellet with ~1/3 volume of O.C.T. embedding medium for frozen specimens (Tissue-Tek brand). Freeze the egg mixture on a microtome chuck (or a quarter) which has been pre-chilled on a block of dry ice. These egg blocks are stored at -80 C. until cutting. We have stored blocks for several weeks. SLICING EGGS We set the microtome for 12-16 m sections at -15 C. We fill a slide with sections and let the sections adhere to the slide on a 37 C hot plate for 30 min. After that the slides are fixed two more times in 4% paraformaldehyde (made fresh) in DEPC M9 or PBS + Mg. The first fixation is on ice. The second is at room temperature with the addition of 0.1% deoxycholate and 0.1% Triton-X100. All fixations are done in glass containers which have been baked in the "hot oven"(250 C for >4 hrs.) to remove RNases. Both fixations are for 15 min. The cellular RNA is acetylated in acetic anhydride/triethanolamine solution. (freshly made and used within 30 minutes) at room temp. for 5 min. 100mM Triethanolamine-HCl pH 7.5 250ml 250 ml DEPC-H(2)0 4.64g Triethanolamine-HCL (Sigma T-1502) 560 I 1ON NaOH add 625 l acetic anhydride immediately before adding to the slides Slides are then rinsed 3 times in 1xPBS+Mg or M9 .Slides are dehydrated in 30%,60%,80%,95% ETOH made in DEPC-H(2)0. We air dry them O/N. Store at -80 C until ready for hybridization. TRANSCRIPTION-ANTISENSE The transcription and hybridization protocols are from Dr. Etsuko Wada, courtesy of Dr. Sandra Petersen, UMC. Some comments may be useful. The lack of any cold nucleotide to supplement the hot label makes the reaction suboptimal in terms of molarity, and causes the probe to be very small (probably 50-75nt). However incorporation is often 95% or better and this small probe doesn't need hydrolysis. Dry down 250 uCi [35S]-CTP in speed-vac (CTP may also be used, cold UTP would then be added.) ADD: 1. 2ul 5X Transcription Buffer (we use that which comes with SP6 ,T7 or T3 polymerase) 2. 1 l 100mM DTT (Made fresh) 3. 0.5 l RNasin 40U/ l 4. 2 l 1OmM ATP 5. 2 l 1OmM UTP (or CTP when using hot UTP) 6. 2 l 1OmM GTP 7. 1 l Linearized DNA Template 1 g/ l 8. 0.5 l RNA Polymerase 20U/ l Gently mix. (Tap tube with finger.) Incubate 30min at 37 C. Add 0.5 l Polymerase and incubate another 30min. Add 89.5 l nuclease-free water. Check % incorporation with PEI chromatography paper. ([35S] count in fluor) Add: 1. 5 l 1 M Tris-HCL pH 8 2. 1 l 1M MgCl(2) 3. 0.5 l tRNA 25 g/ l 4. 0.5 l RNasin 40U/ l 5. 2ul DNase I 1U/ l Gently mix. (DNase is very labile. Do not vortex.) Incubate 30min at 37 C. Run Sephadex G-50 spin column to remove unincorporated counts.

Chamberlin HM et al. (1992) Worm Breeder's Gazette "Cell Ablation Experiments and the Characterization of Cell Fate Specification in the / and / Equivalence Groups of the Male B Lineage"

Chamberlin HM, Sternberg PW

[Worm Breeder's Gazette, 1992]

Cell fate determination in the B lineage of the male tail is due, in part, to cell interactions. Two equivalence groups, alpha/ (B.a(l/r)aa) and gamma/delta (B.a(l/r)pp) are evident from the wild-type cell lineage [Sulston & Horvitz, Dev Biol 82:41-55 (1977)]. For instance, in the alpha/ pair, either B.alaa or B.araa (labeled aa in the figure) can adopt a position anterior to the other. The cell that adopts the anterior position will adopt a fate (i.e., undergo an a lineage), and the posterior cell will adopt ,B fate. Thus, although the cells in each pair have equivalent potential, they adopt different fates depending on their relative positions. This abstract summarizes results of cell ablation experiments that begin to characterize cell fate specification in these two equivalence groups. 1) F and U promote anterior (a and y) fates. Ablation of the male specific blast cells F and U in late L1 males results in abnormal a (7/7 animals) and y (5/7) lineages. Specifically, presumptive alpha's often behave like 's, and presumptive gamma's undergo abnormal truncated lineages. lin-3 ,let-23 ,let-60 ,and lin-45 ,genes identified for their role in hermaphrodite vulval induction, also act in these male tail equivalence groups. Mutations that reduce wild-type function of these genes result in the same lineage defects seen in F-U- animals (see WBG 11.2, p. 103). In addition, the F/U signal is redundant. Lineages in F- or U- animals are generally wild-type (F-: 0/4 a abn; 1/4 alpha abn. U-:V4 gamma abn; 0/4 y abn.). 2) Y.p promotes posterior ( and delta) fates. Ablation of Y.p in late L1 males results in abnormal delta (6/7) and occasionally abnormal (2/7) lineages. Specifically, presumptive delta' s undergo an extra round of division. Y.p may play a minor role in providing posterior information for the alpha/ pair. However, it appears to be neither necessary nor sufficient to specify fate (see section 4). 3) lin-12 acts in the gamma/delta equivalence group? The role of lin-12 in the gamma/delta equivalence group shows similarities to its role in the hermaphrodite vulva. lin-12 (0)mutations result in transformation of delta to gamma [Greenwald et al., Cell 34:435-444(1983)]. Although lin-12 (d)mutations result in no gamma/delta phenotype on their own, ablation of F and U in lin-12 (d)animals results in full transformations of gamma to delta (3/3 transformed). Thus, lin-12 specifies delta fate, but the F/U signal can override lin-12 (d)and induce a normal gamma. In contrast, there is currently no evidence that lin-12 plays any direct role in alpha/ cell fate specification. 4) Interactions between other B.a progeny and the alpha/ equivalence group. B.a progeny play a significant role in specification of fate. Ablation of all B.a progeny except B.a(l/r)aa (i.e., B.a(l/r)pp-, B.a(l/r)pa-, and B.a(l/r)apin late L2 )results in both B.alaa and B.araa adopting alpha fate (4/5 both alpha). However, the presence of any left/right pair of B.a progeny is sufficient to correctly specify fate. In other words, wild-type lineages for B.a(l/r)aa were observed in animals with B.a(l/r)p-(3/3 wt), B.a(l/r)pa-and B.a(l/r)ap (3/3 wt), or B.a(l/r)pp-and B.a(l/r)ap- (2/2 wt) ablations, although occasionally division axes were skewed. 5) Interactions between other B.a progeny and the gamma/delta equivalence group. Experiments to test the interactions between other B.a progeny and B.a(l/r)pp are incomplete. Nevertheless, it is evident that the fates of B.a(l/r)pp (or, possibly their progeny), like B.a(l/r)aa, depend on their B.a neighbors. Gamma/delta are wild type in B.a(l/r)a ablated animals (3/3 wt). However, ablation of B.a(l/r)pa and B.a(l/r)aa results in truncated y lineages (4/4 truncated). Perhaps even more curious, ablation of B.a(l/r)ap and B.a(l/r)pa results in three rounds of division with abnormal axes in both presumptive gamma and delta (2/2 abnormal). It is not likely that this is a transformation of delta to gamma fate. One possible explanation is that B.a(l/r)aa (or their progeny) can actively promote cell division in B.a(l/r)pp (or progeny). In unablated animals, this activity is blocked by B.a(l/r)pa and B.a(l/r)ap. Additional experiments may help clarify the roles of B.a neighbors in specification of gamma and delta fate.

Hishida R et al. (1994) Worm Breeder's Gazette "hch-1 Rescued"

Ishihara T, Kondo K, Katsura I, Hishida R

[Worm Breeder's Gazette, 1994]

hch-l Rescued Ryuichi Katsural I )DNA Research Center, National Institute of Genetics. Mishima, Shizuoka-ken 411, Japan, 2)Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan and 3)Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192, Japan Mutations in the gene hch-l cause defects in both hatching and cell migration (E. Hedgecock et al., Development 100, 365-3~2, 1987). First, hch-l mutants exhibit delayed hatching, because the mutant embryos cannot digest proteins in their own eggshell. Second, the descendants of the QL neuroblast of hch-l mutants fail to migrate posteriorly, but migrate anteriorly, instead. How does hch-l gene function in these quite different systems? We have isolated a new allele of hch-l, utllO, from a mul-6 strain in the course of screening larval lethal mutations that cause gross morphological abnormalities (I. Katsura et al., WBG 12-2 plO8). Recently we cloned a unique Tcl containing fragment (HindIII 3.0kbp) from the genomic DNA of hch-l (utllO). A YAC filter and some cosmid clones were analyzed with a DNA fragment flanking the Tcl as a probe. This probe hybridized to YAC clones Y42D5, YlSF6, Y52Cll, Y43C6 and cosmid clones C42E7, F40ElO, M86, KllE3, TlSAlO, F23Cl. The result is consistent with genetic map position of hch-l gene (near unc-3 X). Since the Tcl insertion site is located near either end of the cosmid clones, we did not try to rescue hch-l worms with the cosmids. Instead, we cloned two DNA fragments containing the Tcl insertion site, a l5kbp PstI-Clal fragment and a 9kbp SacI fragment, from the N2 genomic DNA. These clones together with pRF4 as a dominant Rol marker were injected into hch-l (utl 10) mutants, and the extrachromosomally transformed worms were irradiated with gamma ray to obtain integrative transformants. The integrative transformants containing the Pstl-Clal fragment or the Sacl fragment show no delay of hatching. We are presently trying to rescue with shorter DNA fragments. Total RNA isolated from mixed staged N2 worms was analyzed by Northern blot using the Sacl fragment as a probe. This experiment revealed two transcripts (roughly 3kb and O.5kb in length) in this region. We plan to determine if these RNAs are encoded by hch-l gene and to isolate hch-l cDNA.