Supplemental Methods(1)

Document Sample
Supplemental Methods(1) Powered By Docstoc
					Supplemental Methods Worm culture and strains Culture media (modified NGM, containing no added calcium or magnesium) consisted of the following (per liter): 2 g NaCl, 3.1 g Peptone, 3.1 g KH2PO4, 0.5 g K2HPO4, 20 g Sigma A-7002 agar. After autoclaving and cooling to 55˚ the following was added (per liter): 1.6 ml 5 mg/ ml cholesterol in ethanol, 1.0 ml of 100 mg/ ml Streptomycin in ddH2O, 1.25 ml of 10 mg/ ml mycostatin suspension in ethanol. For spread plates we supplemented modified NGM with 1 g per liter of yeast extract and doubled the peptone. Worm food was strain OP-50 E. coli grown in tryptone broth (5 g NaCl; 10 g tryptone per liter) at 30˚ overnight. 6 cm spread plates were spread with 100 l of OP-50 culture 1 day after pouring and incubated 6 more days at room temperature. Streak plates for standard culture were made by dipping a 6 mm rounded-tip glass rod into OP-50 culture and dragging once across the center of the plate for most of its diameter. For growth assay plates, we spread the plates with 15 l of OP-50 culture, leaving a substantial clear border (~7-10 mm) around the edge to decrease the incidence of worms crawling off the media, and then cured two more days at room temperature with lid side up before storing at 4˚ for up to one month. Locomotion plates were made as described (Miller et al. 1999). We observed and manipulated live animals under Olympus SZX-12 stereomicroscopes equipped with 1.2X, 0.13 NA plan apochromatic objectives. The term “growing culture” used in the below methods means we plated 9 or more L2 or L3 starved larvae (depending on strain growth rate) on spread plates and grew them 6 d (or more for some strains) X 20˚ to make adult F1 progeny that had never been starved. We prepared 24-well culture plates for genetic screens as described (Schade et al. 2005). To titer suspensions of worms for precise plating numbers, we took several 2 – 15 l aliquots immediately after vortexing and added them to wells containing 50 l of 10mM sodium azide in 96-well flat bottom plates. The plate contained a grid drawn on a transparency taped to the bottom to facilitate rapid counting with a finger counter. Other worm culture and manipulation essentially followed previously described methods (Brenner 1974; Stiernagle 2006). References and descriptions of the strains used in this study are cited in the text and/ or figure legends. Complete strain names and genotypes of the major non-wild strains in this study are listed below:

Strain name KG538 KG614 KG545 EG3776 RM2221 KG1020

Genotype egl-30(ad805) egl-30(ad810)/+ egl-30(tg26) egl-30(tg26) unc-73(ox317) egl-8(md1971) egl-8(md1971); ceEx149 [rab-3::egl-8(+) cDNA/ ttx-3::GFP/


rab-3::GFP] 30/15/15 ng/ ul KG1383 JT9887 KG1277 KG1308 RM2425 KG935 KG1278 KG1283 KG1309 KG1358 egl-8(sa47) goa-1(sa734) unc-108(ce361) unc-108(ce361); egl-8(md1971) unc-13(md1072) unc-29(x29) unc-73(ce362) unc-73(ce362) goa-1(sa734) unc-73(ce362)/ dpy-5(e61); egl-8(md1971) unc-73(ce362); ceEx195 [HS::unc-73E cDNA/ myo-3::GFP] 35/12 ng/l KG1382 KG1356 unc-73(ce362); egl-8(md1971) unc-73(ce362); ceIs39 [rab-3::unc-73E cDNA/ ttx-3::GFP/ rab-3::GFP] 50/15/15 ng/ l KG1452 unc-73(ce362); ceEx208 [unc-17::unc-73E cDNA/ unc17::GFP] 35/25 ng/ l KG1397 KG1393 KG1397 XA7300 KG1341 unc-73(ev802) unc-73(ev802)/ dpy-5(e61); egl-8(md1971) unc-73(ev802)/ dpy-5(e61); egl-8(sa47) unc-73(ev802)/ unc-11 dpy-5(e61) unc-73(ev802); egl-8(md1971); qaIs7312 [unc-73D::UNC73D/ F25B3.3::GFP] (also known as unc-73(ev802); egl8(md1971); D1)



unc-73(ev802);qaIs7312 [unc-73D::UNC-73D/ F25B3.3::GFP] (also known as unc-73(ev802); D1)

EG317 EG4719 EG322

unc-73(ox317) unc-73(ox317); egl-8(sa47) unc-73(ox322)

Plasmids KP309, a full-length egl-8 cDNA (Lackner et al. 1999) is gift of Mark Lackner and Joshua Kaplan. To make KG#209 [rab-3::egl-8(+) cDNA] we used Pfu Ultra and primers engineered with restriction sites to clone the egl-8 cDNA coding region from KP309 into Nhe I/ Not I – cut KG#208. KG#208 is a rab-3:: pan-neuronal expression vector identical to KG#59 (Schade et al. 2005), except it contains a Not I site produced by oligonucletide insertion between the Xho I and Bgl II sites in the polylinker. To make KG#281 [rab-3::unc-73e cDNA], we used StrataScript Reverse Transcriptase and a primer engineered with a restriction site to make the full length unc-73e cDNA (the unc-73e isoform we used corresponds to unc-73E as annotated in Wormbase freeze WS170). We then used Accuprime Pfx and primers engineered with restriction sites to amplify and clone the cDNA into Nhe I/ Kpn I cut KG#59. To make KG#94 (unc-17:: expression vector), we used Apa I/ Msc I to cut out the ~1900 bp multi-cloning site + GFP + unc54 3’ control region from RM#349p (unc-17::GFP), leaving the 5900 bp vector fragment containing the 3.2 Kb unc-17 promoter. To this fragment we ligated the 1079 bp Msc I/ Apa I fragment (containing the multi-cloning site and unc-54 3’ control region) cut from pPD96.52 (myo-3:: expression vector; gift of Andrew Fire). To make KG#282 [HS::unc-73e cDNA] and KG#332 [unc-17::unc-73e cDNA], we used Nhe I/ Kpn I to remove the unc-73e cDNA from KG#281 and cloned the insert into the like-digested hsp16-2:: expression vector KG#45 (Reynolds et al. 2005) or the KG#94 unc-17:: expression vector. For the HEK293 cell culture assays, we cloned cDNAs into mammalian expression vectors that use the CMV promoter. To make KG#309 Flag-unc-73e, we used Herculase II polymerase (Stratagene) and primers engineered with restriction sites to amplify the unc-73e cDNA from KG#281 (rab-3::unc-73e cDNA) and cloned it into Eco RI/ Xho I cut pCMV-Tag 2A (Stratagene). The construct encodes an N-terminal Flag tag (DYKDDDDK), then 10 amino acids of MCS sequence before the inframe start of UNC-73E. We made KG#310 Flag-unc-73e [234-577] using similar methods and the same restriction sites. The unc-73e region in KG#310 begins with EKTPEET and ends with GDLSLGA and an artificial stop codon. To make KG#311 egl-30 (Gq Q205L cDNA), we used Herculase II polymerase and primers engineered with restriction sites to amplify the egl-30 Q205L cDNA from KG#49 (HS::egl-30 Q205L cDNA) and cloned it into Nhe I/ Kpn I cut pcDNA3.1 (Invitrogen). We made KG#312 goa-1 (Go Q205L cDNA) using similar methods and the same restriction sites. We used the Quikchange mutagenesis method to introduce the Q205L gain of function mutations into the wild type G cDNAs. In all constructs involving the cloning of PCR fragments, we sequenced the inserts and used clones containing no mutations in the fragment of interest to establish the final plasmid stock. Transgenic strain production

We produced transgenic strains bearing extrachromosomal arrays by the method of Mello et al. (Mello et al. 1991). We used pBluescript carrier DNA to bring the final concentration of DNA in the injection mixture to 175 ng/ l. To make egl-8(md1971); ceEx149 we injected egl-8(md1971) mutants with KG#209 [rab-3::egl-8 cDNA] at 30 ng/ l. To make unc-73(ce362); ceIs39 we injected unc-73(ce362) mutants with KG#281 [rab-3::unc-73e cDNA] at 50 ng/ l. After cloning 12 F2s from one of the stably transmitting lines, one of the lines transmitted the array to 100% of its progeny indicating an integration event had likely occurred. We made unc-73(ce362); ceEx195 by injecting unc-73(ce362) mutants with KG#282 [HS::unc-73e cDNA] at 35 ng/ l. Injection mixtures for ceEx149 and ceIs39 included the co-transformation marker plasmids KG#68 [rab-3::GFP] and KG#67 [ttx-3::GFP] at 15ng/ l each. We used pPD118.20 [myo3::GFP] at 12ng/ l to mark ceEx195. References Brenner, S. 1974. The genetics of C. elegans. Genetics 77: 71-94. Lackner, M.R., Nurrish, S.J., and Kaplan, J.M. 1999. Facilitation of synaptic transmission by EGL-30 Gqand EGL-8 PLC: DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24: 335-346. Mello, C.C., Kramer, J.M., Stinchcomb, D., and Ambros, V. 1991. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10 (12): 3959-3970. Miller, K.G., Emerson, M.D., and Rand, J.B. 1999. Go and diacylglycerol kinase negatively regulate the Gq pathway in C. elegans. Neuron 24(2): 323-333. Reynolds, N.K., Schade, M.A., and Miller, K.G. 2005. Convergent, RIC-8 Dependent G Signaling Pathways in the C. elegans Synaptic Signaling Network. Genetics 169(2): 650670. Schade, M.A., Reynolds, N.K., Dollins, C.M., and Miller, K.G. 2005. Mutations that Rescue the Paralysis of C. elegans ric-8 (Synembryn) Mutants Activate the Gs Pathway and Define a Third Major Branch of the Synaptic Signaling Network. Genetics 169(2): 631-649. Stiernagle, T. 2006. Maintenance of C. elegans. in WormBook (ed. T.C.e.R. Community).


Shared By:
pptfiles pptfiles