AChR subunit constructs
The full-length ACR-2
GFP transgene (pDM1232) was generated by introducing the GFP coding sequence in-frame into sequence of an acr-2
genomic fragment (−3353 to +7776 bp relative to the translational start site) encoding the intracellular loop between TM3 and TM4. The full-length ACR-2(L/S) construct (pBB9) was generated by PCR based site directed mutagenesis using mutant primers and pDM1232 as the template. The Pacr-2
ACR-12 cDNA (pHP3) and Punc-47
ACR-12 cDNA (pBB25) constructs were generated by amplifying the acr-12
cDNA from the EST yk1093d12 (gift of Yuji Kohara) using sequence-specific primers designed to the start and stop of acr-12
and subcloning into the NheI/SacI sites of a plasmid containing an approximately 3.3 kb promoter for the acr-2
gene or into a plasmid containing a 1.3 kb promoter for the unc-47
construct (pPRB5) was generated by subcloning an AgeI/AatII fragment that contained the full length mCherry coding sequence into a vector containing a 1.3 kb promoter for the unc-47
gene. The Pacr-2
GFP (pPRB19) construct was generated by subcloning an AatII/BamHI fragment that contained an approximately 3.3 kb promoter for the acr-2
gene into a vector containing the GFP coding sequence.
C. elegans Strains
C. elegans strains were grown on NGM plates with the OP50 strain of Escherichia coli at 22°C using standard laboratory procedures. Wild type animals are the N2 Bristol strain. All transgenic strains were obtained by microinjection of plasmid DNA into the germ line and data presented are from a single representative transgenic line unless otherwise noted. In all cases, lin-15(n765ts) mutants were injected with the lin-15 rescuing plasmid (pL15EK; 30 ng/μl) with one or more of the following plasmids (30 ng/μl): pBB9, pBB25, pDM1232, pHP3, pPRB4, pPRB5, pPRB14, pPRB19. Multiple independent extragenic lines were obtained for each transgenic strain. Stably integrated lines were generated as necessary by X-ray integration and outcrossed at least four times to wild type. The transgenic strain expressing the integrated ACR-2(L/S) transgene (ufIs25) was outcrossed ten times to wild type. The following strains were used in this study: RB1559 acr-2(ok1887), IZ421 acr-12(ok367), RB2071 ced-3(ok2734), VC1801 cnx-1(ok2234), RB1021 crt-1(ok948), IZ74 unc-29(x29), CB904 unc-38(e264), CB306 unc-50(e306), VC731 unc-63(ok1075), CB883 unc-74(e883), IZ380 ufIs31, IZ814 ufIs25, IZ625 ufIs25;ufIs31, IZ790 ufIs49, IZ627 ufIs42, LX949 vsIs48, IZ924 ufIs25;vsIs48, IZ950 ced-3(ok2734);ufIs25;vsIs48, IZ877 cnx-1(ok2234);ufIs25;vsIs48, IZ926 crt-1(ok948);ufIs25;vsIs48, IZ976 crt-1(ok948);cnx-1(ok2234);ufIs25;vsIs48, IZ971 crt-1(ok948);cnx-1(ok2234);vsIs48, IZ927 crt-1(ok948);cnx-1(ok2234);ufIs42, IZ787 unc-29(x29);ufIs25, IZ928 acr-3(ok2049);ufIs265, IZ929 acr-5(ok180);ufIs25, IZ930 acr-9(ok933);ufIs25, IZ931 acr-19(ok967);ufIs25, IZ932 acr-23(ok2840);ufIs25,IZ446 unc-38(e264);ufIs25, IZ659 unc-50(e306);ufIs25, IZ921 unc-63(ok1075);ufIs25, IZ490 unc-68(e540);ufIs25, IZ604 unc-74(e883);ufIs25, IZ673 acr-12(ok367);ufIs25;ufEx148, IZ937 acr-12(ok367);ufIs25;ufIs60, IZ861 acr-12(ok367);ufIs25;ufEx191.
Genetic screen and identification of suppressors of ACR-2(L/S) induced paralysis
Paralyzed animals expressing the integrated ACR-2(L/S) array (ufIs25
) were mutagenized with 50 mM EMS (Brenner 1974
). Young adult F2 progeny of approximately 20,000 mutagenized animals were washed twice with M9 and transferred to fresh plates. After allowing time for the animals to disperse, moving animals were picked to single plates. Eighty-one candidates suppressors were isolated. A secondary screen showed that fifty-one of these were resistant to the paralyzing effects of levamisole. For genetic complementation tests, males carrying a mutation in candidate levamisole-resistance genes were crossed with hermaphrodites carrying the ACR-2(L/S) transgene and a suppressor mutation. F1 progeny were scored for paralysis. A cross was performed in parallel using N2 males to identify X-linked suppressor mutations and determine dominance/recessivity. The mapping of acr-12
alleles was carried out in the presence of the ACR-2(L/S) transgene. A strain carrying the integrated ACR-2(L/S) transgene (ufIs25
) on LG I was backcrossed 7X to the CB4856 Hawaiian strain. acr-12
alleles were mapped to a region the right of +8 on the X chromosome using the SNP mapping procedure as previously described (Davis et al. 2005
; Wicks et al. 2001
All behavioral analysis was performed with young adult animals (24 hr post-L4) at room temperature (22°C–24°C); different genotypes were scored in parallel, with the researcher blinded to the genotype.
Aldicarb and levamisole assays
Staged populations of adult animals (approximately ten) were transferred to NGM plates containing 1 mM aldicarb (Chem Services) and movement was assessed every 15 minutes for two hours. Data represent the mean ± SEM of at least four assays. For levamisole assays, staged populations of adult animals were scored for paralysis after 120 minutes on plates containing 200 μM levamisole.
Body Bend Analysis
Body bends were scored on unseeded NGM agar. Animals were transferred from their culture plate to an unseeded plate and allowed to crawl away from any food that might have been transferred. The animals were then gently transferred without food to another unseeded plate and allowed to recover for 1 min. After the recovery period the animals were filmed for 5 min using an Imaging Source DMK 21F04 firewire camera and iMovie software.
Confocal microscopy was carried out using a Zeiss Axioskop 2 microscope system and LSM Pascal 5 imaging software (Zeiss). Images were processed using Image J software (open source). Epifluorescent imaging was performed using a Zeiss Axioimager M1 microscope and Axiovision software (Zeiss). Movies and still images for behavioral analyses were obtained using an Olympus SZ61 upright microscope equipped with a Firewire camera (Imaging source). For the developmental timeline, synchronized populations were obtained by bleaching gravid animals on NGM plates seeded with OP50. The resulting progeny were allowed to mature at room temperature. Animals were imaged at 16, 28, 38 and 48 hours after bleaching using wide-field epifluorescent microscopy and the number of surviving cell bodies were counted manually with the researcher blinded to genotype.