Control animals are isogenized (y w ey-FLP GMR-lacZ; FRT80B) unless otherwise indicated. 3L11, 3L12, and 3L13 mutants (y w ey-FLP GMR-lacZ;3L1x FRT80B/TM6B, Tb) were isolated from an ey-FLP ethane methyl sulfonate screen as described previously (Verstreken et al., 2003 (link)) with modifications. cspx1 mutants and UAS-ssp (w;; P{w+UAS-csp-11c/s}, cspu1) flies were provided by K. Zinsmaier (University of Arizona, Tucson, AZ). P-element stocks and deficiencies were obtained from the Bloomington Drosophila Stock Center (Bellen et al., 2004 (link); Parks et al., 2004 (link)), and 3L1 mapping was performed as described previously (Zhai et al., 2003 (link)).
We made a genomic rescue construct by PCR amplifying the 6.5-kb hip14 region from bacterial artificial chromosome clone AC093499. The fragment was cloned into the SalI restriction site of pP{CaSpeR-4} and sequenced. A cDNA construct was made by PCR amplifying hip14 from expressed sequence tag clone LD10758. The fragment was cloned into NotI and XbaI sites of pP{UAST} and sequenced.
To generate genomic GFP-tagged constructs, we first integrated an NheI site just before the ATG start codon (NtermGFP-HIP14) or after the hip14 stop codon (CtermGFP-HIP14) by site-directed mutagenesis (Stratagene). PCR-amplified EGFP sequence was cloned into the NheI site.
We generated chimeric n-SybTMD-CSP constructs by PCR amplifying 111-bp N-terminal and 234-bp C-terminal n-syb sequences from pP{UAST}-syb-GFP and the full-length CSP2 from pP{UAST}-csp2 (provided by K. Zinsmaier). In the next round of PCR, we fused them to generate N-terminal-Syb-csp2-C-terminal-Syb chimeric (SybTMD-csp2). After sequencing, SybTMD-csp2 was cloned into pP{UAST} at NotI and XbaI.
P{w+UAS-SybTMD-csp2} and P{w+UAS-csp2} were expressed using elav-GAL4. For analyses of CSP localization and physiology of third instar larvae, we generated elav-GAL4/+; P{w+UAS-SybTMD-csp2} hip142 FRT80B/Df(3L)brm11 and elav-GAL4/+; hip142 FRT80B P{w+UAS-csp2}/Df(3L)brm11.
We made a genomic rescue construct by PCR amplifying the 6.5-kb hip14 region from bacterial artificial chromosome clone AC093499. The fragment was cloned into the SalI restriction site of pP{CaSpeR-4} and sequenced. A cDNA construct was made by PCR amplifying hip14 from expressed sequence tag clone LD10758. The fragment was cloned into NotI and XbaI sites of pP{UAST} and sequenced.
To generate genomic GFP-tagged constructs, we first integrated an NheI site just before the ATG start codon (NtermGFP-HIP14) or after the hip14 stop codon (CtermGFP-HIP14) by site-directed mutagenesis (Stratagene). PCR-amplified EGFP sequence was cloned into the NheI site.
We generated chimeric n-SybTMD-CSP constructs by PCR amplifying 111-bp N-terminal and 234-bp C-terminal n-syb sequences from pP{UAST}-syb-GFP and the full-length CSP2 from pP{UAST}-csp2 (provided by K. Zinsmaier). In the next round of PCR, we fused them to generate N-terminal-Syb-csp2-C-terminal-Syb chimeric (SybTMD-csp2). After sequencing, SybTMD-csp2 was cloned into pP{UAST} at NotI and XbaI.
P{w+UAS-SybTMD-csp2} and P{w+UAS-csp2} were expressed using elav-GAL4. For analyses of CSP localization and physiology of third instar larvae, we generated elav-GAL4/+; P{w+UAS-SybTMD-csp2} hip142 FRT80B/Df(3L)brm11 and elav-GAL4/+; hip142 FRT80B P{w+UAS-csp2}/Df(3L)brm11.
