End of DInR,from amino acids R to A,is present upstream in the MYC tag. This area includes a PPPP sequence (PP). The following point mutations have been generated: DInRKA (KA) inside the kinase domain; YF (YF) within the Ctail; YF (YF) in the Ctail; Y,F (YF,YF) inside the Ctail; LESL (PL,PL) within the Ctail; YF (YF),YF (YF),YF (YF),YF (YF) and combinations thereof,in NPXY sites in the Ctail. To produce these point mutations,a PCR product on the dinr Ctail from plasmid DInRCKDPAS (Song et al was subcloned in to the vector pSP at its ClaIKpnI web page to yield pSPdinrMT. Sitedirected mutagenesis of pSPdinrMT was carried out by PCR ( C for min; cycles of: C for s,C for min and C for min). The PCR item was digested with DpnI for h at C to destroy any unmutagenized template plasmid present,and was transformed into XL competent cells. All mutations had been confirmed by DNA sequencing. The ClaIKpnI fragments with many dinr mutations had been shuttled from pSPdinrMT into pASOFCT for yeast twohybrid assays. Primer sequences accessible upon request. To produce transgenic Drosophila,fulllength,partial or point mutationcontaining dinr cDNAs were inserted into pUASTdinrMYC,a Pelement vector which consists of a bp area encoding a X Myc tag to create inframe Cterminal fusions. This vector was generated as follows. The AflIINheI fragment in pSPdinr was replaced by the PCR fragment of pSPdinr amplified employing two primers,P and Srf,and digested with AflIINheI to introduce an SrfI site so that the MYC tag could be inserted into the vector. The newly generated plasmid was named pSPdinrM. A MYC tag was excised from the plasmid pSRLhSNT MYC (a present from Dr. Mitch Goldfarb,Hunter College) and subcloned in to the SrfINotI web page of pSPdinrM,producing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20020269 pSPdinrMYC. The dinrMYC cDNA from pSPdinrMYC was subcloned in to the EcoRINotI web-site of pUAST to create thewww.frontiersin.orgJanuary Volume Article Li et al.Segregating Drosophila insulin receptor signalingfulllength pUASTdinrMYC plasmid. dinr cDNAs carrying deletions ( ABC,AB,CD) were inserted into pUASTdinrMYC by replacing the BsiWINotI fragment of pUASTdinrMYC. Point mutations inside the Ctail of dinr,generated in pSPdinrMT,had been moved into pUASTdinrMYC by the replacement of your AflIINotI fragment. To test the a single NPFY motif in the juxtamembrane region,pUASTdinr(JMNPFF)MYC was generated,in which the tyrosine inside the juxtamembrane NPFY motif was changed to a phenylalanine (YF). Sitedirected mutagenesis to alter the TAT codon for tyrosine to the TTT codon for phenylalanine was carried out with typical techniques making use of pSPdinrMYC and Vent polymerase (NEB,Ipswich,MA). Then,a kb fragment spanning the entire dinrMYC coding region,and as a result containing the mutated juxtamembrane NPFF internet site,was released from the mutated pSPdinrMYC plasmid with NotI and EcoRI; this fragment was inserted in to the NotI and EcoRI web-sites of pUASTdinr(Y,,,F)MYC,replacing the entire dinr(Y,,,F)MYC coding area. pUASTdinr(NPXF)MYC was then made by excising a kb fragment containing the mutated NPXF web-sites inside the Ctail from pUASTdinr(Y,,,F)MYC making use of AflII and inserting it into the AflII site of pUASTdinr(JMNPFF)MYC to replace the AflII fragment. The orientation and sequence of every single dinr variant was verified by sequencing.YEAST TWOHYBRID ASSAYSused for analysis. For the lethality rescue analysis,armGALarmGAL;FRTBdinr TMSb,armGFP virgin females have been crossed to UASX(UASX or CyO);dinrex TMSb,armGFP males. Adult progeny that had eclosed were scored for their bristle phenotype: Bay 59-3074 either Sb or.
