ABCMdb

PMID: 10220334

Chen M, Zhang JT
Topogenesis of cystic fibrosis transmembrane conductance regulator (CFTR): regulation by the amino terminal transmembrane sequences.
Biochemistry. 1999 Apr 27;38(17):5471-7., 1999-04-27 [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCC7 p.Lys246Asn ABCC7 p.Arg251Glu ABCC7 p.Gln220Lys ABCC7 p.Arg242Glu ABCC7 p.Arg248Glu ABCC7 p.Glu217Arg Primers carrying various mutations are 5'-AATCTGGAGGTTGTTAAAGGCGTC-3' (E217R/Q220K), 5'-CATCATTTCCCCTAGCCC-3' (R242E), 5'-CT- GATCTTCGTAATTCATCATCAT-3' (K246N/R248E), and 5'-TCCCAGCTTCCTGATCT-3' (R251E). Login to comment 48 ABCC7 p.Gln220Lys ABCC7 p.Glu217Arg To make the CFTR-N4R(-8) and CFTR-N4R(-5) mutants, two PCR reactions were performed as described above using CFTR-N4R(E217R/Q220K) or wild-type CFTR-N4R as templates and the mutant primer 5'-AGGGGAAATGATGATGGAG- TACGAAGATCAGGAAGCTGGGGATATCAG-3'. Login to comment 49 ABCC7 p.Lys246Asn ABCC7 p.Arg251Glu ABCC7 p.Gln220Lys ABCC7 p.Arg242Glu ABCC7 p.Arg248Glu ABCC7 p.Glu217Arg The resulting constructs were named CFTR-N4R(E217R/Q220K), CFTR-N4R(R242E), CFTR-N4R(K246N/R248E), CFTR-N4R(R251E), CFTR-N4R(-8), and CFTR-N4R(-5). Login to comment 50 ABCC7 p.Gln220Lys ABCC7 p.Gln220Lys ABCC7 p.Glu217Arg ABCC7 p.Glu217Arg To remove TM1 and TM2 from CFTR-N4R, CFTR-N4R- (E217R/Q220K), CFTR-N4R(-8), and CFTR-N4R(-5), PCR was performed using a primer 5'-GAGACCATGCA- GATGAGAATAG-3' containing Kozak translation initiation codon and a primer 5'-CACTTTTGCCAACCAG-3' in the glycosylation reporter sequence on templates CFTR-N4R, CFTR-N4R(E217R/Q220K), CFTR-N4R(-8), and CFTR-N4R(-5), respectively. Login to comment 53 ABCC7 p.Lys246Asn ABCC7 p.Lys246Asn ABCC7 p.Gln220Lys ABCC7 p.Arg242Glu ABCC7 p.Arg242Glu ABCC7 p.Arg248Glu ABCC7 p.Arg248Glu ABCC7 p.Glu217Arg The final DNA clones were named CF-TM3,4R, CF-TM3,4R(E217R/Q220K), CF-TM3,4R(-8), and CF-TM3,4R(-5), respectively. To engineer R242E and K246N/R248E mutations into CF-TM3,4R, an EcoRI-EcoNI fragment encoding TM3 and part of TM4 was released from CF-TM3,4R and used to replace the amino terminal-encoding sequence in CFTR-N4R(R242E) and CFTR-N4R(K246N/R248E). Login to comment 54 ABCC7 p.Lys246Asn ABCC7 p.Arg242Glu ABCC7 p.Arg248Glu The resulting constructs were named CF-TM3,4R(R242E) and CF-TM3,4R(K246N/R248E), respectively. To replace TM1 and TM2 of CFTR with that of Pgp, CFTR-N4R(-8) was linearized with XbaI, treated with Klenow DNA polymerase supplemented with dNTP in the absence of dGTP to avoid filling at the G position, and then digested with HindIII. The XbaI-HindIII fragment from CFTR-N4R(-8) and an EcoRI-NciI fragment encoding TM1 and TM2 of Pgp from pGPGP-N3 (12) were ligated into a pGEM-4z vector digested with EcoRI and HindIII. The resulting construct was named P1,2CF3,4R(-8). Login to comment 72 ABCC7 p.Lys246Asn ABCC7 p.Gln220Lys ABCC7 p.Arg242Glu ABCC7 p.Arg248Glu ABCC7 p.Glu217Arg The mutant molecules used were CF-TM3,4R(E217R/ Q220K), CF-TM3,4R(R242E), and CF-TM3,4R(K246N/ R248E). Login to comment