Fig. 1.
Fig. 1. Cloning of B3SAO and B3EQ into the pBabe neo vector. / Locations of the SAO deletion and the E681Q point mutation are indicated by a gray line in the band 3 cDNAs. For the cloning of B3SAO cDNA into pBabe neo, the 750-bp BstXI-SalI fragment of the previously described construct BSXG1.B3SAO11 was first substituted with the 412-bpBstXI-SalI fragment of pBnB3 (A), thus yielding the construct BSXG1*.B3SAO, which contained a SalI restriction site immediately downstream of the B3SAO cDNA in BSXG1. TheEcoRI-SalI fragment of BSXG1*.B3SAO that included 2066 bp of B3SAO cDNA (encoding the 9 amino acid SAO deletion) was then used to replace the EcoRI-SalI fragment of pBnB3 that included a 2093-bp fragment of B3 cDNA (B), thus generating the construct pBnB3SAO (C). Band 3 cDNA that comprised both the Memphis I polymorphism and the E681Q point mutation was cloned into pBn in 3 steps. The previously described BSXG1.B3 construct29 was used as template for site-directed mutagenesis (Sculptur kit; Amersham, Little Chalfont, United Kingdom), with the oligonucleotide 5′-CATATTCCTGCAGTCTCAGATC-3′ as primer (D). The correct mutant clone (designated BSXG1.B3EQ) was identified by PstI restriction digestion and DNA sequencing. When expressed in Xenopusoocytes, the band 3 protein (B3EQ) encoded by this clone did not mediate chloride transport (data not shown). The 1681-bpEcoRI-BstXI fragment of the B3EQ cDNA that spanned band 3 residues L217 to S773 was excised from the BSXG1.B3EQ construct and subcloned into the BSXG1*.B3SAO construct (E) to give BSXG1*.B3EQ. This construct was subjected toEcoRI-SalI restriction digestion, and the 2093-bp fragment that included the E681Q mutation was cloned into pBnB3 (F), thus generating the desired construct, pBnB3EQ (G). The band 3 coding region was verified in all constructs, using a 377 Applied Biosystems automated DNA sequencer. All vectors were linearized by using theScaI restriction site in pBabe prior to transfections.

Cloning of B3SAO and B3EQ into the pBabe neo vector.

Locations of the SAO deletion and the E681Q point mutation are indicated by a gray line in the band 3 cDNAs. For the cloning of B3SAO cDNA into pBabe neo, the 750-bp BstXI-SalI fragment of the previously described construct BSXG1.B3SAO11 was first substituted with the 412-bpBstXI-SalI fragment of pBnB3 (A), thus yielding the construct BSXG1*.B3SAO, which contained a SalI restriction site immediately downstream of the B3SAO cDNA in BSXG1. TheEcoRI-SalI fragment of BSXG1*.B3SAO that included 2066 bp of B3SAO cDNA (encoding the 9 amino acid SAO deletion) was then used to replace the EcoRI-SalI fragment of pBnB3 that included a 2093-bp fragment of B3 cDNA (B), thus generating the construct pBnB3SAO (C). Band 3 cDNA that comprised both the Memphis I polymorphism and the E681Q point mutation was cloned into pBn in 3 steps. The previously described BSXG1.B3 construct29 was used as template for site-directed mutagenesis (Sculptur kit; Amersham, Little Chalfont, United Kingdom), with the oligonucleotide 5′-CATATTCCTGCAGTCTCAGATC-3′ as primer (D). The correct mutant clone (designated BSXG1.B3EQ) was identified by PstI restriction digestion and DNA sequencing. When expressed in Xenopusoocytes, the band 3 protein (B3EQ) encoded by this clone did not mediate chloride transport (data not shown). The 1681-bpEcoRI-BstXI fragment of the B3EQ cDNA that spanned band 3 residues L217 to S773 was excised from the BSXG1.B3EQ construct and subcloned into the BSXG1*.B3SAO construct (E) to give BSXG1*.B3EQ. This construct was subjected toEcoRI-SalI restriction digestion, and the 2093-bp fragment that included the E681Q mutation was cloned into pBnB3 (F), thus generating the desired construct, pBnB3EQ (G). The band 3 coding region was verified in all constructs, using a 377 Applied Biosystems automated DNA sequencer. All vectors were linearized by using theScaI restriction site in pBabe prior to transfections.

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