title: Confirmation of mutant alpha 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats.
abstract: As the sole renal Na,K-ATPase isozyme, the alpha 1 Na,K-ATPase accounts for all active transport of Na+ throughout the nephron. This role in renal Na+ reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)1079-->thymine (T) transversion, resulting in the substitution of glutamine276 with leucine and associated with decreased net 86Rb+ (K+) influx, was identified in Dahl salt-sensitive/JR rat kidney alpha 1 Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T1079 but rather only the wild-type A1079 alpha 1 Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined alpha 1 Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A1079-->T transversion in the Dahl salt-sensitive alpha 1 Na,K-ATPase allele and its transcript, as well as the wild-type A1079 sequence in the Dahl salt-resistant alpha 1 Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T1079 alpha 1 cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type alpha 1 Na,K-ATPase sequence. This identifies a site- and nucleotide-specific Taq polymerase misincorporation, suggesting that a structural basis might underlie a predisposition to nonrandom Taq polymerase errors.
authors.affiliation: Section of Molecular Genetics, Whitaker Cardiovascular Institute, Boston University School of Medicine, MA 02118.
authors.forename: N
authors.initials: N
authors.lastname: Ruiz-Opazo
authors.forename: F
authors.initials: F
authors.lastname: Barany
authors.forename: K
authors.initials: K
authors.lastname: Hirayama
authors.forename: V L
authors.initials: VL
authors.lastname: Herrera
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005796:Genes; D005821:Genetic Techniques; D008025:Ligases; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012965:Sodium Chloride; D008025:Ligases; D000254:Sodium-Potassium-Exchanging ATPase