CONFIRMATION OF MUTANT ALPHA(1) NA,K-ATPASE GENE AND TRANSCRIPT IN DAHL SALT-SENSITIVE/JR RATS

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 glutamine(276) with leucine and associated with decreased net Rb-86(+) (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 T-1079 but rather only the wild-type A(1079) 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 A(1079)-->T transversion in the Dahl salt-sensitive alpha(1) Na,K-ATPase allele and its transcript, as well as the wild-type A(1079) 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 T-1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T-1079 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.