V-H-ATPase consists of at least 10 individual subunits. Isoforms of subunits (70kD, 56kD, 32kD) which are part of the catalytic section of this protein have been described and localized within the kidney. The 16kD subunit forms the transmembranous proton pore and has been cloned from several sources including mouse brain, rat liver and bovine adrenal. We set out to map this subunit within the mouse genome. Using two sets of specific oligonucleotide primers synthesized based on the mouse brain cDNA sequence, PCR was utilized to amplify two segments-the entire coding region (bH, 451bp) and the 3′-untranslated region (sH, 148bp). These two products were subcloned into the plasmid vector pGEM and the DNA sequences were verified. Using bH to probe restriction digested genomic DNA from a (C57BL/6J × Mus spretus) F1 × Mus spretus backcross, chromosomal mapping was performed. RFLP patterns indicated the presence of three independent genes which localized to mouse Chromosomes 6,7 and 17. Using sH as a probe, only two genes were identified. Human mapping studies utilizing DNA from somatic cell hybrids detected two gene loci on Chromosomes 3 and 16. Northern blot analysis using mouse RNA identified a 1.2kb transcript in all tissues tested (kidney, brain, liver, spleen, testis and skeletal muscle), corresponding to the size of the known cDNA. Two larger transcripts (≈3.3kb and ≈5.8kb) were also identified in the kidney. The screening of a mouse kidney cDNA library is underway using bH as a probe. The initial screening detected over 70 positives, several of which have been purified and isolated. DNA sequence analysis found the 3′ end of a 1.6kb clone to be identical to the known sequence and the 5′ end to be completely dissimilar.
We conclude that identification of 3 genetic loci in the mouse genome, the expression of separate RNA transcripts in the kidney and the detection of cDNA clones >1.2kb supports the existence of larger transcripts which may code for isoforms of the 16kD V-H-ATPase subunit. A clone from a kidney cDNA library which differs at the 5′ end may represent an isoform. This may correspond to the band which is not present in the Southern blot chromosomal mapping studies when sH is used as a probe. This work has been funded in part by the Sam and Helen Kaplan Research Fund in Pediatric Nephrology and the Katherine B Richardson Fund.