Gene structure, cDNA cloning, and expression of a mouse mercurial-insensitive water channel
- Univ. of California, San Francisco, CA (United States)
Three cDNAs encoding isoforms of a mercurial-insensitive water channel (mMIWC) were cloned from a mouse brain cDNA library. The predicted proteins had distinct N-terminal sequences and were 32.0 (mMIWC1), 34.3 (mMIWC2), and 37.8 (mMIWC3) kDa. Immunoblot analysis of mouse brain membranes with a C-terminus-derived polyclonal antibody was consistent with the predicted sizes. Expression in Xenopus oocytes indicated that each isoform functioned as a mercurial-insensitive, water-selective channel. Northern blot analysis indicated a major transcript of 5.5 kb in brain > eye > lung {approximately} kidney, and a minor 1.7-kb transcript in heart and muscle. Sequence comparison of mMIWC1 cDNA with a cloned 24-kb mouse genomic DNA indicated three introns (lengths 1.5, 0.5, and 4.0 kb) separating four exons with boundaries at amino acids 127, 182, and 209; analysis of mMIWC2 and mMIWC3 sequences indicated an additional intron at nucleotide -34 upstream from the mMIWC translation initiation site. The mMIWC1 promoter was identified and contained TATA, CAAT, GATA, and AP-2 elements; primer extension revealed mMIWC transcription initiation at 621 bp upstream from the mMIWC1 translational initiation site. Genomic Southern blot analysis revealed a single-copy mMIWC gene. These data indicate the presence of multiple mMIWC isoforms with distinct N-termini encoded by mRNAs produced by distinct transcriptional units and alternative splicing. The genomic cloning of mMIWC represents the first step in the construction of a targeting vector for mMIWC gene knockout. 21 refs., 4 figs.
- OSTI ID:
- 466655
- Journal Information:
- Genomics, Journal Name: Genomics Journal Issue: 3 Vol. 33; ISSN 0888-7543; ISSN GNMCEP
- Country of Publication:
- United States
- Language:
- English
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