Gene structure, cDNA cloning, and expression of a mouse mercurial-insensitive water channel.
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 mMIWC1 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.
Ma
T
T
Department of Medicine, University of California, San Francisco, California, 94143-0521, USA.
Yang
B
B
Verkman
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005091:Exons; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D066298:In Vitro Techniques; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D014158:Transcription, Genetic; D014982:Xenopus laevis
C496021:Aqp1 protein, mouse; D020346:Aquaporins; D017931:DNA Primers; D018076:DNA, Complementary; D007473:Ion Channels; D012333:RNA, Messenger; D051398:Aquaporin 1; C094483:mercurial-insensitive water channel