cDNA cloning, gene organization, and chromosomal localization of a human mercurial insensitive water channel. Evidence for distinct transcriptional units.
Two distinct cDNAs encoding a human mercurial insensitive water channel (hMIWC) were cloned from a fetal brain cDNA library. The longest open reading frame of cDNA clone hMIWC1 encoded 301 amino acids with 94% identity to rat MIWC (Hasegawa, H., Ma, T., Skach, W., Matthay, M. M., and Verkman, A. S. (1994) J. Biol. Chem. 269, 5497-5500). A second cDNA (hMIWC2) had a distinct 5'-sequence upstream from base pair (bp) -34 in clone hMIWC1 and contained two additional inframe translation start codons. Expression of hMIWC cRNAs in Xenopus oocytes increased osmotic water permeability by 10-20-fold in a mercurial insensitive manner. Cell-free translation in a reticulocyte lysate/microsome system generated single protein bands at 30 kDa (hMIWC1) and 32-34 kDa (hMIWC2) without glycosylation. Northern blot and polymerase chain reaction/Southern blot analysis showed expression of mRNA encoding hMIWC in human brain - muscle >> heart, kidney, lung, and trachea. Analysis of hMIWC genomic clones indicated two distinct but overlapping transcription units from which multiple hMIWC mRNAs are transcribed. The promoter region of hMIWC1 was identified and contained TATA, CAAT, AP-1, and other regulatory elements. Primer extension revealed hMIWC1 transcription initiation at 46 bp downstream from the TATA box. There were three introns (lengths 0.9, 0.2, and 6 kilobases) in the hMIWC1 coding sequence at bp 381, 546, and 627. A distinct 5'-sequence in clone hMIWC2 suggested an alternative upstream transcription initiation site. Two alternatively spliced, nonfunctional hMIWC transcripts with exon 3 deletion and partial exon 4 deletion were identified. A poly(A)+ signal sequence was identified at 138 bp downstream of the translation stop codon. Genomic Southern blot analysis indicated the presence of a single copy hMIWC gene; chromosome-specific polymerase chain reaction and in situ hybridization localized hMIWC to human chromosome 18q22. The structural organization of the hMIWC gene represents a first step in definition of hMIWC differential expression, regulation, and possible role in human disease.
Yang
B
B
Department of Medicine, University of California, San Francisco 94143-0521, USA.
Ma
T
T
Verkman
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002874:Chromosome Mapping; D002887:Chromosomes, Human, Pair 18; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005091:Exons; D005260:Female; D005333:Fetus; D006801:Humans; D007438:Introns; D007473:Ion Channels / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D016366:Open Reading Frames; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014981:Xenopus
D017931:DNA Primers; D018076:DNA, Complementary; D007473:Ion Channels; D011994:Recombinant Proteins; C094483:mercurial-insensitive water channel