Isolation of chicken alpha ENaC splice variants from a cochlear cDNA library.
Three splice variants of the alpha subunit of the amiloride-sensitive epithelial sodium channel (alpha ENaC) have been isolated from a chicken cochlear cDNA library. A PCR product, generated from the cochlear library using degenerate primers to regions of homology between the rat alpha ENaC and the degenerin Mec-4, was used as a probe. The three splice variant cDNAs with sizes of 2321, 3399 and 3845 bp correspond to transcripts of 2.5, 3.5 and 3.9 kb as detected by Northern blot analysis. The 3399 bp clone differs from the 2321 clone solely by the addition of 1079 bases in the 3'-non-coding region. Both these cDNAs code for an identical predicted protein of 637 amino acids which has 68% similarity to the rat alpha ENaC, and is probably the chicken homologue of alpha ENaC. The third cDNA of 3845 bp is similar to the 3399 bp clone but includes two exons within the open reading frame. The first of these exons introduces a premature stop codon resulting in a truncated predicted protein of 434 amino acids. Northern blot analysis shows expression of the 2.5 and 3.5 kb transcripts in cochlea and colon, the 2.5 kb transcript in cartilage, whilst the 3.9 kb transcript is only detected in cochlea. No expression is detected in brain, liver, and heart nor, most notably, in lung or kidney.
Killick
R
R
School of Biological Sciences, Sussex University, Falmer, Brighton, UK. r.killick@sussex.ac.uk
Richardson
G
G
D017398:Alternative Splicing; D000584:Amiloride; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003051:Cochlea / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D004848:Epithelium / Q000378:metabolism; D015723:Gene Library; D014644:Genetic Variation; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D014981:Xenopus
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D015222:Sodium Channels; D000584:Amiloride