DETECTION OF TRANSCRIPTS FOR DELAYED RECTIFIER POTASSIUM CHANNELS IN THE XENOPUS-LAEVIS INNER-EAR

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to a mplify sequences for delayed rectifier potassium (drk) channel transcr ipts in Xenopus laevis inner ear and brain. We used degenerate primers that spanned a region between the N-terminal cytoplasmic portion and a region located between the S2 and S3 transmembrane domains of the po tassium channel protein. When inner ear total RNA or brain mRNA was us ed as a template for RT-PCR, a unique product of the expected size (si milar to 560 bp) was observed as a single band after electrophoresis o n agarose gels. The PCR product from reactions using X. laevis genomic DNA as template was similarly sized, indicating a lack of introns in this region. The RT-PCR products from inner ear and brain were isolate d, cloned, and sequenced. Sequence analysis showed that the X. laevis inner ear and brain clones were identical. Sequence alignments of the cloned RT-PCR products with posted GenBank sequences established that the drk sequences from X. laevis inner ear and brain share highest ide ntity with larval X. laevis brain, mouse, rat, and human Kv2 sequences . Positive signals were obtained from inner ear and brain mRNA in Nort hern dot blots hybridized with digoxigenin labeled probes from the inn er ear clone. Taken together, results provide evidence for the express ion of Kv2 sequences in the X. laevis inner ear and brain. (C) 1998 Pu blished by Elsevier Science B.V.