| 1997, 855, Session CC6, Poster |
| RT-PCR analysis of gene expression in the Xenopus laevis inner ear: Actin and delayed rectifier potassium channels |
| C. Trujillo-Provencio, A. Varela-Ramirez, C.E. Williams, J.N. Gladden, *E.E. Serrano (New Mexico State University, Las Cruces, New Mexico) |
Research in our laboratory focuses on the development of the auditory and vestibular systems of the inner ear of Xenopus laevis. We are interested in the cellular and genetic mechanisms that underlie ear maturation. Our long term goal is to identify genes that are expressed during hair cell differentiation. Here we summarize results of experiments where we use an RT-PCR approach to clone actin and potassium (K+) channel transcripts from Xenopus inner ear, brain, and muscle tissue. We designed intron-spanning primers for actin, using sequences for Xenopus alpha-actin or beta-actin. RT-PCR reactions where mRNA from Xenopus brain, muscle, and ear was used as template yielded products of the expected size from all three tissues. The DNA from the RT-PCR reactions was cloned using the pGEM-T vector system (Promega), and then sequenced. Results from BLAST searches suggest that the cloned RT-PCR products are homologous to alpha-actin or beta-actin GENBANK sequences. We amplified K+ channel transcripts using PCR primers for delayed rectifier K+ channels. The primers flanked the N-terminal cytoplasmic region, and a downstream region localized between the S2 and S3 transmembrane domains. When brain mRNA and ear total RNA were used as templates, the RT-PCR products were of the predicted K+ channel size. The DNA was isolated, cloned, and sequenced, and results of a BLAST search indicate that both cloned products have high homology with GENBANK sequences for delayed rectifier K+ channels (Shab; Kv2.1), suggesting that this channel is present in adult Xenopus ear and brain. Northern blot analyses are being conducted to determine the size of actin and K+ channel transcripts. A portion of this research will be presented at the December 1996 Society for Cell Biology Meeting. C. E. W. is the recipient of a Research Fellowship Award from the NASA-New Mexico Space Grant Consortium. |