| Abstract #62, Date 2/14/99, Session E1, Poster (J30) |
| Identification of actin gene family members in the Xenopus laevis inner ear with 3'RACE |
| *J.M. Gladden, E.E. Serrano (New Mexico State University, Las Cruces, NM ) |
| We are studying the expression patterns of actin and ion channel genes in the endorgans of the Xenopus laevis inner ear. Many transcript detection techniques rely on the hybridization of specific RNA probes with homologous mRNA expressed within a cell. It is known that the coding sequence of members within gene families, such as actin or potassium channels, share homology with one another, therefore probes against conserved regions may hybridize to many members of the gene family. Three prime untranslated regions (3’UTR’s) among gene families tend to be less homologous between gene family members than coding sequences. Probes made from 3’UTR sequences will be more selective for a specific gene than probes containing coding. Here we present preliminary results of experiments using 3’ rapid amplification of cDNA ends (3’ RACE) to amplify 3’UTRs of alpha and beta actin sequences from Xenopus laevis inner ears (EAR), isolated sacculae (SAC) and inner ears from which the sacculae have been removed (EAR-SAC). An alpha actin primer was designed to amplify 577bp of the coding region of the XLACTA2 alpha actin gene and its 570bp 3’UTR. The primer also has homology with other actin sequences. A beta actin primer was designed to amplify 25bp of the coding region and the 780bp 3’UTR of XELACTIN5A. In one set of experiments, template mRNA from EAR was used in 3’RACE reactions. At an annealing temperature of 52 oC, two reaction products were detected after the second strand PCR reaction with alpha actin primer. Cloning and sequencing of these products confirmed their identity as two members of the Xenopus alpha actin gene family: XLACASR and XLACTA2. In a second series of experiments, the same amount of mRNA from EAR, SAC, and EAR-SAC was used as template in 3’ RACE. Alpha or beta actin primer was used in the second strand PCR reaction at an annealing temperature of 58oC. Two products were amplified by the alpha actin primer, one corresponding to the expected size for XLACTA2 and another slightly larger product that could be one of several actins. Two products were amplified by the beta actin primer, one of the expected size for XELACTIN5A. In all reactions with the different templates, this product had the most intense band, suggesting that a transcipt present in high abundance had been amplified. The second product was comparable in size to the larger product amplified by the alpha actin primer. Similarly sized products were amplified with both primers from the three different templates. These products are being cloned and sequenced to confirm their identity. Taken together, results suggest that multiple forms of alpha and beta actin are expressed in X. laevis inner ears. |