Abstract
Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.
Highlights
Tropomyosins are rod-like proteins that are associated with actin filament in muscle and nonmuscle cells
In order to detect additional isoforms that might be expressed from the Tm5 gene, we used systematic RT-PCR1 assays to evaluate the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) of the Tm5 gene in the developing and adult rat brain
The 9a ϩ 9d mRNAs produce the same 9a-encoded COOH-terminal amino acid residue proteins as the 9a ϩ 9b mRNAs, and the differences between these variants lie in their 3Ј-UTR regions
Summary
Embryonic and adult rat tissues were collected from Sprague-Dawley rats, immediately frozen in liquid nitrogen, and stored at Ϫ70 °C. Frozen tissues were homogenized in phenol/guanidine isothiocyanate, and RNA was extracted as described [27]
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