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JOURNAL OF EXPERIMENTAL ZOOLOGY 286:149–156 (2000) Heterogeneity of Chicken Slow Skeletal Muscle Troponin T mRNA I. YONEMURA, T. HIRABAYASHI, AND J.-I. MIYAZAKI* Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan ABSTRACT The troponin T (TnT) transcripts in chicken slow skeletal muscle were characterized by S1 nuclease mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5′RACE. We found two kinds of transcripts in the 5′-region, one having the codon for alanine (position 135–137), C (258), and A (262) and the other lacking the codon and having T (258) and G (262) instead of C and A. In the 3′-region, we found four single base substitutions at 703 (T or C), 774 (C or T), 797 (C or T), and 827 (G or A). Four of the six substitutions lead to amino acid changes in chicken sTnT isoforms. We determined the genomic structure of the 3′-region of the chicken sTnT gene. The region includes 7 exons corresponding to position 249–891 of the chicken sTnT cDNA and no alternative exon, showing that the 3′-heterogeneity in sTnT transcripts was due to allelic variation. J. Exp. Zool. 286:149–156, 2000. © 2000 Wiley-Liss, Inc. Skeletal muscle cells are characterized by precise organization of the contractile proteins into the repeating units of overlapping thick and thin filaments, i.e., sarcomeres, that organize in turn myofibrils. The thick filaments are composed primarily of myosin, and major components of the thin filaments are actin, tropomyosin and troponin. These contractile proteins have multiple isoforms that are produced by multigene families and by respective genes through alternative splicing (for review, see Andreadis et al., ’87) and/or use of alternative promoters (Robert et al., ’84; Concordet et al., ’93). Functional significance of these multiple isoforms is not fully clarified so far in spite of much effort devoted for it. Troponin, the key protein of Ca2+-sensitive molecular switching for contraction in vertebrate striated muscle, consists of three subunits, troponin T (TnT), troponin I, and troponin C (for reviews, see Zot and Potter, ’87; Schiaffino and Reggiani, ’96). TnT isoforms are encoded by three genes characteristic of slow skeletal muscle (sTnT), fast skeletal muscle (fTnT), and cardiac muscle (cTnT) (Breitbart et al., ’85; Cooper and Ordahl, ’85; Gahlmann et al., ’87; Smillie et al., ’88; Mesnard et al., ’93). Each gene can generate a variety of transcripts by alternative splicing (Breitbart et al., ’85; Cooper and Ordahl, ’85; Gahlmann et al., ’87; Schachat et al., ’95). Molecular organization of the rat fTnT gene has revealed its capacity to produce 128 different fTnT mRNAs by differential alternative splicing (Med© 2000 WILEY-LISS, INC. ford et al., ’84; Breitbart et al., ’85; Breitbart and Nadal-Ginard, ’87; Morgan et al., ’93). Smiillie et al. (’88) have found four variants of chicken fTnT cDNA, and Schachat et al. (’95) 16 variants of chicken fTnT 5′-cDNA. The chicken cTnT gene generates one embryonic and one adult transcripts, derived from inclusion and exclusion of exon 5, respectively (Cooper and Ordahl, ’85). A more complex alternative splicing pattern involving exons 4 and 12 has been found in the rat cTnT gene (Jin and Lin, ’89; Jin et al., ’92). On the other hand, very poor information is available on the mode of alternative splicing of the sTnT gene. The human sTnT gene can generate three or possibly four transcripts, which differ in the presence or absence of two short inserts (33 and 48 nucleotides) in the 5′- and 3′-end regions (Gahlmann et al., ’87; Samson et al., ’94). Chicken slow skeletal muscle, such as anterior latissimus dorsi (ALD), has been shown so far to contain two kinds of sTnT transcripts differing in inclusion or exclusion of one codon encoding an alanine residue in the 5′-end region (Yonemura et al., ’96). To elucidate the alternative splicing Abbreviations used: ALD, anterior latissimus dorsi; bp, base pair(s); cTnT, cardiac TnT; fTnT, fast muscle TnT; nt, nucleotide(s); PCR, polymerase chain reaction; 5′-RACE, 5′-rapid amplification of cDNA ends; RT-PCR, reverse transcription-PCR; sTnT, slow muscle TnT; TnT, troponin T. *Correspondence to: Jun-Ichi Miyazaki, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. Email: firstname.lastname@example.org Received 22 October 1998; Accepted 11 May 1999 150 I. YONEMURA ET AL. pattern of the sTnT transcripts and to deduce the gene structure it is very important to investigate heterogeneity of sTnT transcripts. In the present work, we perform S1 nuclease mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5′-RACE to search for heterogeneity of chicken sTnT mRNA. We show that 5′ and 3′ heterogeneities in sTnT transcripts are caused by single base substitutions at six positions and by inclusion or exclusion of the codon coding for alanine. We also show the partial genomic structure of the chicken sTnT gene. MATERIALS AND METHODS Materials Adult and one-day-old (H1) white leghorn chickens (Gallus domesticus [L]) were obtained from commercial sources. Slow muscle, anterior latissimus dorsi (ALD), mixed muscle, complexus, and fast muscle, pectoralis major, were dissected out and used for RNA preparation. RNA preparation and reverse transcriptionpolymerase chain reaction (RT-PCR) Total RNA was prepared by using Isogen (Nippon gene) following its protocol. Reverse transcription was performed with 10 µg of total RNA, 0.5 µg of oligo(dT)12–18 and Superscript II (Gibco BRL) at 42°C for 60 min. For PCR of the 5′-region of chicken sTnT, the forward primer, 5′-GGCTCGAGGAGCCAACAGGACCG-3′ (position 1 to 15 of the cDNA + Xho I site), was synthesized based on the chicken sTnT cDNA sequence. The 3′-mixed primer (position 522 to 541) was used as the reverse primer (Yonemura et al., ’96). PCR was performed by 30 cycles of 1 min denaturation at 95°C, 1 min annealing at 60°C and 1 min extension with Taq DNA polymerase (Gibco BRL) at 72°C, followed by final 10 min extension at 72°C. For PCR of the 3′-region of chicken sTnT, the reverse primer, 5′-GGGAATTCGACGAGCAGAGCTTTATTGG-3′ (Primer R, position 871 to 891 of the cDNA + EcoR I site), was also synthesized based on the chicken sTnT cDNA sequence. The 5′-mixed primer (position 248 to 269) was used as the forward primer (Yonemura et al., ’96). PCR was performed by 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 65°C and 1 min extension with Taq DNA polymerase at 72°C, followed by final 10 min extension at 72°C. The 5′-rapid amplification of cDNA ends (5′-RACE) was performed as in Frohman et al. (’88) and Yonemura et al. (’96). Products were cloned in the pBluescript II KS+ phagemid (Stratagene) and sequenced. Antisense DNA probes and S1 nuclease mapping Two clones, stnt-5′R1 and sTnT-a3, were obtained by cloning chicken sTnT cDNA (Yonemura et al., ’96). The former includes 1 to 541, and the latter 78 to 898 of sTnT cDNA. The stnt-5′R1 clone was digested with Xho I and Pst I, and the resulting fragment was subcloned into the Xho IPst I double-digested sTnT-a3 to obtain the full-length cDNA, which did not have three bases (135–137) encoding alanine. This full-length cDNA was cut with Xho I and BamH I and a 690 nt fragment, probe A, was obtained. The probe A was labeled with [32P] ATP using T4 polynucleotide kinase (Nippon gene). Another probe, probe B, was generated by PCR with sTnT-a3 as a template using the 5′-mixed primer as the forward primer (Yonemura et al., ’96) and Primer R as the reverse primer. PCR was performed by 30 cycles of 30 sec denaturation at 95°C, 10 sec annealing at 60°C and 30 sec extension with KOD Dash (TOYOBO) at 74°C, followed by final 10 min extension at 72°C. The probe B was labeled with [32P] dATP using KOD Dash (TOYOBO). Total RNA (5 or 10 µg) was precipitated with 105 cpm of each probe. The pellet was dissolved in 20 µl of the hybridization buffer. Hybridization was performed overnight at 55°C before digestion with S1 nuclease (Gibco BRL). Protected fragments were separated on 6% acrylamide/8 M urea gels, which were exposed to X-ray film (Kodak) with intensifying screen. Cloning of the chicken sTnT gene For PCR of the chicken sTnT gene, the forward primer, 5′-GCAGCCATGTCCGAAGCTGAG-3′ (position 39–62 of the cDNA sequence), was synthesized based on the chicken sTnT cDNA sequence. Primer R was used as the reverse primer. PCR was performed by 30 cycles of 30 sec denaturation at 96°C and 10 min annealing and extension at 74°C with 130 ng of chicken liver genomic DNA using KOD Dash (TOYOBO). Amplified DNA was cloned in the pBluescript II KS+ phagemid (Stratagene) and sequenced. RESULTS Heterogeneity in the 5¢-region of chicken sTnT mRNA We described previously two truncated cDNA clones, stnt-5′R1 and sTnT-a3 (Yonemura et al., ’96). Using a 690 nt antisense probe (probe A) derived from the full-length cDNA, we performed CHICKEN SLOW TROPONIN T mRNA S1 nuclease mapping of total RNA from adult and one-day-old chick (H1) ALD to characterize the 5′-region of chicken sTnT mRNA (Fig. 1). The 690 nt fragment free from S1 nuclease digestion showed that the transcript completely matching with the probe was expressed in both adult and H1 ALD. In addition, two other fragments of 558 nt and about 440 nt were also generated, suggesting that adult and H1 chicken ALD had possibly three kinds of transcripts which differed in at least two positions in the 5′-region. The consistent patterns of S1 nuclease mapping were obtained with several individuals (data not shown). As expected, no protected band was found in adult fast muscle, pectoralis major. The result is consistent with our previous data by Northern blotting (Yonemura et al., ’96). To collect precise information on the sequences which caused the variation in the 5′-region, we performed nucleotide sequencing of RT-PCR and 5′-RACE products from total RNA of adult chicken ALD and neck muscle, complexus, which is known to express sTnT isoforms (Yao et al., ’92). We obtained two RT-PCR products from ALD, A1-1R and Aall-1R, and one 5′-RACE product from complexus, 20-2R (Fig. 2). The 20-2R had GCA coding for alanine at 135 to 137 of chicken sTnT cDNA just as stnt-5′R20b, but A1-1R, Aall-1R and stnt-5′R1 did not (Fig. 2). Furthermore, two single base substitutions were found at 258 and 262. The clones, stnt-5′R20b and 20-2R, had C and A at those positions, respectively, while the remaining clones, stnt-5′R1, A1-1R and Aall-1R, had T and G instead of C and A. Those differences lead to amino acid substitutions of arginine (258) and lysine (262) in the former to tryptophan and arginine in the latter, respectively. Such nucleotide substitutions are also seen in the 5′-region of human sTnT cDNA. Two human sTnT cDNA clones, M1 and MSL-2-27, have G and one clone, H22h, has C at the position 117, resulting in the amino acid changes from glutamic acid in the M1 and MSL-2-27 to aspartic acid in H22h (Gahlmann et al., ’87; Samson et al., ’94). However, we could not find the sequence corresponding to the 33 nt insert reported in the 5′-region of human sTnT cDNA (Gahlmann et al., ’87). Therefore, the determination of sequences showed that the two kinds of transcripts, one having the codon for alanine and C (258) and A (262) and the other lacking the codon and having T (258) and G (262), were expressed in chicken slow skeletal muscle. The results are consistent with the above data from S1 nuclease mapping, showing 151 Fig. 1. S1 nuclease mapping of sTnT mRNA with an antisense probe from the 5′-region of sTnT cDNA. The upper panel shows schematically the structure of sTnT mRNA and the position of the 690 nt antisense probe (probe A) derived from the cDNA clones, stnt-5′R1 and sTnT-a3, with the 32P-labeled 3′-end (closed circle). Total RNA from adult (Ad) chicken ALD, 1-day-old chick (H1) ALD or adult pectoralis major was hybridized to the probe A and digested with S1 nuclease. Protected fragments were separated on the 6% acrylamide/8 M urea gel. No band was detected in fast skeletal muscle, pectoralis major (PM), but three bands of 690 nt, 558 nt and about 440 nt were found in slow skeletal muscle, ALD (A). 152 I. YONEMURA ET AL. Fig. 2. Nucleotide and deduced amino acid sequences of the 5′-region of chicken sTnT cDNA. The cDNA was generated by RT-PCR and 5′-RACE with total RNA from chicken adult ALD and complexus. Two RT-PCR products from ALD, A1-1R and Aall-1R, and one 5′-RACE product from complexus, 20-2R, were sequenced. The remaining stnt-5′R1 and stnt-5′R20b were described previously (Yonemura et al., ’96). The bracket and dashes (-) indicate the absence of GCA coding for alanine and gaps, respectively. The amino acid sequences are indicated in the single-letter code above the nucleotide sequences. Two substitutions at 258 and 262 are marked with asterisks (*) with the corresponding amino acid residues. Only single sequence is shown in 45 to 104, 165 to 224, and 285 to 344 (designated as com.), because the sequence was identical among the clones. that the ca. 440 nt fragment was generated by digestion at 258 and/or 262 and the other fragment of 558 nt by digestion at 135–137. Therefore, incomplete digestion at 258 and 262 might have occurred to produce the 558 nt fragment, because S1 nuclease recognizes normally more than duplex substitutions. Those results showed the heterogeneity in the 5′-region of chicken sTnT mRNA. completely with the probe B (Fig. 3). In addition, minor and smear bands of faster mobilities were found. Similar pattern was detected in gastrocnemius of the 12-day-old embryo (data not shown). In order to characterize those minor protected fragments, we sequenced RT-PCR products of total RNA from adult ALD and complexus (Fig. 4). We found four single base substitutions in the 3′region of sTnT cDNA at 703 (T or C), 774 (C or T), 797 (C or T), and 827 (G or A). The substitutions at 703 and 774 cause the amino acid changes from leucine in the five clones (b20-10, bA3, bA1, b20-4, and b20-3) to proline in the other two clones and from arginine in the two clones (b20-1- and b20-3) to cysteine in the five remaining clones, respectively. Two single base substitutions at 797 and 827 do not cause amino acid changes. The results showed that chicken slow skeletal muscle had at least four kinds of transcripts in the 3′- Heterogeneity in the 3¢-region of chicken sTnT mRNA To search for heterogeneity in the 3′-region of chicken sTnT mRNA, we performed S1 nuclease mapping of total RNA from adult ALD with a 658 nt PCR-produced antisense probe (probe B) which included the 6 nt restriction enzyme site (Xho I or EcoR I) and 2 nt optional sequence at each end. The major protected fragment was 642 nt long, showing that the majority of transcripts matched CHICKEN SLOW TROPONIN T mRNA Fig. 3. S1 nuclease mapping of sTnT mRNA with an antisense probe from the 3′-region of sTnT cDNA. The upper panel shows schematically the structure of sTnT mRNA and the position of the 658 nt antisense probe (probe B) generated by PCR. The probe included the 6 nt restriction enzyme site and 2 nt optional sequence at each end and was 32Plabeled internally. Total RNA from adult chicken ALD (A) was hybridized to the probe B and digested with S1 nuclease, and protected fragments were separated on the 6% acrylamide/8 M urea gel. The major (642 nt) and minor protected fragments (bracket) were found. region. In the 3′-region of human sTnT cDNA, single base substitutions are also seen at positions 648 (G or C) and 649 (C or G) (Novelli et al., ’92). These substitutions cause the amino acid changes from lysine to asparagine (648) and from leucine to valine (649), respectively. However, we could not find the sequence corresponding to the 48 nt insert reported in the 3′-region of human sTnT cDNA (Gahlmann et al., ’87). Structure of the 3¢-region of the chicken sTnT gene To investigate how the heterogeneity in the chicken sTnT transcripts is generated, we deter- 153 mined the partial sequence of the chicken sTnT gene. The size of the chicken sTnT gene was only about 3.5 kb (data not shown), but we encountered serious difficulties in sequencing its 5′-region possibly due to extensive G-C tracts in introns. The 3′-region of the gene was composed of seven exons with the last exon including the stop codon and polyadenylation signal (Fig. 5A). We tentatively designated the exons as exon A to G. There was no alternative exon. Each one of the variable nucleotides at the four positions in the transcripts was found in exon E (T at 703), exon F (C at 774) and exon G (C and G at 797 and 827), suggesting that the 3′-heterogeneity in the transcripts might be derived from allelic variation. We confirmed the absence of the sequence corresponding to the 48 nt insert reported in the 3′-region of human sTnT cDNA (Gahlmann et al., ’87), showing one major difference in the genomic structure between chicken and human sTnT genes. On comparison of the chicken sTnT gene with the quail fTnT gene (Bucher et al., ’89), exons A, B, C, D, E, F, and G in the former had sequence homology to exons 11, 12, 13, 14, 15, 17 and 18 in the latter, respectively (Fig. 5B). However, the length from exons A to G (1547 bp) was considerably shorter than that from exons 11 to 17 (about 7.5 kb) due to highly small introns (84 to 329 bp) in the chicken sTnT gene. DISCUSSION We found the two kinds of transcripts in the 5′-region, one having the codon for alanine (135– 137) and C (258) and A (262) and the other lacking the codon and having T and G instead of C and A. In the 3′-region, we found four single base substitutions at 703 (T or C), 774 (C or T), 797 (C or T), and 827 (G or A). Four out of six substitutions in the 5′- and 3′-regions lead to amino acid changes in chicken sTnT isoforms. Such nucleotide substitutions are also seen in human sTnT (Gahlmann et al., ’87; Samson et al., ’90; Novelli et al., ’92; Samson et al., ’94). However, no single substitution has been reported in fTnT and cTnT cDNAs so far (Garfinkel et al., ’82; Medford et al., ’84; Breitbart et al., ’85; Cooper and Ordahl, ’85; Smillie et al., ’88; Bucher et al., ’89; Jin and Lin, ’89; Jin et al., ’92; Morgan et al., ’93; Wu et al., ’94; Briggs et al., ’94; Schachat et al., ’95; Wang and Jin, ’97) with two exceptions in human fetal and pathological cTnT cDNA (Mesnard et al., ’93, ’95; Townsend et al., ’95). How is the heterogeneity in chicken sTnT then 154 I. YONEMURA ET AL. Fig. 4. Nucleotide and deduced amino acid sequences of the 3′-region of chicken sTnT cDNA. The cDNA was generated by RT-PCR with total RNA from adult chicken ALD and complexus. Two RT-PCR products from ALD, bA1 and bA3, and five RT-PCR products from complexus, b20-1, b20-3, b204, b20-9, and b20-10, were sequenced. Four single base substitutions (marked with asterisks, *) are seen at 703 (T or C), 774 (C or T), 797 (C or T), and 827 (G or A). The substitutions at 703 and 774 cause amino acid changes from leucine to proline and from arginine to cysteine, respectively, but those at 797 and 827 do not. The amino acid sequences are indicated above the nucleotide sequences and amino acid residues are also shown below the sequences at the positions where amino acid changes are found. generated? In the 5′-region, we can consider three possible mechanisms. First, two exons covering 135 to 262 may exist, one having the codon for alanine (135–137) and C (258) and A (262) and the other lacking the codon and having T and G. Two kinds of sTnT transcripts can be generated by mutually exclusive alternative splicing of the two exons. Secondly, when the variable nucleotides in the 5′-region are included in more than two exons, inclusion of one exon may be tightly linked to that of the other(s) resulting in production of only two transcripts. Therefore, alternative exons of each set can be synchronously spliced. Thirdly, we can not rule out completely that two kinds of transcripts may be derived from different alleles of this gene. From the cDNA sequences and sTnT gene structure, we showed that four single base substitu- tions in the 3′-region might be derived from allelic variation. Since human sTnT cDNAs have also several single base substitutions and deletions in the 3′-region (Samson et al., ’90; Novelli et al., ’92), it seems likely that the 3′-region of the sTnT gene could accumulate mutations, possibly suggesting the moderate structural constraints on the COOH region of the sTnT protein. Our data also showed the structural difference between chicken and human sTnT genes, because the chicken sTnT gene did not have the sequence corresponding to the 48 nt insert reported in the 3′-region of human sTnT cDNA (Gahlmann et al., ’87). The structural differences between avian and mammalian genes have also been reported in fTnT (Miyazaki et al., unpublished data; Breitbart and Nadal-Ginard, ’86) and cTnT (Cooper and Ordahl, ’85; Jin et al., ’92). CHICKEN SLOW TROPONIN T mRNA 155 Fig. 5. Structure of the 3′-region of the chicken sTnT gene. (A) The sequence of the 3′-region of the chicken sTnT gene (1547 bp) was determined. It included seven exons, tentatively designated as exons A to G. Capital and lower-case letters indicate exon and intron sequences, respectively. The amino acid sequences are indicated below the nucleotide sequences. Outlined letters in exons E to G indicate the positions of four single base substitutions. The asterisk (*) indicates the stop codon. The bold underline indicates the putative polyadenylation signal. Italic letters within thin underlines indicate the 5′ and 3′ splice sites. (B) Comparison of the 3′-regions between chicken sTnT and quail fTnT genes (Bucher et al., ‘89). 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