Sequences of complete cDNAs encoding four variants of chicken skeletal muscle troponin T.

Conformational Modulation of Troponin T by Configuration of the NH2-Terminal Variable Region and Functional Effects

“…The authenticity of the cloned TnT isoforms was verified by their mobility in SDS-PAGE (Figure 3A) and Western blotting using anti-TnT antibodies (Figure 5C). The results showed that the cloned chicken fast skeletal muscle TnT1-4 isoforms have size differences corresponding to their molecular weight deduced from sequence data (9) and are all recognized by the specific anti-TnT antibody RATnT. Amino acid analysis was used to determine the protein concentration used in the functional assays, and the demonstrated residue ratio agreed with the sequence-deduced amino acid composition (data not shown).…”

“…The primary structure and isoform expression of TnT have been extensively studied in many avian and mammalian species. As the most complex example, the generation of fast skeletal muscle TnT isoforms involves differential splicing of more than six alternative exons (i.e., four to eight, fetal and additional exons found in birds) corresponding to a highly variable NH 2 -terminal region and a pair of mutually exclusive exons (16 and 17) encoding a COOH-terminal variable region (5,(9)(10)(11)(12).…”

Expression, Zinc-Affinity Purification, and Characterization of a Novel Metal-Binding Cluster in Troponin T: Metal-Stabilized α-Helical Structure and Effects of the NH2-Terminal Variable Region on the Conformation of Intact Troponin T and Its Association with Tropomyosin

“…To prepare the nonfusion TnT NH 2 -terminal fragment proteins used in this study, two recombinant plasmids (pTx47 and pTx165) were constructed using the T7 RNA polymerase-based (Studier et al, 1990) pAED4 expression vector for protein expression in E. coli. Using synthetic oligonucleotide primers and a recombinant pAED4 expression plasmid encoding the intact chicken breast TnT1 isoform (Smillie et al, 1988;Jin et al, 1991) as the template, two cDNA fragments encoding NH 2 -terminal 47 (N47) or 165 (N165) amino acids were amplified by polymerase chain reaction (PCR). The N47 DNA fragment was doubledigested with XbaI and EcoRI, and ligated to a similarly digested pAED4 vector.…”

“…The cardiac and skeletal muscle TnTs are encoded by different genes and muscle type specific or developmentally regulated isoforms are expressed from each gene through alternative mRNA splicing (Cooper & Ordahl, 1985;Breitbart & Nadal-Ginard, 1986;Jin et al, 1992). In fast skeletal muscle TnT, alternative mRNA splicing involving a pair of mutually exclusive COOHterminal exons (16/17) and seven (Breitbart & Nadal-Ginard, 1986;Smillie et al, 1988;Briggs & Schachat, 1993) or possibly more (Schachat et al, 1995) exons encoding a variable NH 2 -terminal region produces a large number of protein isoforms. Although the functional significance of TnT isoform diversity is not fully understood, certain pathological conditions have demonstrated a concurrent change in TnT isoform expression (Gulati et al, 1994;Akella et al, 1995;Anderson et al, 1995).…”

“…Interestingly, this NH 2 -terminal region may contain structures independent of TnT's function in the regulation of contraction. Protein (Wilkinson et al, 1984) and cDNA (Smillie et al, 1988) sequencing of the chicken breast muscle fast TnT have revealed an unusual 20 amino acid stretch consisting of four regularly repeated histidine pairs [HE/AEAH] 4 found within the NH 2 -terminal variable region. Recently, this segment (designated Tx) has been shown to be exclusive to TnT isoforms expressed only in the avian orders of Galliformes and Craciformes (Jin & Smillie, 1994).…”