The Molecular Structures, Expression Patterns, and Promoter Analysis of Zebrafish Troponin I Genes
Date Issued
2007
Date
2007
Author(s)
Fu, Chuan-Yang
DOI
zh-TW
Abstract
Troponin I (TnnI), a constituent of the troponin complex on the thin filament, provides a calcium-sensitive switch for striated muscle contraction. Cardiac TnnI is a highly sensitive and specific marker of myocardial injury in the acute coronary syndromes. Although TnnI gene has been identified in birds and mammals as it encodes the isoforms expressed in cardiac muscle, fast skeletal muscle and slow skeletal muscle, there is extremely little known about the developmental regulation of TnnI gene in the lower vertebrates. Firstly, we characterized zebrafish TnnI genes, TnnI1 and TnnI2. In the unrooted radial gene tree for TnnI genes among vertebrates, the zebrafish TnnI1 and TnnI2 genes we cloned correspond to the slow muscle TnnI1 gene and the fast muscle TnnI2 gene of other vertebrates, respectively. Secondly, reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization showed that TnnI1 and TnnI2 transcripts were expressed in the somites at 16 hours post-fertilization (hpf). In addition, we also demonstrated that the expression of TnnI1 and TnnI2 genes was slow- and fast-muscle-specific, respectively, indicating that zebrafish TnnI1 and TnnI2 proteins were slow-TnnI and fast -TnnI, respectively. Interestingly, we also identified a novel TnnI isoform, TnnI-HC (heart and craniofacial), which was expressed in the heart exclusively during early cardiogenesis at 16 hpf, but it was not only in the craniofacial muscle but also in the heart after 48 hpf. Thirdly, in order to go further understanding the regulatory mechanism of TnnI-HC gene, we cloned a 7.6-kb fragment containing a partial TnnI-HC genomic gene from the zebrafish genomic library. Thereafter, we subcloned an upstream 1.4-Kb (from -1400 to +30; -1400/+30) and an intron 1 4-Kb (from +2257 to +6253; +2257/+6253) and we fused them with GFP separately. After microinjection, we found that the -1.4 Kb fragment did not contain any promoter activity. However microinjection of an intron 1 fragment (4-Kb) alone without including -1.4 Kb enabled the expression of GFP in the heart and trunk muscle, but not in the craniofacial muscle. Thus, we speculate that other upstream regulatory segment might play a role in the enhancing the craniofacial muscle expression and repressing the trunk expression of TnnI-HC gene. Deletion analysis in +2257/+6253 segment, we also identified a cis-element located at +2257/+4689 within intron 1 that might control the cardiac expression of zebrafish TnnI-HC gene during early development.
Subjects
斑馬魚
抑制性肌鈣蛋白
啟動子
Zebrafish
Troponin I
Promoter
Type
other
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