Abstract
Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.
Highlights
From the $hfuscle Biology Group, Departments of Animal Science and Biochemistry and Biophysics and the mepartment of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
The location and orientation of set of elastic filaments that connect thick filaments to titin in the sarcomere have been mapped by immunoelectron the Z-line in vertebrate striated muscle myofibrils
Located in the myofibrillar A-band aqjacent to the AII maintaining the positional stability of thick filaments during junction
Summary
The hgtll library was screened as described[38] with polyclonal antibodies to titin, and asingle positive clone(E-2)was isolated and plaque-purified Both the 0.2-kb EcoRIfragment inserted in thisclone and a largerKpnUSstI fragment containing regions of the cloning vector on each side of the cDNA insert were subcloned into pUC118, forming plasmid clones E-2a and E-2b, respectively. Expression and Analysis of Fusion Protein-Plasmid clone TZ-3 was cloneE-2b, a 15-base synthetic nucleotidecomplementary t o the sedigested with PstI, and the resulting 1.3-kb fragment of the cDNA quence of the lacZ gene immediately adjacent to the EcoRI site was insert, designated TP3, was subcloned into PUR vectors (pUR290, synthesized at the Iowa State University Nucleic Acid Center and used pUR291, and pUR292)(46)propagated in E. coli strain TG1. Sis, small-scale liquid cultures of positive clones wereincubated with 2 mM isopropyl-1-thio-P-D-galactopyranosidfeor 3 h, and the insoluble
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