Hum Genet (1991) 88:101-104 [ NSNIN 9 Spfinger-Veflag1991 The human cardiac troponin I locus: assignment to chromosome 19p13.2-19q13.2 Catriona MacGeoch 1, Paul J. R. Barton 2, William J. Vallins 2, Pankaj Bhavsar 2, and Nigel K. Spurr 1 'Human Genetic Resources, Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts. EN6 3LD, UK 2Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW7 6LY, UK Received March 3, 1991 Summary. The three major troponin I isoforms are en- coded by separate genes and are expressed in a muscle- type-specific manner. A human cardiac troponin I cDNA has recently been isolated and used to establish the genomic location of the cardiac troponin I gene locus (designated TNNC1). By somatic cell hybrid analysis, the locus for TNNC1 maps to human chromosome 19 and can be localised to the region p13.2-q13.2 The isolation and cloning of full length human cDNAs for both cardiac TnIc (Vallins et al. 1990) and slow skeletal muscle Tnls (Wade et al. 1990) have re- cently been described, and the TnIs gene (designated TNNS1) has been localised to the ql2-qter region of chromosome 1 (Wade et al. 1990). Here, we report the assignment of the human cardiac troponin I gene locus (designated TNNC1) by somatic cell hybrid analysis. Introduction The effect of Ca 2++ on the interaction of actin and myo- sin in striated muscle is mediated by the troponin com- plex. This is made up of three protein components, troponin C (TnC), troponin I (TnI) and troponin T (TnT); for each of these components, there exist mul- tiple isoforms that are expressed in both a muscle-type- specific and a developmentally regulated manner. Three isoforms of troponin I have been identified and are pre- sent as the major isoforms in cardiac muscle (Tnlc), slow skeletal (TnIs) and fast skeletal (Tnlf) muscle. The amino acid sequence of these isoforms (Leszyk et al. 1988; Koppe et al. 1989; Baldwin et al. 1985; Wilkinson and Grand 1978) indicates that they are encoded by separate genes, several of which have now been cloned from rat, mouse and chick. During the development of the rat heart (Saggin et al. 1989), there is a transition from the expression of TnIs in fetal life, to that of TnIc, which occurs at the time of birth. Cardiac troponin I differs from the skeletal muscle isoforms in that it has an extended N-terminal amino- acid sequence; this extension contains serine residues, which have been shown to be phosphorylated by the action of adrenergic agonists on the heart. Phosphoryla- tion of TnIc influences the Ca+ + binding characteristics of the troponin complex, and a transition from expres- sion of Tnls to TnIc during cardiac development would therefore be expected to influence the ability of the heart to respond to adrenergic stimulation. We are inter- ested in the developmental expression of TnI isoforms in the heart, and in elucidating the mechanisms by which this is regulated. Offprint requests to: N. K. Spurr Materials and methods Cells and DNA preparation Cell lines were cultured at 37~ in a 5% CO2 atmosphere in RPMI supplemented with 10% fetal calf serum, and 1% glutamine. When appropriate, the media were supplemented with HAT (hypoxanthine and thymidine 1%, methotrexate 1%). All hybrids were eharactefised by a combination of karyotypic analysis and by mapped enzyme and DNA markers (Bobrow and Cross 1974; Harris and Hopkinson 1976). The hybrids used are given in Table 1. DNA was prepared on an Applied Biosystems 340A DNA extrac- tor according to the manufacturers recommendations. cDNA probe for the detection of the human cardiac Tnlc gene For detection of the human cardiac troponin I gene, we used a fragment of the previously eharaeterised Tnle eDNA (Vallins et al. 1990). Clone pCTI-3 contains 470 bp of a eDNA sequence from amino acid 77 of the troponin-coding region to the end of the 3' non-coding sequence. Purified insert DNA was prepared from clone pCTI-3 and labelled by random priming. D NA hybridisations Digested DNA was eleetrophoretically separated on 0.8% agarose gels and transferred to Hybond N (Amersham) membranes by transfer in 20 x SSC (Sambrook et al. 1989). Probes were isolated as inserts from low melting point agarose and radio-labelled using a random priming Prime-it kit (Stratagene). Filters were pre- hybridised for 4-6 h in 5 x SSC, 40 mM NaPO4, pH 6.5, 5 • Den- hardt's solution and 0.1 mg/ml denatured salmon sperm DNA at 65"C. DNA probes were then added to the hybridisation buffer plus 10% dextran sulfate. Hybridisations were conducted at 65"C for a minimum of 16 h. After hybridisation, the filters were washed in 2 x SSC, 0.1% SDS at 65~ for 10-15 rain. Results Southern blot hybridisation with clone pCTI-3 resulted in the detection of a single band in human genomic DNA