Close Physical Linkage of Human Troponin Genes: Organization, Sequence, and Expression of the Locus Encoding Cardiac Troponin I and Slow Skeletal Troponin T Paul J. R. Barton, 1 Martin E. Cullen, Philip J. Townsend, 2 Nigel J. Brand, Antony J. Mullen, David A. M. Norman, Pankaj K. Bhavsar, and Magdi H. Yacoub Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom Received September 4, 1998; accepted November 26, 1998 Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I–troponin T gene pair. Here we have inves- tigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and dem- onstrate close physical linkage in two cases through the isolation of individual clones containing a com- plete troponin I–troponin T gene pair. As an initial step toward fully characterizing this pattern of link- age, we have determined the organization and com- plete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also pro- vide the first determination of the structure and se- quence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skel- etal troponin T genes show independent tissue-spe- cific expression. Such close physical linkage has impli- cations for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy. © 1999 Academic Press INTRODUCTION The troponin complex forms the calcium-sensitive molecular switch that regulates striated muscle con- traction in response to alterations in intracellular cal- cium concentration (Tobacman, 1996; Farah and Rein- ach, 1995). It is located on the thin filament of the sarcomere and is composed of three subunits: troponin C, the calcium binding subunit; troponin T, which is involved in the attachment of the complex to tropomy- osin; and troponin I, the inhibitory subunit. Multiple isoforms of each of these subunits have been identified and are expressed with distinct patterns of tissue spec- ificity and developmental regulation (Parmacek and Leiden, 1991; Schiaffino et al., 1993). In total, eight troponin genes have been identified in the human ge- nome as summarized in Table 1. Two of these, cardiac troponin T and cardiac troponin I, have been directly implicated in familial hypertrophic cardiomyopathy (Thierfelder et al., 1994; Kimura et al., 1997), an auto- somal dominant disease associated with myofibrillar disarray within the myocardium. Based on mapping studies, we have previously re- ported that the genes for the troponin I and troponin T subunits of the complex are organized as paralogous pairs at three dispersed chromosomal sites, each con- taining a troponin I–troponin T gene pair (Barton et al., 1997). In contrast, the two genes encoding fast skeletal and cardiac/slow-skeletal troponin C are not linked to each other or to other troponin genes (Townsend et al., 1997a,b). While all sarcomeric pro- teins, including isoforms of actin, myosin, myosin light chain, and tropomyosin, are encoded by multigene fam- ilies, only the myosin heavy chain genes have been previously identified as being linked. These are grouped at two loci, one containing the - and -cardiac genes (MYH6 and MYH7) (Saez et al., 1987) and the other containing the skeletal muscle genes (MYH1- MYH5), (Soussi-Yanicostas et al., 1993; Yoon et al., 1992), and probably originated through a process of tandem gene duplication. The functional significance of myosin gene clustering remains unclear. For example, the organization of the skeletal muscle cluster does not correlate directly with their developmental order of expression (Yoon et al., 1992) as is seen, for example, with globin genes (Crossley and Orkin, 1993). For the troponin I and T genes, the situation is different from that of myosin as pairing occurs between genes that Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AJ011712 and AJ011713. 1 To whom correspondence should be addressed. Telephone: (+44) 171 351 8184. Fax: (+44) 171 376 3442. E-mail: p.barton@ic.ac.uk. 2 Current address, Molecular Cardiology, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, UK. Genomics 57, 102–109 (1999) Article ID geno.1998.5702, available online at http://www.idealibrary.com on 102 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.