Hum Genet (1992) 88: 69%698
© Springer-Verlag1992
Assignment of the slow troponin T (TNNT1) gene to chromosome 19 using polymerase chain reaction G. Novelli 1'a, M. Gennareili 1'2, M. Rocchi 3, and B. Dallapiccola 1'2 • 1Dipartimento di Sanit~ Pubblica e Biologia Cellulare, Universit~ "Tor Vergata", Roma Universit~t di Urbino, Urbino, Italy 2C.S.S., Hospital, IRCCS, Sangiovanni, Rotondo, Italy 3Istituto di Genetica, Universit~ di Bari, Bari, Italy Received June 5, 1991 / Revised October 21, 1991
Summary. Confirmation that the slow troponin (TNNT1) gene lies on chromosome 19 has been obtained by means of the polymerase chain reaction and somatic cell hybrids.
porting further examination of this protein as a candidate gene for DM. We report here the chromosome location of the human TNNT1 gene using the pobwnerase chain reaction (PCR; Innis et al. 1990).
Introduction
Materials and methods
Troponin T (TNNT1), is a tropomyosin-binding component of the troponin complex (subunits I, C and T), and is effective during muscle contraction (Squire 1981). The genomic organization of this gene family is poorly understood. However, the structure of the isoform I gene has been determined (Baldwin et al. 1985), and a c D N A clone for the human slow skeletal muscle TNNT1 form has been reported by Gahlman et al. (1987). This c D N A has provided the first nucleotide sequence information about TNNT1 and has shown that a single slow TNNT1 gene is present in the human genome. Recently, a new c D N A clone corresponding to the slow TNNT1 gene has been isolated by substraction hybridization of a c D N A muscle library using a myotonic dystrophy (DM) muscle c D N A (Samson et al. 1990). This clone has been provisionally mapped onto chromosome 19 (Ropers and Pericak-Vance 1990), probably in the D M region, sup-
Two different oligomers designed on the basis of the originally published TNNT1 cDNA were used to amplify a genomic fragment of 1045 bp extending from the carboxyl portion of the TNNT1 coding sequence ot the immediately 3' untranslated region of the cDNA (Gahlman et al. 1987). The forward primer, TrozlA, was: 5'-GCTGTACAACCGCATCAG-3'; the reverse primer, Troz2A, was: 5'-GCATGGGACAAACACTCC-3'. This pair fails to demonstrate specificTNNT1 PCR products in hamster DNA. The specificity of our hybrid PCR product was evaluated by restriction analysis and direct sequencing (G.Novelli, M. Genarelli, F. Sangiuolo, L. d'Agruma, B. Dallapiccola, in preparation). This primer pair was used to amplify hybrid DNA from a panel of 19 humanhamster hybrid cell lines, and human and hamster control DNA.
* Present address and address for offprint requests: B. Dallapiccola, c/o Laboratorio Centrale C.R.I., Servizio di Genetica, Via Ramazzini 15, 1-00151 Roma, Italy
1 2
3
4 5
6
7
8
9
10 11 12 13 t4 15
Results and discussion The results obtained by PCR using the T r o z l A and Troz2A primers are shown in Fig. 1, and summarized in Table 1. Preliminary experiments have demonstrated the validity of the PCR strategy for synthesizing specific TNNT1 c D N A from m R N A in human skeletal muscle
16 17 18 19 21 22 23 24
1045 bp
Fig. 1. Analysis of PCR products for human TNNT1 in somatic cell hybrids on 2.0% agarose gel. Lane 2 CHO DNA; lane 3 human genomic DNA; lane 8 HY.22AZA1 hybrid; lane 19 YC2T1 hybrid; lane 21 Y.XY.8F6 hybrid; lanes 1, 16, and24pBR328 DNA digested with BglI and HinfI; lanes 4-7, 9-15, 17, 18, 20, 22, 23
amplified DNA from hybrids without chromosome 19
698 Table 1. Segregation of human TNNT1 in human-hamster hybrids. + / - , Scoring of the presence or the absence of human chromosome; (+), only part of chromosome present because of deletion Hybrid
Human chromosome TNNT11
HY.11AZA1 Y2C2T1 Y.XY.8F6 HY.19.16T3D HY.31.24E HY.60A HY.70b2 HY.75E1 HY.94A HY.95A1 HY.95B HY.95S HY.137J HY. t37K HY. 166T4 RJ.387.51T5 RJ.387.58 Y.173.5CT3 Y.XY.8FT7
+ + +
2
3
4
5
6
7
8
9
10
11
+ + +
+
+ + +
+
+
+ + + +
+ +
+ +
14
15
+ +
+ + +
18
19 20
+
+ +
+ + +
+ + +
+
+ +
+
+ +
+
+
+
+
+ +
+
+ +
+
+ +
+ + +
Acknowledgements. This work was supported by the Italian C.N.R. (P.F. Ingegneria Genetica no. 91.00037.PF99 and P. F. Biotecnologie e Biostrumentazione) and by the financial support of Telethon - Italy to the project "Molecular study of the DM gene".
References Baldwin AS, Kittler ELW, Emerson CP (1985) Structure, evolution, and regulation of a fast skeletal muscle troponin I gene. Proc Natl Acad Sci USA 82 : 8080-8084
Note added in proof. An additional member of the troponin gene family has been assigned to chromosome 19 (MacGeoch C, Barton PJR, Vallins WJ, Bhavsar P, Spurr NK, 1991). The human cardiac troponin I locus: assignment to chromosome 19p13.2-19q13.2. Hum Genet 88:101-104
22
+
+ +
+
X
Y
+ +
+ + +
(+) + + (+) (+) + + + (+) + + + + + +
+
+ +
+
+ +
(+)
+
+
+
+
+
+
(Novelli et al. 1991). T h e 1045-bp f r a g m e n t expected was strongly seen only i n hybrids H Y . 2 2 A Z A 1 , Y C 2 T 1 , a n d Y . X Y . 8 F 6 , all of which r e t a i n e d c h r o m o s o m e 19 (Fig. 1). T h e 1045-bp f r a g m e n t was n o t d e t e c t e d in the h a m s t e r D N A s a m p l e ; i n s t e a d , several low a n d higher m o l e c u l a r - w e i g h t b a n d s were amplified. T h e p r e s e n c e of the T N N T 1 1045-bp f r a g m e n t o n l y in hybrids r e t a i n i n g h u m a n c h r o m o s o m e 19, a n d the a b s e n c e in all o t h e r hybrids e x a m i n e d supports the p r o v i s i o n a l a s s i g n m e n t ( R o p e r s a n d P e r i c a k - V a n c e 1990) of the T N N T 1 g e n e to c h r o m o s o m e 19. This study also confirms the usefulness of P C R for testing the p r e s e n c e of specific h u m a n chrom o s o m e s , or c h r o m o s o m a l regions, in s o m a t i c cell hybrids.
21
+
+
+
+
17
+
+
+
16
+ +
+ +
+
+ + + + +
13
+ +
+ +
+
+
+ +
+
+ +
12
+ +
Gahlmann R, Troutt AB, Wade RP, Gunning P, Kedes L (1987) Alternative splicing generates variants in important functional domains of human slow skeletal troponin T. J Biol Chem 262 : 16122-16126 Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR protocols - a guide to methods and applications. Academic Press, San Diego Novelli G, GennarelIi M, Zelano G, Sangiuolo F, Dallapiccola B (1992) PCR amplification of the coding sequence for the human N-terminal slow skeletal troponin T. Ital J Biochem (in press) Ropers HH, Pericak-Vance MA (1990) Report of the committee on the genetic constitution of chromosome 19. (Human Gene Mapping 10.5 :update to the 10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 55 : 218-228 Samson F, Lee JE, Hung WY, Potter TG, Herbstreith M, Roses AD, Gilbert JR (1990) Isolation and localization of a slow troponin (TnT) gene on chromosome 19 by substracfion hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA. J Neurosci Res 27:441-451 Squire J (1981) The structural basis of muscular contraction. Plenum, New York, pp 193-200
© Springer-Verlag1992
Assignment of the slow troponin T (TNNT1) gene to chromosome 19 using polymerase chain reaction G. Novelli 1'a, M. Gennareili 1'2, M. Rocchi 3, and B. Dallapiccola 1'2 • 1Dipartimento di Sanit~ Pubblica e Biologia Cellulare, Universit~ "Tor Vergata", Roma Universit~t di Urbino, Urbino, Italy 2C.S.S., Hospital, IRCCS, Sangiovanni, Rotondo, Italy 3Istituto di Genetica, Universit~ di Bari, Bari, Italy Received June 5, 1991 / Revised October 21, 1991
Summary. Confirmation that the slow troponin (TNNT1) gene lies on chromosome 19 has been obtained by means of the polymerase chain reaction and somatic cell hybrids.
porting further examination of this protein as a candidate gene for DM. We report here the chromosome location of the human TNNT1 gene using the pobwnerase chain reaction (PCR; Innis et al. 1990).
Introduction
Materials and methods
Troponin T (TNNT1), is a tropomyosin-binding component of the troponin complex (subunits I, C and T), and is effective during muscle contraction (Squire 1981). The genomic organization of this gene family is poorly understood. However, the structure of the isoform I gene has been determined (Baldwin et al. 1985), and a c D N A clone for the human slow skeletal muscle TNNT1 form has been reported by Gahlman et al. (1987). This c D N A has provided the first nucleotide sequence information about TNNT1 and has shown that a single slow TNNT1 gene is present in the human genome. Recently, a new c D N A clone corresponding to the slow TNNT1 gene has been isolated by substraction hybridization of a c D N A muscle library using a myotonic dystrophy (DM) muscle c D N A (Samson et al. 1990). This clone has been provisionally mapped onto chromosome 19 (Ropers and Pericak-Vance 1990), probably in the D M region, sup-
Two different oligomers designed on the basis of the originally published TNNT1 cDNA were used to amplify a genomic fragment of 1045 bp extending from the carboxyl portion of the TNNT1 coding sequence ot the immediately 3' untranslated region of the cDNA (Gahlman et al. 1987). The forward primer, TrozlA, was: 5'-GCTGTACAACCGCATCAG-3'; the reverse primer, Troz2A, was: 5'-GCATGGGACAAACACTCC-3'. This pair fails to demonstrate specificTNNT1 PCR products in hamster DNA. The specificity of our hybrid PCR product was evaluated by restriction analysis and direct sequencing (G.Novelli, M. Genarelli, F. Sangiuolo, L. d'Agruma, B. Dallapiccola, in preparation). This primer pair was used to amplify hybrid DNA from a panel of 19 humanhamster hybrid cell lines, and human and hamster control DNA.
* Present address and address for offprint requests: B. Dallapiccola, c/o Laboratorio Centrale C.R.I., Servizio di Genetica, Via Ramazzini 15, 1-00151 Roma, Italy
1 2
3
4 5
6
7
8
9
10 11 12 13 t4 15
Results and discussion The results obtained by PCR using the T r o z l A and Troz2A primers are shown in Fig. 1, and summarized in Table 1. Preliminary experiments have demonstrated the validity of the PCR strategy for synthesizing specific TNNT1 c D N A from m R N A in human skeletal muscle
16 17 18 19 21 22 23 24
1045 bp
Fig. 1. Analysis of PCR products for human TNNT1 in somatic cell hybrids on 2.0% agarose gel. Lane 2 CHO DNA; lane 3 human genomic DNA; lane 8 HY.22AZA1 hybrid; lane 19 YC2T1 hybrid; lane 21 Y.XY.8F6 hybrid; lanes 1, 16, and24pBR328 DNA digested with BglI and HinfI; lanes 4-7, 9-15, 17, 18, 20, 22, 23
amplified DNA from hybrids without chromosome 19
698 Table 1. Segregation of human TNNT1 in human-hamster hybrids. + / - , Scoring of the presence or the absence of human chromosome; (+), only part of chromosome present because of deletion Hybrid
Human chromosome TNNT11
HY.11AZA1 Y2C2T1 Y.XY.8F6 HY.19.16T3D HY.31.24E HY.60A HY.70b2 HY.75E1 HY.94A HY.95A1 HY.95B HY.95S HY.137J HY. t37K HY. 166T4 RJ.387.51T5 RJ.387.58 Y.173.5CT3 Y.XY.8FT7
+ + +
2
3
4
5
6
7
8
9
10
11
+ + +
+
+ + +
+
+
+ + + +
+ +
+ +
14
15
+ +
+ + +
18
19 20
+
+ +
+ + +
+ + +
+
+ +
+
+ +
+
+
+
+
+ +
+
+ +
+
+ +
+ + +
Acknowledgements. This work was supported by the Italian C.N.R. (P.F. Ingegneria Genetica no. 91.00037.PF99 and P. F. Biotecnologie e Biostrumentazione) and by the financial support of Telethon - Italy to the project "Molecular study of the DM gene".
References Baldwin AS, Kittler ELW, Emerson CP (1985) Structure, evolution, and regulation of a fast skeletal muscle troponin I gene. Proc Natl Acad Sci USA 82 : 8080-8084
Note added in proof. An additional member of the troponin gene family has been assigned to chromosome 19 (MacGeoch C, Barton PJR, Vallins WJ, Bhavsar P, Spurr NK, 1991). The human cardiac troponin I locus: assignment to chromosome 19p13.2-19q13.2. Hum Genet 88:101-104
22
+
+ +
+
X
Y
+ +
+ + +
(+) + + (+) (+) + + + (+) + + + + + +
+
+ +
+
+ +
(+)
+
+
+
+
+
+
(Novelli et al. 1991). T h e 1045-bp f r a g m e n t expected was strongly seen only i n hybrids H Y . 2 2 A Z A 1 , Y C 2 T 1 , a n d Y . X Y . 8 F 6 , all of which r e t a i n e d c h r o m o s o m e 19 (Fig. 1). T h e 1045-bp f r a g m e n t was n o t d e t e c t e d in the h a m s t e r D N A s a m p l e ; i n s t e a d , several low a n d higher m o l e c u l a r - w e i g h t b a n d s were amplified. T h e p r e s e n c e of the T N N T 1 1045-bp f r a g m e n t o n l y in hybrids r e t a i n i n g h u m a n c h r o m o s o m e 19, a n d the a b s e n c e in all o t h e r hybrids e x a m i n e d supports the p r o v i s i o n a l a s s i g n m e n t ( R o p e r s a n d P e r i c a k - V a n c e 1990) of the T N N T 1 g e n e to c h r o m o s o m e 19. This study also confirms the usefulness of P C R for testing the p r e s e n c e of specific h u m a n chrom o s o m e s , or c h r o m o s o m a l regions, in s o m a t i c cell hybrids.
21
+
+
+
+
17
+
+
+
16
+ +
+ +
+
+ + + + +
13
+ +
+ +
+
+
+ +
+
+ +
12
+ +
Gahlmann R, Troutt AB, Wade RP, Gunning P, Kedes L (1987) Alternative splicing generates variants in important functional domains of human slow skeletal troponin T. J Biol Chem 262 : 16122-16126 Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR protocols - a guide to methods and applications. Academic Press, San Diego Novelli G, GennarelIi M, Zelano G, Sangiuolo F, Dallapiccola B (1992) PCR amplification of the coding sequence for the human N-terminal slow skeletal troponin T. Ital J Biochem (in press) Ropers HH, Pericak-Vance MA (1990) Report of the committee on the genetic constitution of chromosome 19. (Human Gene Mapping 10.5 :update to the 10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 55 : 218-228 Samson F, Lee JE, Hung WY, Potter TG, Herbstreith M, Roses AD, Gilbert JR (1990) Isolation and localization of a slow troponin (TnT) gene on chromosome 19 by substracfion hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA. J Neurosci Res 27:441-451 Squire J (1981) The structural basis of muscular contraction. Plenum, New York, pp 193-200
