Pergamon Neuromusc. Disord., Vol.3, No. 5/6, pp. 519-524, 1993 Elsevier Science Ltd Printedin Great Britain.All rights reserved 096~8966/93 $6.00 + .00 EXPRESSION OF THE TRANSCRIPTS INITIATED IN THE 62nd INTRON OF THE DYSTROPHIN GENE M. LAMBERT,* P. CHAFEY,* J. P. HUGNOT,* A. KOULAKOFF,t Y. BERWALD-NETTER,t C. BILLARD,~ G. E. MORRIS,§ A. KAHN,* J. C. KAPLAN* and H. GILGENKRANTZ* *ICGM, U129, INSERM, 24 rue du Fb St Jacques, 75014 Paris, France; tBiochimie Cellulaire, Collrge de France, 11 place Marcelin Berthelot, 75005 Paris, France; :~Neurologie Prdiatrique, Centre G. de Clocheville, 49 bd Brranger, 37044 Tours cedex, France; §Research Division, North East Wales Institute, Deeside, Clwyd, U.K. Abstract--The pattern of expression of two distal transcripts initiated in the 62nd intron of the dystrophin gene was investigated under different circumstances: (i) during the development of different rat tissues these transcripts and Dp71, a protein encoded by one of them, increased with brain development and decreased with muscle development; (ii) in cultured glial and neuronal cells, the distal promoter was coactivated with tissue-specific upstream promoters, the muscle- type promoter in glial cells and the brain-type promoter in neuronal cells, which suggests that activity of the upstream promoter does not interfere with activity of the distal promoter; (iii) in lymphoblasts of DM D patients with various deletions of the dystrophin gene, the most distal of which included the 56th intron, the production of the distal transcript was not perturbed. Key words: DMD, apodystrophin, developmental expression, quantitative PCR. INTRODUCTION The dystrophin gene encodes a variety of transcripts that are directed by at least five different promoters [1-6]. One of these species, of 4.8 kb, is initiated in the 62nd intronic sequence of the dystrophin gene and begins with a specific first exon which is absent from the dystrophin cDNA. It encodes a 71 kDa protein also coined apodystrophin 1, which shares the last two domains of the dystrophin protein, and is expressed in all analysed tissues except in adult muscle [6, 7]. It has been reported that the last 13 amino acids of adult muscle dystrophin are replaced in this protein [8], as in the fetal dystrophin isoform [9], by 31 different amino acids which confer hydrophobicity to this region. The carboxy-terminal domain of all these proteins seems particularly conserved among species [10] and shows a high degree of similarity with only one other known protein, DRP (dystrophin related protein), also called utrophin [11], which is particularly abundant in fetal tissues. More recently, a shorter mRNA, a 2.2 kb species, initiated at the same specific exon as the apodystrophin 1 RNA, has been described [12]. The 4.8 kb and 2.2 kb species seem, therefore, to be directed by the same promoter. The predicted protein encoded by the 2.2 kb form, called apodystrophin 3, should be almost exclusively composed of the third domain of dystrophin, which has been suggested to interact with a glycoprotein complex associated with the sarco- lemmal membrane [13]. The function of all these isoforms is not known. In this study, we describe the pattern of expression of apodystrophin 1 and 3 (1/3) mRNA and of the 71 kDa protein during development of rat brain, heart, liver and muscle. We also investigate whether in a given non- muscle cell type, namely glial and neuronal cells, both dystrophin and apodystrophin 1/3 tran- scripts are co-expressed. Finally, we report the lack of impact of various DMD gene deletions located upstream from exon 56 on the apodys- trophin expression in lymphoblasts from these patients. MATERIALS AND METHODS cDNA synthesis cDNA synthesis was performed on 0.9/ag of total RNA of each specimen. An exogenous sample of reference RNA (either 100 ng of total rat liver RNA for human cells or 100 ng of total RNA HepG2 cells RNA for rat tissues) was added as a source of internal standard. Total RNA was reverse transcribed into cDNA using a random hexamer primer procedure [14] as previously described [15]. For each reaction, a negative control consisting in omitting the reverse transcriptase step, was performed. 519