Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia. The mutation responsible for DM has been identified as the amplification of a polymorphic (CTG)n repeat in the 3' untranslated region of a gene encoding a serine/threonine kinase (DMK). We have produced a polyclonal rabbit antibody preparation against a fusion protein encoding the C-terminal amino acids 471-629 of the human DMK gene. This antibody specifically detects products of both full length and truncated human DMK genes expressed in bacteria and in insect cells. On immunoblots, we observed protein species of approximately 74 and 82 kDa in cardiac muscle, skeletal muscle, ependyma and choroid plexus. By immunofluorescence, DMK was found to localize post-synaptically at the neuromuscular junction of skeletal muscle, at intercalated discs of cardiac tissue and at the apical membrane of the ependyma and choroid plexus. We have also detected two to three species (approximately 45-50 kDa) in other regions of the brain. Synaptic localization of DMK in the cerebellum, hippocampus, midbrain and medulla was noted. These results suggest that DMK plays a specialized role in intercellular communication.

Myotonic dystrophy is caused by the expansion of a CTG repeat found in the 3-untranslated region of the myotonic dystrophy kinase. The mechanism of disease and the role of the kinase are currently obscure. Here we begin the investigation of domain structure/function correlations to aid in determining its normal function.Expressed full-length protein and protein truncated before a C-terminal hydrophobic domain were compared. In vitro, signal peptide function and protection of kinase by microsomal membranes were absent; thus, it is not translocated, as previously proposed. However, full-length kinase expressed in insect cells was found in fractions enriched for membranes and decorated mitochondria. The truncated form was found primarily in the cytosol. The kinase was present as two self-associated, disulfide-linked complexes. The majority of fulllength kinase was found in the larger of the two complexes, while almost all of the truncated form was found in the smaller. Thus, the C-terminal region confers a higher order of self-association. Furthermore, fulllength kinase expressed in COS-1 cells was present as high molecular weight complex, while the truncated form was present as monomer species. These experiments indicate that the myotonic dystrophy kinase is not membrane-integrated, but that it may have a molecular organization which favors peripheral association with membranes.

Myotonic dystrophy (DM1) is a multisystemic disorder caused by a CTG repeat expansion within the 3'-UTR of the DMPK gene. DM1 is characterized by delayed muscle development, muscle weakness and wasting, cardiac conduction abnormalities, cognitive defects and cataracts. Recent studies have demonstrated that the disease mechanism involves a dominant gain-of-function conferred upon mutant transcripts by expanded repeats. However, further attempts to model aspects of DM muscle pathology in cultured myoblasts suggest that 3'-UTR sequences flanking the CTG repeat tract are also required for full expression of the disease phenotype. Here, we report that overexpression of the DMPK 3'-UTR including either wild-type (11) or expanded (91) CTG repeats results in aberrant and delayed muscle development in fetal transgenic mice. In addition, transgenic animals with both expanded and wild-type CTG repeats display muscle atrophy at 3 months of age. Primary myoblast cultures from both 11 and 91 repeat mice display reduced fusion potential, but a greater reduction is observed in the 91 repeat cultures. Taken together, these data indicate that overexpression of the DMPK 3'-UTR interferes with normal muscle development in mice and that this is exacerbated by inclusion of a mutant repeat. This suggests that the delayed muscle development in DM1 involves an interplay between the expanded CTG repeat and adjacent 3'-UTR sequences.

Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5 to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Cotransfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10 -20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to upregulate transcription of this gene in myoblasts.

Myotonic dystrophy is caused by a (CTG) n trinucleotide repeat expansion located in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). To date, the disease mechanism has proven elusive. The mutation would not be expected to affect kinase function and yet the disease is inherited in a dominant fashion. Mutant DMPK transcripts have been demonstrated to be retained in affected cell nuclei which could reduce DMPK protein levels and cause disease by haploinsufficiency. An alternate hypothesis is that the expansion confers a toxic gain of function on the transcript. In previous studies, various 52-55 kDa proteins have been detected using antisera targeted against DMPK and a decline of two of these candidates in disease tissues was reported. Current information now suggests that these proteins are not products of the myotonic dystrophy gene. We have characterised an antiserum which has been confirmed to recognise authentic 71 and 80 kDa isoforms of DMPK. Determination of the kinase levels in disease tissues with controls for patient age and tissue integrity demonstrates a modest overexpression in adult patients. In tissues from severely affected congenital patients only a slight decline is seen. This data argues against DMPK haploinsufficiency as a disease mechanism.