Structural diversity despite strong evolutionary conservation in the 5'-untranslated region of the P-type dystrophin transcript.

Analysis of the 5'-flanking regions of the Purkinje (P-) dystrophin genes and mRNAs in different species revealed strong sequence conservation but functional diversity. Multiple transcription initiation sites were identified in cerebella and muscles, tissues expressing P-dystrophin. The predominant initiation site was conserved, with another muscle-specific site located upstream. Despite sequence homology, significant tissue- and species-specific structural diversity in the P-type 5'-ends exists, including alternative splicing within the 5'-untranslated region combined with alternative splicing of intron 1. One amino terminus is conserved in mammals and, to a lesser extent, in chicken. However, alternative usage of ATG codons may result in a choice of N-termini or translation of short upstream ORFs in different species. Promoter activity of a fragment upstream of the cap site was shown by transient expression in myoblasts and in vivo following intramuscular injection. It is tissue- and developmentally regulated. Analysis of promoter deletions suggests the existence of negative regulatory elements in the proximal region.

Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein.

Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at least 8 contractile protein genes, most commonly beta myosin heavy chain, myosin binding protein C, and cardiac troponin T. Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion. To investigate the functional properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in Escherichia coli, purified, and combined with human cardiac troponins I and C to reconstitute human cardiac troponin. Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments motile and the velocity of motile filaments at pCa 9. (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocity of actin-tropomyosin filaments than wild-type troponin (12.3%). These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca(2+). However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca(2+) indicated an opposite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%). These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease.

Differential expression of syntrophins and analysis of alternatively spliced dystrophin transcripts in the mouse brain.

Expression of syntrophin genes, encoding members of the dystrophin-associated protein complex, was studied in the mouse brain. In the hippocampal formation there is distinctive co-localization of specific syntrophins with certain dystrophin isoforms in neurons, e.g. alpha1-syntrophin with the C-dystrophin in CA regions and beta2-syntrophin with the G-dystrophin in the dentate gyrus. Expression of the alpha1-syntrophin is predominant in CA regions and the olfactory bulb and it is also present in the cerebral cortex and the dentate gyrus. The beta2-syntrophin mRNA is most abundant in the dentate gyrus and is also evident in the pituitary, the cerebral cortex and in Ammon's horn and in traces in the caudate putamen. The choroid plexus was labelled by both alpha1- and beta2-syntrophin-specific probes. The expression of syntrophins in the brain correlates with expression of dystrophins and dystroglycan. There are brain areas such as the cerebral cortex where several different syntrophins and dystrophins are expressed together. Syntrophin expression co-localizes with utrophin in the choroid plexus and caudate putamen. Finally, no syntrophin was detected in the cerebellar Purkinje cells where the specific dystrophin isoform (P-type) is present. This specific distribution of syntrophins in the brain is particularly interesting, as muscle syntrophin interacts with neuronal nitric oxide synthase. This may suggest that the dystrophin-associated protein complex may be involved in synaptic organisation and signal transduction machinery in both muscle and neurons. The dystrophin isoform, with exons 71-74 spliced out and hence lacking syntrophin binding sites, had been believed to be predominant in the brain, but our analyses using in situ hybridization, S1 nuclease protection and the semi-quantitative polymerase chain reaction revealed that this alternatively spliced mRNA is a minor, low abundance form in the brain.