Abstract
alpha-Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a dystrophin-associated and dystrophin-related protein. Knockout of the gene in the mouse results in muscular dystrophy. The control of the alpha-dystrobrevin gene in the various tissues is therefore of interest. Multiple dystrobrevin isoforms differing in their domain content are generated by alternative splicing of a single gene. The data presented here demonstrate that expression of alpha-dystrobrevin from three promoters, that are active in a tissue-selective manner, also plays a role in the function of the protein in different tissues. The most proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding sites, is restricted to brain. Promoter C, which contains multiple consensus myogenic binding sites, is up-regulated during in vitro myoblast differentiation. Interestingly, the organization and the activity of the alpha-dystrobrevin promoters is reminiscent of those in the dystrophin gene. Taken together we suggest that the multipromoter system, distributed over a region of 270 kilobases at the 5'-end of the alpha-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages.
Highlights
Dystrobrevin, a dystrophin related protein, was originally identified from the Torpedo californica electric organ as an 87-kDa phosphoprotein associated with the cytoplasmic face of the postsynaptic membrane [1, 2]
The electric organ, skeletal muscle, and brain, and it has been postulated to play a role in synaptic structure and function, because it copurifies with the acetylcholine receptors and rapsyn from the electric organ membranes
The genetic basis of this isoform diversity and additional alternative splicing was resolved by the determination of the genomic organization of the coding region of a single gene on mouse chromosome 18 [6]
Summary
Characterization, and Sequencing of Genomic Clones—To determine the order and estimate the distance between the 5Ј-UTR exons, a BAC (mouse 129/sv) genomic library was screened (Research Genetics, Huntsville, AL) by a PCR approach using primer sets specific for the 5Ј-UTR exon A (RnpAf, 5Ј-GGGGGAAAAGAATCTGACTCTGT3Ј; RnpAr, 5Ј-CTGCTTTTGTAGTTCACGCAC-3Ј), exonB (RnpBf, 5Ј-GTGCGTGCGCGTCCGTGG-3Ј; RnpBr, 5Ј-CTCGCCAAACTCTTAGAAGGTG-3Ј), exonC (RnpCf, 5Ј-GCATCTGCCAGTGGGACTTC-3Ј; RnpCr, 5Ј-CCAGGTACAGCATCCTTTCTTC-3Ј), exon D (UTRDf1, 5Ј-GATAAATAGGATTTACAAGCC-3Ј; UTRDr, 5Ј-GAAGAGACAGCATGGACTTT-3Ј); and coding exon (DB3f, 5Ј-ACATAGAACTCAACGTGGCC-3Ј; DB3r1, 5Ј-GTGGATTTGGTGAGTGGTTG-3Ј). To isolate genomic DNA upstream and downstream of the 5Ј-UTR exons E, F, and G, which were detected in a minority of ␣-dystrobrevin cDNAs, a YAC vectorette library of the YAC clone ICRFy902M0312Q was screened by PCR using hemi-nested primer sets made from the sequences of exon E, F, and G (see Fig. 1) and a universal vectorette primer as described previously [6]. Vectorette PCR products were cloned into pGEM-T vector (Promega) and sequenced using Sp6 and T7 primers. Construction of Promoter-Reporter Fusion Plasmids—Mouse genomic DNA and the primer set ProAf/ProAr (5Ј-CGCGGATCCTCCAGTGAGGGAAGGCAG-3Ј, 5Ј-CGCGGATCCCTCTAACTCTTCCGCAG-3Ј) was used to amplify a 1068-bp product spanning the 858-bp flanking region and the first 210 bp of exon A. Immunoblot Analysis—Western blots using the primary antibody -CT-FP (diluted 1:1000) and Horseradish peroxidase-conjugated secondary antibody, donkey anti-mouse (diluted 1:5000; Jackson ImmunoResearch Laboratories, Inc.) were performed as described previously [24]
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