cDNA cloning of mouse nebulin - Evidence that the nebulin-coding sequence is highly conserved among vertebrates

Nebulin is a family of giant myofibrillar proteins with molecular masses ranging over 700-900 kDa, Using a human nebulin cDNA probe, we isolated three nebulin cDNA clones from a mouse skeletal muscle cDNA library. These three clones, labeled 8c, 7a and 4b, carry inserts of 2.0, 3.0 and 3.5 kb, respectively. In Northern blots, each insert detected the same approximate to 25 kb message from Skeletal muscle as the human nebulin probe. while detecting no messages from cardiac muscle. Sequence data in combination with reverse-transcriptase PCR indicates that clones 7a and 8c overlap to form 4076 bp contiguous sequence. Alignment with the published full-length human nebulin sequence indicates that clone 4b overlaps with clone 7a over 1596 bp. However, after the first 798-bp overlap, the sequence of these two mouse nebulin clones diverge, suggesting that they derive from distinct transcripts encoding isoforms of mouse nebulin. The mouse nebulin clones encode a series of approximate to 245-residue super repeats, each of which can be subdivided into seven approximate to 35-residue, weakly repeating modules centered around a conserved tyrosine residue, consistent with the human nebulin sequence. The mouse nebulin clones align along the central third of the full-length human sequence, corresponding to super repeats 8-16 of the 22 super repeats found in human nebulin. The translated sequence is greater than 90% identical to the human sequence, with the exception of a 200-amino-acid region at the C-terminus of clone 4b, which is less than 60% identical. In genomic Southern blots, a mouse nebulin probe detected a homologous sequence in a wide variety of vertebrate species under stringent conditions. However, no significant hybridization was observed to genomic DNA from invertebrates and microorganisms, even under very low stringency. The sequence and Southern-blot data suggest that the nebulin sequence is highly conserved among vertebrate species.