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 mass es ranging over 700-900 kDa, Using a human nebulin cDNA probe, we isol ated three nebulin cDNA clones from a mouse skeletal muscle cDNA libra ry. 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 t he same approximate to 25 kb message from Skeletal muscle as the human nebulin probe. while detecting no messages from cardiac muscle. Seque nce data in combination with reverse-transcriptase PCR indicates that clones 7a and 8c overlap to form 4076 bp contiguous sequence. Alignmen t with the published full-length human nebulin sequence indicates that clone 4b overlaps with clone 7a over 1596 bp. However, after the firs t 798-bp overlap, the sequence of these two mouse nebulin clones diver ge, suggesting that they derive from distinct transcripts encoding iso forms of mouse nebulin. The mouse nebulin clones encode a series of ap proximate to 245-residue super repeats, each of which can be subdivide d into seven approximate to 35-residue, weakly repeating modules cente red around a conserved tyrosine residue, consistent with the human neb ulin 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 sequen ce is greater than 90% identical to the human sequence, with the excep tion of a 200-amino-acid region at the C-terminus of clone 4b, which i s less than 60% identical. In genomic Southern blots, a mouse nebulin probe detected a homologous sequence in a wide variety of vertebrate s pecies under stringent conditions. However, no significant hybridizati on was observed to genomic DNA from invertebrates and microorganisms, even under very low stringency. The sequence and Southern-blot data su ggest that the nebulin sequence is highly conserved among vertebrate s pecies.