Fig. 2. Cloning of muscle supervillin (archvillin) and northern analyses. (A) Diagram showing the coding regions (boxes), the 5'- and 3'-UTRs, and the sequences used as an archvillin-specific probe (probe 1) and as a probe for both archvillin and supervillin messages (probe 2). Lines represent partial sequences derived from gene-specific primers (no arrows) and 5'- (left arrows) and 3'- (right arrows) RACE products from skeletal muscle cDNAs. Archvillin-specific sequences in the 5'-halves of the coding regions (gold), predicted nuclear targeting signals (blue), and coding sequences for the highly conserved villin/gelsolin homology regions at the 3'-ends of the coding regions (pink) are indicated. Asterisks on clones M03 and M08 denote the location of an additional 23 bp of sequence. Consensus sequences of these human and murine cDNAs are available (accession nos AF109135, AF317422), as is the potential alternatively spliced murine cDNA sequence (AF317423). (B) Multiple tissue northern blot (Clontech) containing 2 µg per lane of poly(A)⁺ RNA from heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7) and pancreas (lane 8) after hybridization with ³²P-labeled probe 2. The blot was exposed to XAR/MS film at –80°C with an intensifying screen for 24 hours. (C) Lanes from the same northern blot containing heart (lane 1) and skeletal muscle RNA (lane 6) after exposure to film at –80°C for only 3 hours. (D) The same northern blot after stripping and re-probing with archvillin-specific probe 1. (E) Hybridization with ß-actin probe to show equal RNA loading. Migration positions of archvillin message are shown (AV, asterisks), as are the positions of supervillin mRNA (SV, arrowheads).

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