Subtractive cDNA cloning as a tool to analyse secondary effects of a muscle disease. Characterization of affected genes in the myotonic ADR mouse.
In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.
Schleef
M
M
University of Bielefeld, Developmental Biology Unit, Germany.
Zühlke
C
C
Schöffl
F
F
Jockusch
H
H
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis*; D015536:Down-Regulation / Q000502:physiology; D015723:Gene Library; D051379:Mice; D008818:Mice, Neurologic Mutants; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics; D009135:Muscular Diseases / Q000235:genetics* / Q000378:metabolism; D009222:Myotonia / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000096:biosynthesis
D018118:Chloride Channels; D018076:DNA, Complementary; D009124:Muscle Proteins; D012313:RNA