Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin.
Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis. Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins. Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA. The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers. Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way. Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence). This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization.
Sedgwick
S G
SG
Genetics Division, MRC National Institute for Medical Research, Mill Hill, London, UK.
Nguyen
T M
TM
Ellis
J M
JM
Crowne
H
H
Morris
G E
GE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D016189:Dystrophin / Q000235:genetics* / Q000276:immunology; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics
D000911:Antibodies, Monoclonal; D003062:Codon; D004251:DNA Transposable Elements; D016189:Dystrophin; D000939:Epitopes