Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin

Abstract

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.

Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes

“…2A). This 427-kDa band was also detected with another mAb, MANDYS8 (21), which recognizes an epitope in the mid-rod region of dystrophin molecule (data not shown). In addition to the 427-kDa band, MANDRA1 detected a predominant 75-kDa band only on a blot of the astrocyte homogenate, suggesting that the protein band represented Dp71, a small-type isoform of dystrophin (6)(7)(8)(9).…”

The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression

“…Transposon sequencing and mutagenesis system Generation of TnlOOO transposon insertions into target plasmids and use as a sequencing strategy was essentially as described (30). An extension of this method was used to locate the dbf3-1 rescuing activity to the SSBJ gene within the seventh plasmid (31).…”

SPO12andSIT4suppress mutations inDBF2, which encodes a cell cycle protein kinase that is periodically expressed

“…SDB22 was isolated as a single clone with an insert of approximately 22kb. To locate the suppressor gene within this the plasmid was subjected to Tnl000 mutagenesis in E. coli [ 16] and a library of randomly disrupted plasmids was transformed into LI 19-7D. Subsequent replica-plating of the transformants onto YPD at 37°C and 25°C indicated those plasmids where suppressor function had been disrupted.…”