Cloning and characterization of alternatively spliced isoforms of Dp71.
Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS)
Austin
R C
RC
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Howard
P L
PL
D'Souza
V N
VN
Klamut
H J
HJ
Ray
P N
PN
D000328:Adult; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000650:Amnion / Q000166:cytology / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary; D049030:Dystroglycans; D016189:Dystrophin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D018076:DNA, Complementary; D016189:Dystrophin; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; C085339:apo-dystrophin 1; D049030:Dystroglycans