Binding of tissue-specific forms of alpha A-CRYBP1 to their regulatory sequence in the mouse alpha A-crystallin-encoding gene: double-label immunoblotting of UV-crosslinked complexes.
The alpha A-CRYBP1 regulatory sequence (alpha A-CRYBP1RS), at nucleotides -66 to -57 of the mouse alpha A-crystallin-encoding gene (alpha A-CRY) promoter, is an important control element involved in the regulation of mouse alpha A-CRY expression. The gene encoding a protein (alpha A-CRYBP1) that specifically binds to the alpha A-CRYBP1RS sequence has been cloned from a cultured mouse lens cell line. In the present study, we have used an antibody (specific to the alpha A-CRYBP1 protein and made against a synthetic peptide) to directly identify UV-crosslinked protein-DNA complexes via a double-label immunoblotting technique. Multiple alpha A-CRYB1 antigenically related proteins interacted with alpha A-CRYBP1RS in nuclear extracts from both a cloned mouse lens cell line (alpha TN4-1) that expresses alpha A-CRY and a mouse fibroblast line (L929) that does not express the gene. Two sizes (50 kDa and 90 kDa) of proteins reacting with the alpha A-CRYBP1-specific Ab were detected in both cell lines and, in addition, a > 200-kDa protein reacting with the Ab was unique to the fibroblast line. Thus, alpha A-CRYBP1 antigenically related proteins interact with alpha A-CRYBP1RS regardless of alpha A-CRY expression. Moreover, differential processing of the alpha A-CRYBP1 protein and/or alternative splicing of the alpha A-CRY transcript may affect expression of alpha A-CRY.
Kantorow
M
M
Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, MD 20892.
Becker
K
K
Sax
C M
CM
Ozato
K
K
Piatigorsky
J
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003459:Crystallins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
D000906:Antibodies; D003459:Crystallins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins