Murine transcription factor alpha A-crystallin binding protein I. Complete sequence, gene structure, expression, and functional inhibition via antisense RNA.
alpha A-crystallin binding protein I (alpha A-CRYBP1) is a ubiquitously expressed DNA binding protein that was previously identified by its ability to interact with a functionally important sequence in the mouse alpha A-crystallin gene promoter. Here, we have cloned a single copy gene with 10 exons spanning greater than 70 kb of genomic DNA that encodes alpha A-CRYBP1. The mouse alpha A-CRYBP1 gene specifies a 2,688-amino acid protein with 72% amino acid identity to its human homologue, PRDII-BF1. Both the human and the mouse proteins contain two sets of consensus C2H2 zinc fingers at each end as well a central nonconsensus zinc finger. The alpha A-CRYBP1 gene produces a 9.5-kb transcript in 11 different tissues as well as a testis-specific, 7.7-kb transcript. alpha A-CRYBP1 cDNA clones were isolated from adult mouse brain and testis as well as from cell lines derived from mouse lens (alpha TN4-1) and muscle (C2C12). A single clone isolated from the muscle C2C12 library contains an additional exon near the 5'-end that would prevent production of a functional protein if the normal translation start site were utilized; however, there is another potential initiation codon located downstream that is in frame with the rest of the coding region. In addition, we identified multiple cDNAs from the testis in which the final intron is still present. Finally, we used an antisense expression construct derived from an alpha A-CRYBP1 cDNA clone to provide the first functional evidence that alpha A-CRYBP1 regulates gene expression. When introduced into the alpha TN4-1 mouse lens cell line, the antisense construct significantly inhibited expression from a heterologous promoter that utilized the alpha A-CRYBP1 binding site as an enhancer.
Laboratory of Molecular and Developmental Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892.
J P
JP
Brady
M
M
Kantorow
C M
CM
Sax
D M
DM
Donovan
J
J
Piatigorsky
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C091656:Hivep1 protein, mouse; D016372:RNA, Antisense; D014157:Transcription Factors