Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.
Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.
Sax
C M
CM
Laboratory of Molecular and Developmental Biology, NEI, NIH, Bethesda, MD 20892.
Ilagan
J G
JG
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D003459:Crystallins / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D007908:Lens, Crystalline / Q000378:metabolism*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D016385:TATA Box; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003459:Crystallins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase