Molecular analyses of carbonic anhydrase-II expression and regulation in the developing chicken lens.
The expression of carbonic anhydrase-II (CA-II) in the developing chicken lens was examined and compared with that in the retina of the chicken embryo. CA-II expression was measured by immunohistochemistry and radioimmunoassay during development, and CA-II mRNA was quantified by Northern blot and densitometric scanning and localized by in situ hybridization. A functional promoter of the chicken CA-II gene was identified by transfection of primary embryonic chicken lens epithelial cells and analyzed in deletion mutants. The results establish that CA-II makes up about 0.1% of the total soluble protein of the embryonic chicken lens, an amount insufficient to make it a candidate for an enzyme crystallin in this species. Lens fiber differentiation coincided with a loss of CA-II mRNA and protein; by contrast, CA-II persisted in the epithelial cells of the embryonic and mature lens. This and previous studies showed that CA-II amounts to as much as 3% of the protein of the embryonic chicken retina and follows a different developmental time course of expression; like the lens, CA-II decreases until day 10 in the embryonic retina, but, unlike the lens, it increases thereafter and plateaus at hatching. Progressive deletions of the 5' flanking regions (from position -1314 to +32) of the CA-II gene fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene resulted in a gradual loss of promoter activity, consistent with an additive effect of putative cis-regulatory elements found in many crystallin genes. These experiments provide the foundation for a molecular analysis of the developmental and differential regulation of the CA-II gene in lens and retina.
Buono
R J
RJ
Whitney Laboratory, Department of Anatomy and Cell Biology, University of Florida, St. Augustine 32086.
Linser
P J
PJ
Cuthbertson
R A
RA
Piatigorsky
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D002642:Chick Embryo; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation / Q000235:genetics; D007908:Lens, Crystalline / Q000196:embryology* / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D016385:TATA Box
D012333:RNA, Messenger; D002256:Carbonic Anhydrases