Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism.
Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.
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Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Beebe
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D000818:Animals; D001483:Base Sequence; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003459:Crystallins / Q000235:genetics*; D004848:Epithelium / Q000502:physiology; D005786:Gene Expression Regulation; D007438:Introns; D007553:Isotope Labeling / Q000379:methods; D007908:Lens, Crystalline / Q000196:embryology / Q000502:physiology*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D002247:Carbon Isotopes; D003459:Crystallins; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012313:RNA