Isolation and characterization of nucleolin gene as one of the vitamin A-responsive genes in airway epithelium by a palindromic primer-based mRNA differential display method.
A palindromic primer-based mRNA differential display method has been used to isolate various vitamin A-responsive genes from primary cultures of monkey tracheobronchial epithelial cells. This method, as compared with the original mRNA differential display (mDD) method described by Liang and Pardee, used only one arbitrarily designed primer instead of two in the polymerase chain reaction. The single-primer mDD method has several advantages over the two-primer mDD system, especially in the reamplification and the selection of 5'-end cDNA clone. To verify the usefulness of this approach, one of these differential display bands, M34, was initially chosen for further amplification and cloning. The clone derived from the M34 band has a DNA sequence with > 90% homology to the human nucleolin gene. Furthermore, DNA sequencing confirms that both 5' and 3' ends of the insert of M34 contain the invertly repetitive nucleotide sequence that was used to direct this cloning. Nucleolin is a multifunctional phosphoprotein that plays an important role in ribosome biogenesis and mRNA stability. Northern blot analysis demonstrated that in addition to the elevation by vitamin A, the level of nucleolin message is significantly higher in fetal than in adult tracheobronchial epithelial cultures. Furthermore, in situ hybridization demonstrated that the amount of nucleolin message is significantly higher in both basal and ciliated cell types than in mucous and intermediary cell types. These results support the feasibility that the single-primer mDD technique can be used to isolate vitamin A-responsive genes with a palindromic nature.
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California Regional Primate Research Center, University of California, Davis 95616, USA.
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Wu
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001980:Bronchi / Q000166:cytology; D002478:Cells, Cultured / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D004847:Epithelial Cells; D000882:Haplorhini; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D009697:Nucleolus Organizer Region / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins; D017422:Sequence Analysis, DNA; D014132:Trachea / Q000166:cytology; D014801:Vitamin A / Q000235:genetics*; D000097796:Nucleolin
D017931:DNA Primers; D016373:DNA, Antisense; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014801:Vitamin A; D000097796:Nucleolin