ISOLATION AND CHARACTERIZATION OF NUCLEOLIN GENE AS ONE OF THE VITAMIN-A-RESPONSIVE GENES IN AIRWAY EPITHELIUM BY A PALINDROMIC PRIMER-BASED MESSENGER-RNA DIFFERENTIAL DISPLAY METHOD

A palindromic primer-based mRNA differential display method has been u sed to isolate various vitamin A-responsive genes from primary culture s of monkey tracheobronchial epithelial cells. This method, as compare d with the original mRNA differential display (mDD) method described b y 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 t he reamplification and the selection of 5'-end cDNA clone. To verify t he usefulness of this approach, one of these differential display band s, M34, was initially chosen for further amplification and cloning. Th e clone derived from the M34 band has a DNA sequence with > 90 % homol ogy to the human nucleolin gene. Furthermore, DNA sequencing confirms that both 5' and 3' ends of the insert of M34 contain the invertly rep etitive nucleotide sequence that was used to direct this cloning. Nucl eolin is a multifunctional phosphoprotein that plays an important role in ribosome biogenesis and mRNA stability. Northern blot analysis dem onstrated that in addition to the elevation by vitamin A, the level of nucleolin message is significantly higher in fetal than in adult trac heobronchial epithelial cultures. Furthermore, in situ hybridization d emonstrated that the amount of nucleolin message is significantly high er in both basal and ciliated cell types than in mucous and intermedia ry cell types. These results support the feasibility that the single-p rimer mDD technique can be used to isolate vitamin A-responsive genes with a palindromic nature.