Am. J. Respir. Cell Mol. Biol., Vol 15, No. 3, Sep 1996, 398-403.
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
PM Reddy, G An, YP Di, YH Zhao and R Wu
California Regional Primate Research Center, University of California, Davis 95616, USA.
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.
