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

P. M. Sekhar Reddy, Gang An, Yuan Pu Di, Yu Hua Zhao, Reen Wu

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5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)398-403
Number of pages6
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume15
Issue number3
StatePublished - 1996

Fingerprint

Gene Expression Profiling
Vitamin A
Epithelium
Genes
Display devices
Messenger RNA
Cloning
Organism Cloning
Clone Cells
Nucleic Acid Repetitive Sequences
Phosphoproteins
RNA Stability
DNA Sequence Analysis
Ribosomes
Northern Blotting
Polymerase chain reaction
DNA sequences
Haplorhini
In Situ Hybridization
Cell culture

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

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title = "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",
abstract = "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.",
author = "{Sekhar Reddy}, {P. M.} and Gang An and Di, {Yuan Pu} and Zhao, {Yu Hua} and Reen Wu",
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T1 - 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

AU - Sekhar Reddy, P. M.

AU - An, Gang

AU - Di, Yuan Pu

AU - Zhao, Yu Hua

AU - Wu, Reen

PY - 1996

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N2 - 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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