Abstract
Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy. We isolated the rat homologue of plunc, a murine gene highly expressed in lung and nasopharyngeal regions, by this method. Rat plunc encodes a 270-amino acid protein containing a putative signal peptide. plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization. plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis. Antibodies against Plunc detected a 31-kDa protein in lung, heart, and spleen. Rat nasal epithelium displayed robust immunoreactivity that was highly localized to the microvilli layer of respiratory epithelium. The expression of plunc was up-regulated after bulbectomy in respiratory epithelium. We also detected secreted plunc in rat and human mucus. Sequence and homology analyses suggest that Plunc is a member of the secretory gland protein family with putative bactericidal/bacteriostatic function. This is the first protein found in respiratory epithelium whose expression is regulated by olfactory neuronal injury and may provide protection against infection subsequent to injury.
Highlights
Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy
Rat plunc encodes a 270amino acid protein containing a putative signal peptide. plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization. plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis
Plunc Is Identified as an RNA Transcript That Is Up-regulated after Bulbectomy—Total RNA isolated from normal nasal epithelium and from nasal epithelium of bulbectomized rats was used as the tester and driver for suppression hybridization (SSH), respectively
Summary
Experimental Animals and Tissue Preparation—All experimental protocols were approved by The Johns Hopkins University Institutional Animal Care and Use Committee, and all applicable guidelines from the National Institutes of Health Guide for the care and use of laboratory animals were followed. The membranes were hybridized under 5ϫ SSC, 5ϫ Denhardt’s, 1% SDS, and 100 g/ml denatured salmon sperm DNA, with equivalent amounts of RsaI (a four-base cutter, as used for the construction of the initial subtracted library)-digested 32P-labeled double-stranded cDNA of equal specific activity (2.2 ϫ 106 cpm/ml) derived from driver and tester mRNA, respectively. Slides were post-fixed in Bouin’s for 5 min at room temperature, washed twice with TBS, incubated with 0.1 M triethanolamine buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride (Sigma Chemical Co.) for 10 min, and pre-hybridized at 37 °C for 10 min with 2ϫ SSC containing 50% (v/v) deionized formamide. Section slides were incubated overnight at 4 °C in PBS containing the affinity-purified Plunc antibody at a dilution of 1:50. Heuristic searches were performed to identify the best tree for 11 taxa. 500 bootstrap replicates were performed
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