Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR.
Keshav S., McKnight AJ., Arora R., Gordon S.
Differential display polymerase chain reaction (DD-PCR) is a powerful technique for comparing gene expression between cell types, or between stages of development or differentiation. Differentially expressed genes may be cloned and analysed further. Here we extend the use of DD-PCR to analyse differences in gene expression between two complex epithelia: that of the small intestine and of the large intestine. The aim of this study was to identify genes expressed preferentially in Paneth cells. Paneth cells are secretory epithelial cells putatively involved in host defense and regulation of crypt cell proliferation and are found at the base of the small intestinal crypts adjacent to the stem cell zone. Of 34 clones that were analysed, partial sequencing identified two clones related to known Paneth cell products: a homologue of secretory phospholipase A2 (clone B1) and a homologue of a neutrophil defensin (clone C5). B1 was strongly expressed in Paneth cells, as demonstrated by in-situ hybridization. B1 was also expressed at a lower level in the large intestinal epithelium. A full length B1 cDNA clone was isolated and sequenced, and shown to be highly homologous to type II secretory phospholipase A2 genes, and almost identical to the enhancing factor gene and the putative gene for the MOM-1 locus. B1 expression is limited to the intestinal tract, and we propose that it be designated intestinal phospholipase A2, or i-PLA2. The method we describe is well suited to the rapid identification of genes expressed exclusively or predominantly in Paneth cells.