Mega base map of the epidermal growth factor (EGF) receptor gene flanking regions and structure of the amplification units in EGF receptor-hyperproducing squamous carcinoma cells

Abstract

We have established a mega base scale physical map of the 5'- and 3'-flanking regions of the epidermal growth factor (EGF) receptor gene using CpG-recognition rare-cutting restriction enzymes and pulsed-field gel electrophoresis. In this map, a methylation-free CpG island (HTF island) is located within an 8-kilobase pair (kb) EcoRI fragment which includes exon 1 of the EGF receptor gene. From this HTF island, a 390-kb NotI fragment was identified as the longest 5'-flanking region and a 540-kb MluI fragment as the longest 3'-flanking region. Utilizing this map information, we have analyzed the structure of the flanking regions of amplified EGF receptor genes which are found in various squamous carcinoma cells. Among seven cell lines tested, four cell lines carrying EGF receptor genes in amounts more than 20 times that of normal cells showed amplification together with large 5'- and 3'-flanking regions. The amplified 5'-flanking regions were rearranged in different forms but were distinct in each cell line. The amplified 3'-flanking regions were at least 540 kb in size and common to all the cell lines, except that A431 had rearrangement points within 540 kb downstream of the HTF island. Thus, the size of amplification units appears to be large and different in each cell line.