THE RAT N-RAS GENE - INTERFERENCE OF PSEUDOGENES WITH THE DETECTION OF ACTIVATING POINT MUTATIONS

The PCR technique in combination with selective hybridization to mutat ion specific oligonucleotides, is a widely used methodology for the de tection of activating point mutations in ras oncogenes. In the present paper we demonstrate for the N-ras gene of the rat that processed pse udogenes do interfere with this method. A first indication for this in terference came from the sequence analysis of cloned PCR fragments of exon 1, amplified with primers derived from previously reported exon s equences of the mouse N-ras gene. Between different clones originating from one PCR reaction, a marked sequence heterogeneity is observed an d this is shown to be the result of the presence of at least two diffe rent processed pseudogenes of the rat N-ras gene. These two pseudogene s, together with the wildtype N-ras gene and a small 3' part of the un r gene, were eventually cloned and their genomic organization and nucl eotide sequences determined. Furthermore, representative examples of t he confounding effects of these pseudogenes on the screening for activ ating point mutations are presented. Taken together, our results demon strate that intron-specific amplification is a prerequisite for the un ambiguous detection of activating point mutations in the N-ras gene of the rat.