IDENTIFICATION OF EXONS IN A NOVEL EMBRYONAL CARCINOMA LOCUS USING THE GRAIL PROGRAM

Embryonal carcinoma (EC) cells have proven to be of particular value i n studies of both oncogenesis and mammalian development, as well as in evaluating the relationship between these two phenomena. Infection of the EC cell line, NR1-0, with a defective retrovirus containing a neo mycin resistance cassette (Neo(r)), produced a mutant cell line: NR1-6 . Genetic analysis of this variant cell line indicates that there is o nly a single insertion site. Interestingly, however, the NR1-6 cell li ne is unique in its morphology, tumorigenicity, and differentiative po tential (1). We have sequenced over 18 kb from the regions flanking th e retroviral insertion which we then analyzed using the computer progr ams GCG and BLAST. Although homology was found to 4 B1 repeat elements (approximately 150 bp long) and a novel CA/GT dinucleotide repeat, no homology was found to any known genes (2). Furthermore, attempts to i dentify potential exons or transcripts using various molecular techniq ues and the above mentioned computer programs were all negative. Most recently we employed the GRAIL (Gene Recognition and Analysis Internet Link) computer program which was specifically designed to identify po tential exons (3). Analysis with this program identified 5 exon candid ates: two characterized as excellent (>90% probability) and three as m arginal (>60% probability). Using reverse transcriptase (RT)-PCR we ha ve demonstrated that the two 'excellent' and one of the 'marginal' exo n candidates identified by GRAIL are expressed as mRNA in the mutant c ells. Sequencing of these PCR products indicates that the mRNA is iden tical to the genomic DNA sequence. Thus, we have found that GRAIL prov ides an efficient, reliable means of identifying real exons within lon g regions of novel genomic DNA.