Identification of exons in a novel embryonal carcinoma locus using the GRAIL program

Embryonal carcinoma (EC) cells have proven to be of particular value in 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 neomycin resistance cassette (Neo(r)), produced a mutant cell line: NR1-6. Genetic analysis of this variant cell line indicates that there is only a single insertion site. Interestingly, however, the NR1-6 cell line is unique in its morphology, tumorigenicity, and differentiative potential (1). We have sequenced over 18 kb from the regions flanking the retroviral insertion which we then analyzed using the computer programs 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 identify potential exons or transcripts using various molecular techniques 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 potential exons (3). Analysis with this program identified 5 exon candidates: two characterized as excellent (>90% probability) and three as marginal (>60% probability). Using reverse transcriptase (RT)-PCR we have demonstrated that the two 'excellent' and one of the 'marginal' exon candidates identified by GRAIL are expressed as mRNA in the mutant cells. Sequencing of these PCR products indicates that the mRNA is identical to the genomic DNA sequence. Thus, we have found that GRAIL provides an efficient, reliable means of identifying real exons within long regions of novel genomic DNA.