Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

Determining Gene Expression Using Orion Methyl-Filtered Sequences

Farzaneh Rostami¹ , Leigh-Emma Burnley¹ , Irfan Gunduz¹ , Michael Gore² , Luca Rossi¹ , Charles Opperman³ , Steven Lommel³ , Alec Hayes¹

¹  Philip Morris USA, 4201 Commerce Road, Richmond, VA 23234

²  Lancaster Labs, 2425 New Holland Pike, Lancaster PA 17605

³  North Carolina State University, CBNP, 840 Main Campus Drive, 1400 Partners II, Raleigh, NC 27606

The bulk of the repetitive, intergenic, non-coding DNA in plants, such as tobacco, is hyper-methylated, and therefore distinguishable from genic regions, which are in general hypo-methylated. Methyl-filtration is a cloning strategy that enriches for the coding regions of the genome. The Tobacco Genome Initiative (TGI) is utilizing the methyl-filtered library sequencing approach as one approach to discover genes of Nicotiana tabacum. Of the unique sequences identified from the methyl-filtered library, approximately 18% were homologous to known genes in the NCBI non-redundant protein database. Of the remaining 82%, 17% contained open reading frames (ORF’s) exceeding 90 predicted amino acids, indicating that they could be coding sequences. In order to provide experimental data to confirm that these sequences were expressed genes, a pool of mRNA transcripts from multiple tobacco tissue sources, developmental stages and environmental exposures, was amplified by reverse-transcription PCR (RT-PCR). Primers were designed for 112 sequences with long ORF’s. Nearly 60% of these sequences were amplified from this pool, indicating gene expression. The results from this experiment suggest that methyl-filtration is an effective tool for unique gene discovery.