CD-TAGGING - A NEW APPROACH TO GENE AND PROTEIN DISCOVERY AND ANALYSIS

We describe a new method for gene discovery and analysis, CD-tagging, that puts specific molecular tags on a gene, its transcript and its pr otein product. The method has been successfully tested in two organism s, the haploid unicellular alga Chlamydomonas reinhardtii and the meta zoan Drosophila melanogaster. The method utilized a specially designed DNA molecule, the CD-cassette, that contains splice acceptor and dono r sits surrounding a short open reading frame. Insertion of the CD-cas sette into an intron in a target gene introduced a new exon, represent ed by the open reading frame of the CD-cassette, surrounded by two fun ctional hybrid introns. As a result (i) the gene is tagged by a specif ic nucleotide sequence, (ii) the mRNA is tagged by a specific nucleoti de sequence and (iii) the protein is tagged by a specific peptide sequ ence. Because these tags are unique, specific nucleotide or antibody p robes can be used to obtain and/or analyze the gene, transcript or pro tein. As a gene discovery technology, CD-tagging has two unique advant ages: 1) Genes can be identified through a primary screen at the prote in level, and so the very process by which a gene is identified provid es specific empirical information about its biological function. 2) Th e cassette arms, which are spliced out of the transcript of the target gene, are available to carry a wide variety of DNA sequences, such as genes encoding drug resistance that can be used to select for the pre sence of the CD-cassette in the genome.