PI's abstract) This project addresses the function of genomic imprinting and the underlying regulatory mechanisms. The goals of the project include: 1) Identification of additional mouse imprinted genes in order to establish the basis for comparative work in the human genome. Using the Restriction Landmark Genome Scanning (RLGS) method, candidate loci were identified which show allele-specific methylation; a common feature of imprinted genes that led to the cloning of novel imprinted genes. It is now planned to clone 11 additional candidate loci for preliminary characterization and to identify the adjacent gene in large genomic clones for one of them. 2) A detailed characterization of the newly identified imprinted region on mouse chromosome 9 including the paternally expressed Grf1 (guanine nucleotide exchange factor) gene. Grf1 is the mouse homologue to the yeast CDC25 gene which acts as a Ras activator and thus plays an important role in cell cycle regulation. The genomic structure of the Grf1 gene within a 500 kbp contig of genomic clones will be analyzed. These experiments will include the characterization of the exon-intron structure of Grf1, identification of additional allele-specific methylation sites and exon-trapping experiments to identify novel coding sequences. 3) A detailed study on the expression of the different alternatively spliced forms of Grf1 transcripts, including the identification and analysis of the promoter for the largest, paternally expressed Grf1 transcript.
