International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

A9 Genomic Organization of the Imprinted Mouse Gene, RasGrf1

Aranzazu de la Puente and Christoph Plass. Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, 420 West 12th Ave., Columbus, Ohio 43210

Normal mammalian development requires the expression from both maternal and paternal alleles. Genomic imprinting results in allele-specific expression of certain genes from either the maternal or the paternal allele. Imprinted genes are marked before fertilization in a way that either the maternal or the paternal alleles are transcriptionally silenced in the offspring. This is due in most cases to methylation of the cytosine in the CG dinucleotides mainly located in CpG-island. Preliminary, studies in the lab using Restriction Landmark Genomic Scanning with methylation sensitive enzymes (RLGS-M) identified an imprinted locus Irlgs-3 on mouse chromosome 9 (9E3-F1). This locus contains the paternally expressed RasGrf1 (Guanine nucleotide Release Factor-1), the mouse homologue of CDC25 in Saccharomyces cerevisiae. In order to study the mechanisms underlying the regulation of imprinted expression we studied the genomic structure of RasGrf1. Several BAC clones were identified by screening of a mouse BAC library (RPCI-22 129/SvEvTACBr) with pooled RasGrf1 cDNA probes generated by RT-PCR with primer pairs designed from the published cDNA sequence (L20899). Positive BAC clones were confirmed by hybridization with specific primer. The exon-intron structure of RasGrf1 was identified by sequencing of the BAC clones with cDNA specific primers. RasGrf1 contains 26 exons and 25 introns, spanning approximately 142kb of genomic DNA. Intron sizes have been determined by long-range PCR with primer pairs located at the ends of exons. All the intron-exon junctions have been sequenced and follow the canonical AC-GT rules. There is a single polyadenylation signal (AATATA) which contains a mismatch with respect to consensus (A to T conversion at the fifth position). We are currently on the way to identify the promoter region of RasGrf1 by primer extension and 5'RACE. Since imprinted genes are found in clusters, a BAC contig has been developed across the RasGrf1 region (approximately 500kb), in order to isolate novel imprinted genes in this region.

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