Plant and Animal Genomes Conference 2006, San Diego, CA. Abstract P805.
Ataxia-telangiectasia (AT) is recessive autosomal disorder involving cerebellar degeneration, immune deficiencies, cancer predisposition, chromosomal instability and radiation sensitivity. The majority of AT patients carry truncating mutations in ATM gene resulting in prematurely terminated proteins that are highly unstable. To develop relevant porcine model of AT, we sequenced ATM transcripts, characterized the 5'-untranslated region (5' UTR), identified splice variants and developed constructs for gene targeting. Similar to humans, porcine ATM gene exhibits extensive alternative splicing. In contrast with humans it involves not only the 5' UTR but also coding regions. Six splice variants with 3 alternative exons were observed in 5' UTR, three of them spliced out the first coding exon, altering the translation start and giving rise to the putative protein lacking the N-terminus substrate binding domain. Real time PCR analysis revealed variable levels of ATM expression in 24 different tissues. Although each splice variant was ubiquity expressed in all tissues studied, differences in the relative abundances of specific 5' UTRs were detected. The impact of the longest splice variant was not less than 60% of total ATM transcript in each tissue monitored. Finally, to knock-out the ATM gene by introducing truncation mutation shortly after the start codon, we developed two gene targeting constructs for both known and putative translation start sites, according to splice variants detected. The similarity the porcine gene to human in extensive alternative splicing could impact in creation more relevant model for AT than in mouse. (This work was supported in part by USDA/NRI-CSREES grant AG2001-35205-11698 and USDA-ARS AG58-5438-2-313 and AT Children's Project).