Construction of physical map and comparison of genome sequences of F344 and LEC rats to identify radiation susceptibility gene

The Long Evans Cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA double-strand break repair and is a model for hepatocellular carcinogenesis. Although radiation susceptibility is controlled by a recessive gene on rat chromosome 4, it has not been identified. We previously determined that the region associated with radiation susceptibility is located in a 1.2-Mb on chromosome 4 using LEC congenic lines and recipient Fischer 344 (F344). Comparison of the coding sequences for seven known genes in the region between F344 and LEC rats showed no changes in deduced amino acid sequences, suggesting that radiation susceptibility gene is not known genes. In the present study, we constructed physical map and compared genome sequences of F344 and LEC rats to identify the radiation susceptibility gene by a positional cloning approach. We first constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We demonstrated that defective DNA repair in LEC is fully complemented by a 200-kb BAC, 65K18 in transient and stable transfected LEC cells. Further genetic analysis determined that the radiation susceptibility region is located in a 129-kb region of 65K18. We constructed the Fosmid library of the LEC genome and isolated four clones completely covering the region. We compared genome sequences of the region between F344 and LEC and found that an intronless Rpl36a gene is inserted into the LEC genome, but not into F344. The gene expression of Rpl36a of LEC cells was lower than that of F344 cells, suggesting that the intronless Rpl36a in the region is a pseudogene like other intronless genes. It is possible for the insertion of the intronless Rpl36a to disrupt a gene or to change the expression of a gene. We mapped four expressed sequence tags (ESTs) in the region. The expression of three ESTs was not different between F344 and LEC cells, but the remaining one was not expressed in LEC cells. To determine whether the EST is associated with radiation susceptibility, we have isolated a full-length sequence and performed a functional analysis. The identification of the radiation susceptibility gene will potentiate our understanding of the molecular mechanisms of radiation-induced DNA damage repair. １３ｔｈ International Congress of Radiation Reserc