Abstract
Many types of DNA damage block replication because replicative DNA polymerases are unable to bypass altered bases. To overcome this, cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. The TLS polymerases are also characterized in terms of their low-fidelity on undamaged DNA, leading to the production of mutation. One of the most interesting studies for TLS polymerases is to know their role on biological consequence at tissue or whole-body level. For this purpose Medaka is most suitable because both of classical and modern molecular genetics are available. Recently a general reverse genetics method was reported, which can identify mutations in genes that are known only by their sequence. The method, called TILLING (Targeting Induced Local Lesions IN Genome), includes random mutagenesis, followed by screening for induced mutations in target genes at the genomic DNA level. Adult male Medaka were mutagenized with ENU and then outcrossed with female to generate F1 progeny for the library. We established 5771 ENU-mutagenized F1 male fishes. To construct a library, genomic DNA and testis samples were isolated and cryopreserved from each F1 fish. A pilot screening revealed that the average per-base mutation frequency was 1 in 350kbp. Rev1 protein is an essential player in the production of both spontaneous and DNA damage-induced mutations. We have identified 17 mutations in Rev1 gene, which include 2 nonsense and 15 missense mutations. Summary of the screening and the phenotype of these mutants will be described.
