Autonomously replicating sequences (ARSs) are thought to occur within, or adjacent to, the matrix attachment regions (MARs). To identify fish ARSs, MARs of the mud loach fish were obtained from nu-clear matrices using a modified LIS method. These DNA fragments were screened for their ability to act as ARSs by being cloned into the ARS cloning vector, pURY19, and transformed into Saccharomyces cere-visiae. Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive con-trol vector, pURY19-2 mm, which contained the 2 mm circle origin of yeast. In particular, one clone, pURY19- ARS223, was 18 times more efficient in back-transfor-ming E. coli than the positive control vector. Therefore, ARS223, which has strong ARS activity in yeast, could be a good candidate for inclusion in expression vehi-cles that are used to transfect fish cell lines or embryos. A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus se-quences, and are predicted to have hairpin loop struc-tures, or curved or kinked DNA. In addition, the MAR-Finder program suggested that ARSs also con-tain MAR motifs. These include AT tracts, ORI pat-terns, kinked DNA, ATC tracts, and Topoisomerase II consensus sequences. The in vitro matrix binding assay confirmed that all of the cloned ARSs could associate with the nuclear matrix. This indicates that ARSs ele-ments may be located in or near the MARs. This is the first study that has identified and characterized ARSs in fish.

Keywords: Matrix, Autonomously Replicating Sequences (ARSs)