Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis).
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 nuclear 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 cerevisiae. Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive control vector, pURY19-2 microm, which contained the 2 microm circle origin of yeast. In particular, one clone, pURY19-ARS223, was 18 times more efficient in back-transforming 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 vehicles that are used to transfect fish cell lines or embryos. A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus sequences, and are predicted to have hairpin loop structures, or curved or kinked DNA. In addition, the MAR-Finder program suggested that ARSs also contain MAR motifs. These include AT tracts, ORI patterns, 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 elements may be located in or near the MARs. This is the first study that has identified and characterized ARSs in fish.
Lim
Hak-Seob
HS
Department of Biotechnology, Pukyong National University, Busan, Korea.
Kim
Moo-Sang
MS
Park
Jin-Young
JY
Choi
Kang-Eun
KE
Hwang
Jee-Youn
JY
Kim
Dong Soo
DS
Lee
Hyung-Ho
HH
D000818:Animals; D003001:Cloning, Molecular; D003531:Cypriniformes / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D006801:Humans; D015530:Nuclear Matrix / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004247:DNA