title: Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis).
abstract: The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.
authors.affiliation: Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea.
authors.forename: Moo-Sang
authors.initials: MS
authors.lastname: Kim
authors.forename: Hak-Seob
authors.initials: HS
authors.lastname: Lim
authors.forename: Sang Jung
authors.initials: SJ
authors.lastname: Ahn
authors.forename: Yong-Kee
authors.initials: YK
authors.lastname: Jeong
authors.forename: Chul Geun
authors.initials: CG
authors.lastname: Kim
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003531:Cypriniformes / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D045170:Matrix Attachment Regions / Q000235:genetics*; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014162:Transfection
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins