Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis).
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.
Kim
Moo-Sang
MS
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea.
Lim
Hak-Seob
HS
Ahn
Sang Jung
SJ
Jeong
Yong-Kee
YK
Kim
Chul Geun
CG
Lee
Hyung Ho
HH
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
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins