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.

Changes in antioxidant defense systems by 2,2',5,5'-tetrachlorobiphenyl exposure in neuronal SK-N-MC cells.

Polychlorinated biphenyls (PCBs) are known to alter the mammalian antioxidant defense system. To determine whether similar detoxification processes are activated in human neuronal cells, we investigated activities of antioxidant enzymes and the glutathione status (i.e., the levels of reduced and oxidized glutathione, GSH and GSSG) in human neuronal SK-N-MC cells exposed to 2,2',5,5'-tetrachlorobiphenyl (PCB 52). Upon PCB 52 treatment, time- and concentration-dependent inhibitions of cell viability were observed. PCB 52 did not affect GSH contents upon increasing the concentration up to 15 microg/ml, but significant depletions in GSH were observed at the concentrations of 20 and 25 microg/ml. PCB 52 exposure increased GSSG levels in the SK-N-MC cells, while GSH levels were decreased, and these changes naturally modified the GSSG/GSH ratios. Cytosolic glutathione S-transferase (GST) activity with 1-chloro-2,4-dinitrobenzene as substrate was enhanced by two-fold in neuronal cells after exposure to PCB 52 versus controls. In contrast, neuronal cells showed a sustained decrease in glutathione peroxidase activity with increasing concentrations of PCB 52, and a sustained decrease in Cu/Zn-superoxide dismutase (SOD) activity with increasing concentrations of PCB 52. Catalase activity was increased until 12 h after exposure to PCB 52, but was decreased 24 h after exposure. Overall, these results imply a major effect of PCB 52 on GSH status and upon the activities of antioxidant enzymes in human neuronal SK-N-MC cells, and upon the overall process of detoxification in human neuronal cells.

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.