Related Experiment Videos
Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis)
Hak-Seob Lim1, Moo-Sang Kim, Jin-Young Park
1Department of Biotechnology, Pukyong National University, Busan, Korea.
Abstract:
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.
Insights
Researchers identified novel autonomously replicating sequences (ARSs) in mud loach fish, which are crucial for DNA replication. These fish ARSs, located near matrix attachment regions (MARs), show high efficiency for potential use in fish gene expression vectors.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Autonomously replicating sequences (ARSs) are essential DNA elements for replication initiation.
- ARSs are often found associated with nuclear matrix attachment regions (MARs).
- Identifying fish ARSs is crucial for developing gene delivery systems in aquaculture and research.
Purpose of the Study:
- To identify and characterize autonomously replicating sequences (ARSs) in the mud loach fish.
- To investigate the association of fish ARSs with matrix attachment regions (MARs).
- To evaluate the potential of identified fish ARSs for use in gene expression vectors.
Main Methods:
- Isolation of mud loach fish nuclear matrix DNA fragments.
- Cloning fragments into an ARS-deficient yeast vector (pURY19) for functional screening.
- Transformation of yeast (Saccharomyces cerevisiae) and bacteria (E. coli) to assess ARS activity.
- DNA sequence analysis and MAR motif prediction.
- In vitro matrix binding assays.
Main Results:
- Sixteen fish ARSs were successfully isolated from mud loach fish MARs.
- Most isolated ARSs exhibited higher transformation efficiency than the positive control in yeast.
- One clone, pURY19-ARS223, showed 18-fold higher efficiency in E. coli.
- Sequence analysis revealed conserved ARS elements, potential hairpin structures, and MAR motifs.
- All identified ARSs demonstrated binding to the nuclear matrix in vitro.
Conclusions:
- This study provides the first identification and characterization of ARSs in fish.
- Fish ARSs are associated with MARs, suggesting a conserved genomic organization.
- The identified ARS223 is a promising candidate for enhancing expression vectors used in fish cell transfection and gene expression studies.