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[Autonomic replication of plasmids containing DNA fragments from rat liver alpha-polymerase complex]
Abstract:
We have cloned a number of plasmids pori, containing DNA fragments derived from rat liver alpha-polymerase complex. Colony hybridization identified 30 clones with homologies to origin SV40. Five clones of pori were examined in transfected V79 hamster cells. Plasmid DNA was isolated at time intervals after transfection and screened by the CfuI resistance assay or by the bromodeoxyuridine substitution. We have identified two plasmids pori22 and pori32 that can support plasmid replication in mammalian cells. Thus, at least two DNA fragments from rat liver alpha-polymerase complex act as origins of replication in a mammalian system. Five days after transfection of pori32 Hirt the supernatant contains the molecules of RNA which hybridize with pUC19. Our results indicated that pori32 has an origin of replication and promotor of transcription.
Insights
Researchers identified two rat liver DNA fragments that function as replication origins in mammalian cells. These fragments, pori22 and pori32, also show potential for transcriptional promotion, advancing our understanding of DNA replication and gene expression.
Area of Science:
- Molecular Biology
- Genetics
- Mammalian Cell Biology
Background:
- The rat liver alpha-polymerase complex is crucial for DNA replication.
- Understanding origins of replication is key to controlling DNA synthesis in mammalian systems.
Purpose of the Study:
- To identify DNA fragments from the rat liver alpha-polymerase complex that can function as origins of replication in mammalian cells.
- To investigate the potential of these fragments as transcriptional promoters.
Main Methods:
- Cloning of plasmid DNA fragments from rat liver.
- Colony hybridization to identify homologous sequences to SV40 origin.
- Transfection of V79 hamster cells with identified plasmids.
- Screening for plasmid replication using CfuI resistance assay and bromodeoxyuridine substitution.
- RNA isolation and hybridization to detect transcriptional activity.
Main Results:
- Thirty plasmid clones (pori) showed homology to the SV40 origin.
- Two plasmids, pori22 and pori32, successfully supported plasmid replication in transfected mammalian cells.
- pori32 demonstrated an origin of replication and a promoter of transcription, evidenced by RNA hybridization.
Conclusions:
- At least two DNA fragments from the rat liver alpha-polymerase complex function as functional origins of replication in a mammalian system.
- The plasmid pori32 possesses both replication origin and transcriptional promoter activities.
- These findings contribute to the understanding of DNA replication mechanisms and gene regulation in mammalian cells.