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Gene A protein interacting with recombinant plasmid DNAs containing 25-30 b.p. of the phi X174 replication origin
Abstract:
Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the 30 b.p. origin region of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR). Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe. Approximate lengths of the inserts were determined by restriction enzyme analysis. Exact length and orientation of each insert was determined by DNA sequencing. Plasmid DNA with an insert homologous to the first 25 b.p. of the phi X174 origin is not nicked by the gene A protein. However, plasmid DNA containing the 27 b.p. fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b.p. or the complete 30 b.p. conserved origin region of the isometric phages.
Insights
Researchers synthesized DNA fragments of bacteriophage phi X174 origin and cloned them. The gene A protein nicked DNA containing 27-30 base pairs of the origin, but not 25 base pairs.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The bacteriophage phi X174 origin of replication is a critical regulatory region.
- Understanding the minimal sequence required for replication initiation is essential for viral DNA replication studies.
Purpose of the Study:
- To determine the precise DNA sequence requirements for recognition and nicking by the bacteriophage phi X174 gene A protein.
- To identify the minimal functional origin of replication for bacteriophage phi X174.
Main Methods:
- Synthetic oligodeoxyribonucleotides were used to construct double-stranded DNA fragments.
- Fragments were cloned into the kanamycin resistance gene of pACYC177.
- Antibiotic selection and filter-hybridization were employed for transformant selection.
- Restriction enzyme analysis and DNA sequencing were used to characterize the inserts.
Main Results:
- Plasmid DNA with a 25 bp insert homologous to the phi X174 origin was not nicked by the gene A protein.
- Plasmid DNA containing 27 bp, 28 bp, or the complete 30 bp origin fragment was efficiently nicked by the gene A protein.
- The orientation of the 27 bp fragment did not affect nicking by the gene A protein.
Conclusions:
- The minimal functional origin of replication for bacteriophage phi X174 requires at least 27 base pairs.
- The gene A protein's nicking activity is highly sensitive to the precise length of the origin sequence.