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Process of attachment of phi X174 parental DNA to the host cell membrane
Abstract:
The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.
Insights
Researchers isolated the phi X174 DNA-membrane complex from E. coli, identifying its location and a key protein. This complex is crucial for phi X174 DNA replication, acting as a template in the cytoplasmic membrane.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Bacteriophage phi X174 is a small, single-stranded DNA virus.
- Understanding viral DNA replication mechanisms is crucial in molecular biology.
Purpose of the Study:
- To isolate and characterize the phi X174 DNA-membrane complex.
- To elucidate the role of the membrane complex in phi X174 DNA replication.
Main Methods:
- Isopycnic sucrose gradient centrifugation
- Zone electrophoresis
- Pulse-labeling and chase experiments
Main Results:
- The phi X174 DNA-membrane complex was isolated from E. coli.
- Two distinct membrane fractions (intermediate and cytoplasmic) containing the complex were identified.
- A specific 80,000 MW membrane-bound protein was found in the complex.
- Radioactivity from pulse-labeled DNA migrated from the intermediate to the cytoplasmic fraction.
- Parental replicative form I (RFI) DNA was associated with the membrane complex.
Conclusions:
- The phi X174 DNA-membrane complex plays a role in viral DNA replication.
- Conversion of single-stranded DNA to RFI occurs at a wall/membrane adhesion region.
- The cytoplasmic membrane fraction is involved in serving as a template for progeny DNA replication.