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Identification of the E. coli dnaJ gene product
Abstract:
We have previously shown that a mutation (groPC259) in the E. coli dnaJ gene renders the cell inviable at high temperatures and arrests bacteriophage lambda DNA replication at all temperatures (Sunshine et al., 1977). We have isolated lambda dnaJ+ transducing phages both by in vitro cloning and by abnormal excision of a lambda dnaK transducing phage integrated near the dnaJ locus. The dnaJ gene product has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells by lambda dnaJ+ derivative phages. It is a polypeptide chain with an apparent molecular weight of 37,000-daltons. This has been verified by the fact that a transducing phage carrying an amber mutation in the dnaJ gene fails to induce the synthesis of the 37,000-dalton polypeptide chain upon infection of sup+ bacteria, but does so upon infection of supF or supD bacteria.
Insights
A mutation in the E. coli dnaJ gene causes cell inviability and halts bacteriophage lambda DNA replication. Researchers identified the dnaJ gene product as a 37,000-dalton polypeptide chain.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- A mutation in the E. coli dnaJ gene (groPC259) leads to inviability at high temperatures and arrests bacteriophage lambda DNA replication.
- Previous research established the critical role of the dnaJ gene in cellular processes and viral replication.
Purpose of the Study:
- To isolate lambda dnaJ+ transducing phages.
- To identify and characterize the dnaJ gene product.
- To confirm the identity of the dnaJ gene product using genetic complementation.
Main Methods:
- Isolation of lambda dnaJ+ transducing phages via in vitro cloning and abnormal phage excision.
- Identification of the dnaJ gene product using SDS-PAGE following infection of UV-irradiated E. coli.
- Verification using amber mutations in the dnaJ gene and different suppressor strains (sup+, supF, supD).
Main Results:
- Successfully isolated lambda dnaJ+ transducing phages.
- Identified a 37,000-dalton polypeptide as the dnaJ gene product.
- Confirmed the dnaJ gene product's identity by its absence in sup+ bacteria and presence in supF/supD bacteria infected with an amber mutant dnaJ phage.
Conclusions:
- The dnaJ gene product is a 37,000-dalton polypeptide.
- This polypeptide is essential for E. coli viability and bacteriophage lambda DNA replication.
- The genetic and biochemical data confirm the identification of the dnaJ gene product.