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Abstract:
We have cloned the phage Mu A gene, with and without the gene ner, under the control of the pL promoter of phage lambda in a multicopy plasmid vector. We demonstrate that plasmid-carrying cells are able to support growth of superinfecting Mu A am phages in a temperature-dependent fashion in a host strain carrying a defective lambda prophage which specifies the cI857-coded lambda repressor. In addition, we show that the presence of the ner gene reduces the efficiency of plating of the superinfecting phage. Analysis of proteins specified by the cloned Mu fragments indicates that two proteins, 70 and 33 kDal, are synthesized. The level of synthesis, compared to that of the vector-encoded beta-lactamase, was found to increase with temperature. This indicates that their transcription is driven by the pL promoter. The Mr of the 70-kDal protein is identical to that previously observed for pA.
Insights
Researchers cloned the phage Mu A gene and ner gene using a plasmid vector. The presence of the ner gene reduced phage growth, demonstrating its regulatory role in phage Mu replication.
Area of Science:
- Molecular Biology
- Bacteriophage Genetics
- Gene Expression Regulation
Background:
- Phage Mu is a versatile temperate bacteriophage with a complex replication cycle.
- Understanding the regulatory mechanisms of phage Mu gene expression is crucial for deciphering its life cycle.
- The roles of the A gene and ner gene in phage Mu replication require further elucidation.
Purpose of the Study:
- To clone and express the phage Mu A gene and the ner gene in a heterologous system.
- To investigate the functional role of the cloned genes in supporting phage Mu replication.
- To analyze the impact of the ner gene on the efficiency of phage Mu infection.
Main Methods:
- Cloning of phage Mu A and ner genes into a multicopy plasmid vector under the control of the lambda phage pL promoter.
- Transformation of E. coli host strains carrying a defective lambda prophage.
- Assessing the growth of superinfecting Mu A am phages at different temperatures.
- Analysis of protein synthesis using SDS-PAGE and comparison with beta-lactamase expression.
Main Results:
- Plasmid-carrying cells supported the growth of superinfecting Mu A am phages in a temperature-dependent manner.
- The presence of the ner gene significantly reduced the efficiency of plating of superinfecting phages.
- Analysis revealed the synthesis of two proteins (70 and 33 kDa) from the cloned Mu fragments, with increased expression at higher temperatures, indicating pL promoter-driven transcription.
- The 70 kDa protein's molecular weight matched that of the previously identified pA protein.
Conclusions:
- The cloned phage Mu A gene, under pL promoter control, can functionally complement Mu A am mutations.
- The ner gene acts as a negative regulator, inhibiting the growth of superinfecting Mu A phages.
- This study provides evidence for temperature-dependent transcriptional regulation of phage Mu genes via the lambda pL promoter.