A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect

Area of Science:

• Molecular Biology
• Genetics

Background:

• The pBR322 plasmid is a commonly used cloning vector.
• Plasmid copy number is tightly regulated by specific RNA molecules and proteins.
• Transposon Tn5 can be used for random mutagenesis to study gene function.

Purpose of the Study:

• To investigate the mechanism behind the high copy number phenotype of the pLO88 plasmid mutant.
• To identify the specific Tn5 insertion site and its role in altering pBR322 copy number regulation.
• To elucidate the molecular interactions leading to increased plasmid replication at elevated temperatures.

Main Methods:

• Random mutagenesis of pBR322 using transposon Tn5.
• Isolation and characterization of high copy number plasmid mutants.
• Nucleotide sequencing of the Tn5 insertion site.
• Deletion analysis of Tn5 sequences.
• Analysis of RNA transcripts involved in plasmid copy number control.

Main Results:

• A high copy number plasmid mutant, pLO88, was isolated.
• Tn5 insertion occurred within an open reading frame encoding a negative regulator of pBR322 copy number.
• The high copy number phenotype was dependent on Tn5 insertion and the presence of specific Tn5 sequences.
• An outwardly directed Tn5 promoter initiated a transcript (RNA X) that interfered with normal plasmid replication control at temperatures >= 37°C.

Conclusions:

• The high copy number of pLO88 is caused by a Tn5 insertion that disrupts a negative regulator of pBR322.
• Specific Tn5 sequences and an outward-directed promoter are crucial for the observed phenotype.
• A novel regulatory mechanism involving RNA X interfering with RNA I/RNA II interactions at elevated temperatures was identified.