Plasmid vectors for positive selection of DNA inserts controlled by the lambda pL promoter, repressor and...

Area of Science:

• Molecular Biology
• Bacteriophage Genetics

Background:

• Hybrid plasmids formed from pBR322/pOP203-3 and lambda DNA fragments exhibit host lethality.
• Bacterial host death is linked to the expression of a specific lambda gene, likely the kil function.

Purpose of the Study:

• To investigate the mechanisms of host lethality mediated by lambda DNA in hybrid plasmids.
• To develop novel vector plasmids for direct positive selection of cloned DNA fragments.

Main Methods:

• Construction of hybrid plasmids using pBR322/pOP203-3 and lambda EcoRI-D fragments.
• Analysis of surviving bacterial hosts to identify genetic modifications in plasmids (deletions, insertions, point mutations).
• Development and characterization of new vector plasmids (pKL1, pHA10) for positive selection.

Main Results:

• Expression of a lambda gene within hybrid plasmids leads to bacterial host death.
• Surviving plasmids show deletions of the lambda kil gene or transcriptional blocking insertions.
• The constructed vectors pKL1 and pHA10 facilitate direct positive selection of cloned fragments.
• These vectors are effective for cloning N-unresponsive termination signals using BamHI and its isoschizomers.

Conclusions:

• The lambda kil gene function is responsible for host lethality in specific hybrid plasmids.
• Vector plasmids pKL1 and pHA10 provide a direct positive selection system for molecular cloning.
• These vectors are valuable tools for studying gene regulation, particularly N-unresponsive termination signals.