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Related Experiment Videos

In vivo packaging of bacteriophage lambda monomeric chromosomes

L C Thomason1, D S Thaler, M M Stahl

  • 1Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.

Journal of Molecular Biology
|March 21, 1997
PubMed
Summary

This study investigates lambda DNA packaging, revealing that cellular nucleases may prevent monomer packaging in vivo. A specific phage mutant (plm1) significantly enhances in vivo monomer packaging, suggesting replication intermediates can be substrates.

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Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • A discrepancy exists between in vivo and in vitro requirements for lambda DNA packaging, with concatemers needed in vivo but monomers packaging in vitro.
  • Cellular nucleases might degrade DNA intermediates, limiting in vivo monomer packaging.
  • Previous studies showed enhanced monomer packaging in ExoV- hosts.

Purpose of the Study:

  • To investigate the role of cellular nucleases in lambda DNA packaging in vivo.
  • To identify genetic factors influencing the packaging of monomeric lambda DNA.
  • To elucidate the mechanism of lambda DNA packaging, particularly concerning concatemers and monomers.

Main Methods:

  • Comparative packaging assays in wild-type and mutant bacterial hosts (ExoV-, sbcB, sbcC).

Related Experiment Videos

  • Isolation and characterization of a phage mutant (plm1) with increased in vivo monomer packaging.
  • Genetic mapping, sequencing, and structural analysis of the plm1 mutation in the Cro protein.
  • Density transfer experiments to assess DNA synthesis and replication intermediates in packaged DNA.

    • Main Results:

    • Enhanced packaging of lambda monomers was observed in an ExoV- host.
    • A novel phage mutant, plm1, exhibited a 1000-fold increase in in vivo monomer packaging.
    • The plm1 mutation alters Ala29 to Ser in the Cro protein's DNA binding domain.
    • Packaging of both wild-type and plm1 lambda DNA was facilitated by limited DNA synthesis, but packaged chromosomes were not fully replicated concatemers.
    • Packaged DNA in plm1 mutants appeared to originate from replication intermediates, not concatemers formed by replication, recombination, or annealing at cos sites.

    Conclusions:

    • Cellular nucleases likely contribute to the requirement for concatemers in vivo by degrading monomeric DNA intermediates.
    • The plm1 mutation in the Cro protein enhances in vivo monomer packaging, suggesting a role for replication intermediates in packaging.
    • Packaging of lambda DNA may not strictly require canonical concatemers, and replication intermediates can serve as substrates, particularly in specific mutant contexts.