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M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates
Abstract:
M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.
Insights
New M13 cloning vectors enable the selection of DNA sequences that initiate DNA synthesis on single-stranded templates. These vectors facilitate the identification and genetic analysis of DNA replication origins and initiation determinants.
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- M13 cloning vectors are essential tools for molecular biology research.
- Understanding DNA replication origins is crucial for various biological processes.
- Previous methods for studying initiation determinants were limited.
Purpose of the Study:
- To develop novel M13 cloning vectors for selecting DNA sequences that direct DNA synthesis initiation on single-stranded templates.
- To identify and characterize DNA sequences responsible for initiating DNA replication.
- To enable functional dissection of replication origins and genetic analysis of initiation determinants.
Main Methods:
- Construction of M13 deletion mutants with engineered receptor sites for foreign initiation determinants.
- Introduction of EcoRI linker sequences into deletion sites of M13 mutants.
- Cloning of DNA fragments from bacteriophage G4 RF and Co1E1 DNA into M13 vectors.
- Selection of clear-plaque isolates based on restored phage growth and plaque morphology.
- Analysis of initiation capability by inserting DNA fragments into M13 vectors.
Main Results:
- Developed M13 cloning vectors derived from deletion mutants defective in single-strand to replicative form conversion.
- Successfully introduced receptor sites for foreign initiation determinants into M13 vectors.
- Identified specific DNA sequences from bacteriophage G4 RF and Co1E1 DNA that restore normal phage growth and plaque type.
- Selectively cloned the Co1E1 HaeIII-E fragment using the M13 delta E101 vector.
- Demonstrated that both H and L strands of the Co1E1 HaeIII-E fragment contain initiation determinants.
Conclusions:
- The developed M13 cloning vectors provide a novel method for selecting DNA sequences with initiation capabilities.
- These vectors facilitate the functional dissection of replication origins and identification of DNA sequences involved in discontinuous DNA synthesis.
- Plaque morphology serves as a rapid indicator for assessing initiation capability and performing genetic analysis of initiation determinants.