Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts.

M A Alting-Mees; J M Short

doi:10.1016/0378-1119(93)90256-3

Home/Archive/PubMed

PubMed · 7506693

Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts.

M A Alting-Mees·J M Short

View original source ↗Export metadata ↓

Abstract

A new class of hybrid vectors, 'polycos' vectors, incorporate a phage lambda cos site and filamentous phage origin to allow high-efficiency cloning via in vitro lambda packaging extracts. The head-filling mechanism of cos site recognition by lambda packaging proteins permits concatemers of several of these small cos-containing vectors to be packaged per phage particle. Excision of vector monomers after infection is accomplished via a lambda ZAP-like M13 excision process. This system has the advantage of adapting high-efficiency lambda packaging extracts to M13 and phagemid cloning vectors.

Explore related subjects

Bacteriophage M13 Bacteriophage lambda Base Sequence Cloning, Molecular Cosmids DNA Restriction Enzymes Epitopes Genetic Vectors Molecular Sequence Data Recombinant Fusion Proteins Journal Article Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

M A Alting-Mees, J M Short. 1993-12-27. Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts.. https://doi.org/10.1016/0378-1119(93)90256-3

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Unit-length, single-stranded circular DNAs of both polarity of begomoviruses are generated in Escherichia coli harboring phage M13-cloned begomovirus genome with single copy of replication origin.

Replication of genomic DNAs of plant-pathogenic begomoviruses has been demonstrated in prokaryotes, which supported the possibility of analyzing DNA replication process of begomoviruses in bacteria. However, previous studies indicated that the replication of begomovirus DNAs in prokaryotes requires tandem constructs of viral genomes with at least two copies of the origin of replication (ori). In this study, phage M13 vector harboring the unit-length genome with only a single copy of ori of a mono-partite begomovirus, Ageratum yellow vein virus PD isolate (AYVV-[PD]), was constructed and used to investigate the replication of AYVV-[PD] DNAs in Escherichia coli. The generation of single-stranded, circular DNAs (sscDNAs) corresponding to the unit-length AYVV-[PD] genome of both polarity was observed and verified. Replication-associated (Rep) protein of AYVV-[PD] was detected only in bacteria generating the corresponding sscDNAs, whereas disruption of the Rep gene abolished the phenomenon. The results suggested that a single copy of ori is sufficient for the prokaryotes to support the generation of unit-length, genomic sscDNAs of begomoviruses, which requires the presence of functional Rep protein.

Bacteriophage M13↗

PubMed2006

Diagnosing viruses by the rolling circle amplified synthesis of DNAzymes.

Circular DNA is used as a template for the amplified detection of M13 phage ssDNA by a rolling circle amplification (RCA) process that synthesizes DNAzyme chains, thus enabling the colorimetric or chemiluminescent detection of the analyte.

Bacteriophage M13↗

PubMed2006

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein.

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.

Bacteriophage M13↗