PubMed · 8959715
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D Dottavio. 1996. Epitope mapping using phage-displayed peptide libraries.. https://doi.org/10.1385/0-89603-375-9%3A181
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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.
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.
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.