Phagemid DNA Research Articles

13] Site-directed mutagenesis of single-stranded and double-stranded DNA by phosphorothioate approach

Publisher Summary Oligonucleotide-directed mutagenesis allows the introduction of almost any precisely defined mutation into a cloned, sequenced gene. The mutation may comprise single or multiple mismatches or it may involve the insertion or deletion of a large number of bases. The chapter describes a number of methods for the efficient production of site-directed mutations. The gapped duplex, uracil-containing template, and coupled primer approaches have all been used to improve the basic method described in detail. However, these methods are limited to protocols using single-stranded vectors, involve the transfection of heteroduplex DNA, and very often require plaque purification steps. The original phosphorothioate-based mutagenesis method has undergone a number of improvements and has several advantages over the other methods addressed in the chapter. Finally, the phosphorothioate approach is not limited to the use of single-stranded or phagemid DNA but has been extended to double-stranded DNA vectors.

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.

Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85

Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.

A surface expression vector for antibody screening

To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili. Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG. Ab::plll was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences. It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen. The phagemid DNA is even smaller than that ofwt fd phages and can easily be propagated in plasmid form. Most importantly, its Ab:: pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants. After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.

Resolution of sequencing ambiguities: a universal FokI adapter permits Maxam-Gilbert re-sequencing of single-stranded phagemid DNA

We propose a method to resolve ambiguities encountered when single-stranded (ss) phagemid DNA templates are sequenced by the dideoxy method. A single oligodeoxyribonucleotide (oligo) is synthesized with the following features : ( i) the 20 nucleotides (nt) at the 5′ -end form a double-stranded hairpin containing a FokI restriction site, exactly as previously described by Podhajska and Szybalski [Gene 40 (1985) 175–182]; ( ii)the 23 nt at the 3′-end hybridize to the (+)strand of ss phagemid DNA in the region complementary to the M 13 universal sequencing primer. In a simple one-tube set of reactions, ss phagemid DNA is annealed to this oligo, cleaved by FokI at a unique site outside the vector multiple cloning site and then labelled at this unique site by Klenow polymerase and [α- 32P]dCTP. These reactions provide a convenient route by which Maxam-Gilbert chemical degradation sequencing methods can be used to resolve ambiguities encountered in the dideoxy-sequencing of a unidirectional deletion series already prepared in popular phagemid vectors. A single oligo allows labelling of all members of a deletion series. A second universal oligo allows the same set of reactions to be applied to inserts cloned into ( −)strand phagemids.

Lambda phagemids and their transducing properties

Two recombinant λ DNAs, λgt : ̈ pMB9 and λNM : ̈ pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lgt::pMB9 phage producing A repressor cIts ranges from 10 −7 to 10 −2 transductant colonies per input phage, depending on the temperature and strain used, while λNM::pBR322 phage carrying imm 21 transduces with a frequency of up to 1. This means that each λNM : ̈ pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with λgt : ̈ pMB9 was obtained when E. coli cells lysogenic for λ were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all λgt : ̈ pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from λNM : ̈ pBR322 has not been observed.