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PubMed1994

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.

Bacteriophage lambda↗
PubMed1985

The DNA between Rz and cosR in bacteriophage lambda is nonessential.

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.

Bacteriophage lambda↗
PubMed1984

Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene.

A set of plasmids has been constructed that carry a constitutive lamB gene (LamBc phenotype) from Escherichia coli and that confer functional phage lambda receptors to bacteria other than E. coli. This E. coli LamBc strain has been selected to escape both maltose-inducible and glucose-repressible control. Constitutivity results from an IS-3 insertion, carrying a mobile promoter, proximal to lamB. The LamBc DNA has been cloned into both broad and narrow host-range plasmids, and the resulting pTROY plasmids have been transferred to diverse bacteria. Both Salmonella typhimurium/pTROY and Klebsiella pneumoniae/pTROY strains efficiently adsorb phage lambda; Pseudomonas aeruginosa/pTROY strains do not. Introduction of a functional E. coli LamB protein into foreign bacterial will allow these bacteria carrying pTROY plasmids to be infected by phage lambda recombinant DNA libraries, phage lambda::Tn insertion mutagenesis vectors, and in vivo lambda-packaged cosmids.

Bacterial Outer Membrane Proteins↗
PubMed1986

Direction of travel of RecBC recombinase through bacteriophage lambda DNA.

We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction. The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange. In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end.

Bacteriophage lambda↗
PubMed1981

Cloning and characterization of a transcription termination signal in bacteriophage lambda unresponsive to the N gene product.

The pHA10 plasmid was designed for the cloning and selection of transcriptional termination signals. This study demonstrates the use of pHA10 as a cloning vehicle in the selection and characterization of an N-unresponsive terminator. Following the random cloning of lambda DNA, and N-unresponsive transcription terminator was isolated. Hybridization of in vivo 32P-labeled RNA to various DNA fragments, using the Southern (1975) blotting technique, revealed that the transcriptional barrier that cannot be overridden in the presence of N is located close to the end of gene J of lambda DNA. The terminator determines the 3'-end of the pL operon and prevents the transcription of the anti-sense DNA strand of lambda late genes. The primary structure and other characteristics of this terminator are now under investigation.

Bacteriophage lambda↗
PubMed1995

The Escherichia coli heat shock response and bacteriophage lambda development.

The Escherichia coli/bacteriophage lambda genetic interaction system has been used to uncover the existence of various biological machines. The starting point of all these studies was the isolation and characterization of E. coli mutants that blocked lambda growth, and the corresponding lambda compensatory mutations. In this manner, the lambda N-promoted transcriptional anti-termination machine was discovered composed of the NusA/NusB/NusE/NusG host proteins. In addition, the DnaK and GroEL chaperone machines were discovered composed of DnaK/DnaJ/GrpE and GroES/GroEL heat shock proteins. The individual members of the DnaK and GroEL chaperone machines have been conserved throughout evolution in both function and structure. Their biological roles include a direct involvement in lambda DNA replication and morphogenesis, the protection of proteins from aggregation, the disaggregation of various protein aggregates, the manipulation of protein structure and function, as well as the autoregulation of the heat shock response. The evolution of lambda to extensively rely on the status of the heat shock response of E. coli is likely linked to its lytic versus lysogenic choice of lifestyle. The bacteriophage T4 gp31 protein has been purified and shown to substitute for many of GroES' co-chaperonin activities.

Bacteriophage lambda↗
PubMed1974

Bacteriophage lambda having EcoRI endonuclease sites only in the nonessential region of the genome.

A derivative of lambda b221 that has lost by mutation all EcoRI restriction sites has been isolated by alternative growth on restrictive and nonrestrictive strains. It has an efficiency of plating equal to 1 on the restrictive strain. Genetic cross of this bacteriophage with lambda plac5 imm21 gave rise to recombinants of intermediate restricting ratios. The analysis of the EcoRI endonuclease-cleaved DNA by polyacrylamide gel electrophoresis, compared with the genetic results, has permitted identification of EcoRI endonuclease cleavage sites in the recombinants. The genotypes are: lambda plac5 CI857 sRIlambda3(0)sRIlambda2(0)sRIlambda1(0) and lambda plac5 CI857 sRIlambda2(0)sRIlambda1(0). The remaining cleavage sites, respectively, sRIlac sRIlambda4 and sRIlac sRIlambda4 sRIlambda3, are all located in a region nonessential for bacteriophage multiplication. The involvement of these mutant bacteriophages as vector for foreign genes are discussed.

Binding Sites↗
PubMed1991

Isolation and preliminary characterization of Escherichia coli mutants resistant to lethal action of the bacteriophage lambda P gene.

Both spontaneous and NTG-induced mutants of Escherichia coli 594 insensitive to the lethal action of lambda P gene were isolated and called rpl (resistant to P lethality). These mutants were of two types, showing different phenotypes. On type I rpl mutants, lambda cl- and lambda v1v3 did not plate, while lambda vir, lambda cl- c17, lambda imm434, and lambda imm21 did; plasmid pMR45 carrying the lambda P gene could not complement lambda imm21P- phage in type I mutants. On the other hand, the type II rpl mutants support the growth of all the above phages including lambda cl-. Neither type of rpl mutation affects growth of the bacteria.

Bacteriophage lambda↗