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PubMed1985

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.

Animals↗
PubMed1994

Bacteriophage lambda N-dependent transcription antitermination. Competition for an RNA site may regulate antitermination.

Bacteriophage lambda controls the expression of its early genes in a temporal manner by a series of transcription termination and antitermination events. This antitermination requires the lambda N protein as well as host proteins called Nus, and cis-acting sites called nut. Following transcription of the nut site, N and Nus proteins bind to the nut RNA and modify the transcription complex to a termination-resistant form. The nut site is a composite of at least two components; one is the boxB hairpin structure which interacts with N. The other is boxA, a nine-nucleotide sequence upstream of boxB. To understand more about the formation of the antitermination complex, we have characterized the effect of point mutations in and deletions of boxA on antitermination. Point mutations in boxA were found to either enhance or reduce N-mediated antitermination. Several boxA deletions, on the other hand, had little effect on antitermination other than to eliminate the requirement for the NusB host protein. To explain these observations, we propose that at least two factors compete to interact with boxA, NusB and an inhibitor of the antitermination reaction. In addition, we propose that NusB is required to prevent the inhibitor from binding at boxA. The results with various nusB and boxA mutations can be explained by this model of competition between NusB and an inhibitor for boxA RNA.

Bacterial Proteins↗
PubMed1993

Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.

Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.

Bacteriophage lambda↗
PubMed2002

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein.

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.

Amino Acid Motifs↗
PubMed1999

Regulation of bacteriophage lambda development by guanosine 5'-diphosphate-3'-diphosphate.

On infection of its host, Escherichia coli, bacteriophage lambda can follow one of two alternative developmental pathways: lytic or lysogenic. Here we demonstrate that the "lysis-versus-lysogenization" decision is influenced by guanosine tetraphosphate (ppGpp), a nucleotide that is synthesized in E. coli cells in response to amino acid or carbon source starvation. We found that the efficiency of lysogenization is the highest at ppGpp concentrations somewhat higher than the basal level; too low and too high levels of ppGpp result in less efficient lysogenization. Maintenance of the already integrated lambda prophage and phage lytic development were not significantly influenced in the host lacking ppGpp. We found that the level of HflB/FtsH protease, responsible for degradation of the CII protein, an activator of "lysogenic" promoters, depends on ppGpp concentration. The highest levels of HflB/FtsH was found in bacteria lacking ppGpp and in cells bearing increased concentrations of this nucleotide. Using lacZ fusions, we investigated the influence of ppGpp on activities of lambda promoters important at the stage of the lysis-versus-lysogenization decision. We found that each promoter is regulated differentially in response to the abundance of ppGpp. Moreover, our results suggest that the cAMP level may influence ppGpp concentration in cells. The mechanism of the ppGpp-mediated control of lambda development at the stage of the lysis-versus-lysogenization decision may be explained on the basis of differential influence of guanosine tetraphosphate on activities of p(L), p(R), p(E), p(I), and p(aQ) promoters and by dependence of HflB/FtsH protease level on ppGpp concentration.

ATP-Dependent Proteases↗
PubMed1988

Preparation of bacteriophage lambda DNA using the TL-100 ultracentrifuge.

A procedure for the preparation of DNA from bacteriophage lambda is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is of good purity and is easily cuttable by restriction enzymes.

Bacteriophage lambda↗
PubMed1979

Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.

Bacteriophage lambda↗
PubMed1981

The R gene product of bacteriophage lambda is the murein transglycosylase.

The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage lambda R and lambda R+ were compared by polyacrylamide gel electrophoresis. lambda R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 KD corresponding to the molecular weight of murein transglycosylase - a bacteriolytic enzyme from lambda lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was undetectable in induced cultures of E. coli W3350 su degrees (lambda cI857 Ram 5) and C600 (lambda cI857 acR301), while it was present in a lambda Rz mutant lysate. We conclude that the transglycosylase is the R gene product.

Bacteriophage lambda↗
PubMed1993

A cryptic promoter in the O(R) region of bacteriophage lambda.

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

Bacteriophage lambda↗
PubMed1995

Transcription termination signals in the nin region of bacteriophage lambda: identification of Rho-dependent termination regions.

The approximately 3-kb nin region of bacteriophage lambda, located between genes P and Q contains transcription termination signals as well as 10 open reading frames. Deletions in the nin region frees phage growth from dependence on the lambda-encoded N-transcription antitermination system, conferring a Nin phenotype (N-independence). A subregion of nin, roc, is defined by a 1.9-kb deletion (delta roc) which partially frees lambda growth from the requirement for N antitermination. The roc region has strong transcription termination activity as assayed by a plasmid-based terminator testing system. We report the following features of the roc region: the biologically significant terminators in the roc region are Rho dependent, deletion analysis located the biologically significant termination signals to a 1.2 kb-segment of roc, and analysis of other deletions and point mutations in the roc region suggested at least two biologically significant regions of termination, tR3 (extending from bp 42020 to 42231) and tR4 (extending from bp 42630 to 42825).

Bacteriophage lambda↗
PubMed1983

Separate sites for binding and nicking of bacteriophage lambda DNA by terminase.

The cohesive end site (cos) is the site of action of bacteriophage lambda terminase, the enzyme that introduces staggered nicks to generate the 12-base cohesive ends of mature lambda DNA. Deletion mutations that remove the lambda cohesive end sequence have been isolated after in vitro mutagenesis. The deletions were obtained by digesting the DNA of a cos duplication phage with S1 nuclease to remove the cohesive ends and adjacent base pairs, followed by blunt end ligation and DNA packaging into phage particles. cos2 is the result of a 22-base-pair deletion that exactly removes the segment of rotational symmetry that includes the cohesive end sequence. The cos2 mutation abolishes nicking by terminase but does not affect terminase binding. We conclude that cos contains two sites that interact with terminase: cosN, the nicking site; and cosB, a binding site for terminase.

Bacteriophage lambda↗