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PubMed1988

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column.

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.

Bacteriophage lambda↗

PubMed1997

A folded monomeric intermediate in the formation of lambda Cro dimer-DNA complexes.

The folding, dimerization and DNA binding equilibria of the bacteriophage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is substantially populated under conditions used for the formation of dimer-DNA complexes. Although Cro dimers are the only DNA-bound species observed in electrophoretic mobility shift assays, cooperativity in Cro-DNA binding isotherms shows that the predominant free protein species is monomeric at nanomolar concentrations. Micromolar dissociation constants for Cro dimers have been measured in the absence of DNA by sedimentation equilibrium and gel filtration chromatography. Denaturation of Cro dimers in the 10 to 100 micromolar concentration range by guanidine hydrochloride (GdnHCl) is well modeled as a two-state process, with folded dimers and unfolded monomers as the only significantly populated species. However, linear extrapolation of this composite unfolding and dimer dissociation free energy predicts a nanomolar dissociation constant in the absence of denaturant. This extrapolation is clearly inconsistent with the DNA binding and hydrodynamic measurements. Our interpretation of these results is that the monomeric species detected in DNA binding and hydrodynamic experiments is predominantly folded. The stability of the folded monomeric species can be calculated as the difference between the dimerization free energy determined from hydrodynamic measurements and the folding free energy extrapolated from GdnHCl denaturation. The calculated stability of the Cro F58W monomer is greater than that of the wild-type Cro monomer. Thus, residue 58, which makes critical intermolecular contacts across the dimer interface, is also involved in intramolecular stabilization of the monomeric intermediate.

Bacteriophage lambda↗

PubMed1998

Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity.

The overall affinity of the bacteriophage lambda Cro repressor for its operator DNA site is limited by dimer dissociation at submicromolar concentrations. Since Cro dimer-operator complexes form at nanomolar concentrations of Cro subunits where free dimers are rare, these dimers must bind with compensating high affinities. Previous studies of the covalent dimer Cro V55C suggest little change in DNA binding affinity even though the dimeric species is quantitatively populated; this is an apparent contradiction to the expectation of high intrinsic dimer-DNA affinity. In contrast to the disulfide linkage at the center of the dimer interface in Cro V55C, polypeptide linkers that join the two subunits allow single-chain Cro repressors to bind operator DNA with picomolar affinities. A series of five single-chain Cro repressors have been expressed from fused tandem cro genes. Each contains a peptide linker of 8-16 hydrophilic residues that connects the C-terminus of one subunit to the N-terminus of the next. All bind to operator DNA with at least 100-fold higher affinity than Cro V55C. Proteins containing the longest and shortest linkers have been purified and characterized in detail. Both exhibit similar CD spectra to wild-type Cro and enhanced thermal stability. Sedimentation equilibrium experiments show that single-chain Cro repressors do not associate at concentrations up to 30 microM. The rate of dissociation of Cro-DNA complexes is almost unchanged by covalent linkage. Biophysical characterization of Cro variants such as these, where DNA binding is uncoupled from subunit assembly, is necessary for a quantitative understanding of the structural and energetic determinants of DNA recognition in this simple model system.

Bacteriophage lambda↗

PubMed1991

Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like rap mutants, prevented lambda growth and were killed by transcription of cloned bar sites; (iii) sequencing a 1600 bp DNA fragment comprising both loci revealed an ORF located within the limits set by a complementation analysis and encoding a putative polypeptide of 21 kDa; (iv) cloning and sequencing of rap and pth(ts) mutant DNAs both revealed single nucleotide transitions from the wild type ORF sequence, resulting in Arg134 to His and Gly101 to Asp changes respectively. Analysis of plasmid-directed proteins identified a polypeptide of approximately 21 kDa; the N-terminal sequence, amino acid composition and isoelectric point of this protein match those expected from the ORF nucleotide sequence. We propose that Pth activity, directly or indirectly, is the target for lambda bar RNA leading to rap cell death.

Bacteriophage lambda↗

PubMed1981

Resolution of cointegrates between transposons gamma delta and Tn3 defines the recombination site.

Transposition of the genetically related insertion elements gamma delta and Tn3 is thought to involve two steps. In the case of transposition from one replicon to another, the first step is fusion of the parent and target replicons with the element appearing in direct orientation at the two junctions. In a subsequent reaction, the cointegrate structure is resolved via a site-specific recombination event. I have constructed two plasmids, each carrying segments of gamma delta and Tn3, that contain the internal resolution site. The tnpR gene product encoded by either Tn3 or gamma delta mediates intramolecular recombination between these two sites. The product of this recombination is a hybrid region that contains gamma delta and Tn3 sequences fused at the point of crossover. DNA sequence analysis of such recombinants indicates that the recombination occurs within a 19-base-pair (bp) region of exact homology between gamma delta and Tn3. The site lies in the 160-bp center intercistronic region, 50 bp before the beginning of the tnpA gene. My results therefore suggest a model for the coupled regulation of the repressor (tnpR) and the transposase (tnpA) genes and site-specific recombination of transposition intermediates. The Tn3/gamma delta recombination system and bacteriophage lambda integration are compared.

Bacteriophage lambda↗

PubMed1988

Recognition of ill-defined signals in nucleic acid sequences.

A set of programs has been developed for the definition and handling of nucleic acid sequence consensus information. The sequences of known genetic control signals are combined in a matrix. The origins and positions of the signals are recorded. Old matrices can be updated dynamically: new signals are included and obsolete ones deleted. Matrices of several different types are computed optionally. Several of these matrices can be combined to find possible new signals. The use of matrices allows the exact quantification of signal qualities. The described programs are part of a program library named GENEXPERT. Application examples given are the search for tRNA genes and the search for promoters in the bacteriophage lambda genome.

Algorithms↗

PubMed1995

Circuit simulation of genetic networks.

Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. Because of the many parallels in the function of these biochemically based genetic circuits and electrical circuits, a hybrid modeling approach is proposed that integrates conventional biochemical kinetic modeling within the framework of a circuit simulation. The circuit diagram of the bacteriophage lambda lysislysogeny decision circuit represents connectivity in signal paths of the biochemical components. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda.

Bacteriolysis↗

PubMed1992

Effect of Escherichia coli nusG function on lambda N-mediated transcription antitermination.

The Escherichia coli Nus factors act in conjunction with the bacteriophage lambda N protein to suppress transcription termination on the lambda chromosome. NusA binds both N and RNA polymerase and may also interact with other Nus factors. To search for additional components of the N antitermination system, we isolated host revertants that restored N activity in nusA1 mutants. One revertant, nusG4, was mapped to the rif region of the E. coli chromosome and shown to represent a point mutation near the 3' end of the nusG gene. The nusG4 mutation also suppressed nusE71 but not nusASal, nusB5, nusC60 (rpoB60), or nusD026 (rho026). However, nusG+ expressed from a multicopy plasmid suppressed nusD026 and related rho mutants for both lambda and phage T4 growth. These results suggest that NusG may act as a component of the N antitermination complex. In addition, the data imply a role for NusG in Rho-dependent termination.

Bacterial Proteins↗

PubMed1992

The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase.

The bacteriophage lambda gene Q transcription antiterminator modifies RNA polymerase during an extended pause in elongation at nt +16 and +17 of the phage late gene promoter transcript. We show here that Q binds a specific DNA site between the -10 and -35 elements of the promoter as it interacts with the enzyme. We show that the pause must reflect a specialized elongation structure that is receptive to modification by Q, because Q does not bind to RNA polymerase stopped artificially after transcribing 16 nt of mutant DNA that does not encode the natural pause. Footprinting shows that RNA polymerase in the paused complex makes distinctive interactions with DNA in the region where Q binds; binding of Q, in turn, changes the footprint both at the Q-binding site and in the transcription bubble. Binding of Q to the paused transcription complex is stabilized by the transcription factor NusA, as expected from the dependence of lambda Q-mediated antitermination on NusA.

Bacteriophage lambda↗

PubMed1980

The lambda phage att site: functional limits and interaction with Int protein.

Site specific integrative recombination of bacteriophage lambda involves unequal partners. The minimal phage att site is composed of approximately 240-base pairs and four distinct binding sites for Int protein, at least three of which are crucial for function. This 'donor site' recombines efficiently with a smaller 'recipient site' that lacks the extensive interactions with Int protein.

Attachment Sites, Microbiological↗

PubMed1983

Expression of the phage lambda recombination genes exo and bet under lacPO control on a multi-copy plasmid.

The bacteriophage lambda genes exo and bet, whose products (lambda exonuclease and beta protein, respectively; Red phenotype) mediate homologous recombination of lambda phages, have been cloned under lacPO lacIq control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in lambda exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of lambda Red- phages in vivo was similarly inducible. Only one out of 25 bet delta plasmids (constructed by a variety of in vitro techniques) expressed lambda exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. When lambda bet3 phages (Exo-Bet-) infected IPTG-induced recA3 bacteria containing exo+bet+ plasmids, recombination frequencies were not more than twice those typical for infection of plasmid-free recA3 cells with exo+bet+ phages, even in the case of IPTG induction sufficient to elevate the production of lambda exonuclease about 100-fold. Even when plasmid induction was delayed till as late as 50 min after infection, recombination was significant. Preliminary experiments suggest that these plasmids encode a polypeptide with Gam activity that corresponds to the 98-amino acid "shorter" open reading frame assigned to gam by Sanger et al. (J. Mol. Biol. 162 (1982) 729-773).

Bacteriophage lambda↗

PubMed1996

[Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein].

Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag. The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively. The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg. Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles.

Amino Acid Sequence↗

PubMed1982

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome.

A recombinant bacteriophage lambda, lambda argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.

Bacterial Proteins↗

PubMed1991

3'-end formation at the phage lambda tR1 rho-dependent transcription termination site.

The rho-dependent transcription terminator tR1 of bacteriophage lambda stops RNA synthesis downstream of the major rightward promoter, PR, shortly after the cro gene. Terminated transcripts produced in a purified in vitro transcription system display a heterodisperse set of 3' termini, occurring in clusters located at +290-300, 308-312, 340-345, 385-390, and 440-450 nucleotides from the transcription start site [Morgan, W.D., Bear, D.G., & von Hippel, P.H. (1983) J. Biol. Chem. 258, 9553-9564]. However, transcripts from the same promoter in vivo have been reported to end primarily at +310-312 [Court, D., Brady, C., Rosenberg, M., Wulff, D. L., Behr, M., Mahoney, M., & Izumi, S. (1980) J. Mol. Biol. 138, 231-254]. In order to understand the nature of this discrepancy, we have carried out a comparative analysis of lambda PR transcription products produced in translationally active S30 cell extracts, in a purified in vitro system and in vivo. RNAs from the cell extracts coupled to translation show primarily three PR-derived transcripts beginning at one predominant 5' end and terminating at +263, 308, and 318. Sites +263 and +308 appear to be RNA processing sites. S1 nuclease mapping studies of RNAs produced in vivo show one 5' end and two 3' termini ending at +263 and 311; the +263 site is the predominant 3' end. When transcripts produced in a purified in vitro transcription system are incubated in the S30 cell extract under various conditions, the RNAs are degraded to two primary products with lengths of 263 and 308-311 nt.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗

PubMed2005

Polarity within pM and pE promoted phage lambda cI-rexA-rexB transcription and its suppression.

The cI-rexA-rexB operon of bacteriophage lambda confers 2 phenotypes, Imm and Rex, to lysogenic cells. Immunity to homoimmune infecting lambda phage depends upon the CI repressor. Rex exclusion of T4rII mutants requires RexA and RexB proteins. Both Imm and Rex share temperature-sensitive conditional phenotypes when expressed from cI[Ts]857 but not from cI+ lambda prophage. Plasmids were made in which cI-rexA-rexB was transcribed from a non-lambda promoter, pTet. The cI857-rexA-rexB plasmid exhibited Ts conditional Rex and CI phenotypes; the cI+-rexA-rexB plasmid did not. Polarity was observed within cI-rexA-rexB transcription at sites in cI and rexA when CI was nonfunctional. Renaturation of the Ts CI857 repressor, allowing it to regain functionality, suppressed the polar effect on downstream transcription from the site in cI. The second strong polar effect near the distal end of rexA was observed for transcription initiated from pE. The introduction of a rho Ts mutation into the host genome suppressed both polar effects, as measured by its suppression of the conditional Rex phenotype. Strong suppression of the conditional Rex[Ts] phenotype was imparted by ssrA and clpP (polar for clpX) null mutations, suggesting that RexA or RexB proteins made under conditions of polarity are subject to 10Sa RNA tagging and ClpXP degradation.

Bacteriophage lambda↗

PubMed1997

Inhibition of Escherichia coli protein synthesis by abortive translation of phage lambda minigenes.

Escherichia coli mutants defective in peptidyl-tRNA hydrolase activity are unable to maintain bacteriophage lambda vegetative growth. Phage mutants, named bar, overcome the host limitation to support viral growth. Multicopy expression of lambda wild-type bar regions is deleterious to hydrolase-defective cells because it provokes arrest of protein synthesis. We noticed that the bar regions include minigenes whose transcripts would contain a Shine-Dalgarno-like sequence appropriately spaced for translation from a two codon open reading frame. To investigate the mechanism of bar inhibition, we asked if transcripts of the barI region function as mRNAs in their ribosomal interactions. We found that bar-containing RNA associates with ribosomes, forms ternary initiation complexes, yields a toeprint signal, and can be removed from ribosomes by run-off translation, as authentic mRNA. Since bar-containing RNA has the properties of a messenger, we propose that its translation leads to drop-off and accumulation of peptidyl-tRNA in pth-defective cells. Starvation of the tRNA(s) sequestered in pepidyl-tRNA(s) eventually causes inhibition of protein synthesis.

Bacterial Proteins↗

PubMed1990

Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method.

A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates. The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones. A phage "spot-blot" analysis was employed to quickly screen these potential recombinants. This eliminated 9 of the 21 clones as the result of false positive signals. The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure. The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis. The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library. Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified. The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation. Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library.

Bacteriophage lambda↗

PubMed1997

Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.

The terminase enzyme from bacteriophage lambda is responsible for excision of a single genome from a concatameric DNA precursor and its insertion into an empty viral procapsid. The enzyme possesses a site-specific endonuclease activity which is responsible for excision of the viral genome and the formation of the 12 base-pair single-stranded "sticky" ends of mature lambda DNA. We have previously reported a kinetic analysis of the endonuclease activity of lambda terminase which showed an enzyme concentration-dependent change in the kinetic time course of the reaction [Tomka, M. A., & Catalano, C. E. (1993b) J. Biol. Chem. 268, 3056-3065]. We presented a model which suggested that the rate-limiting step in the nuclease reaction was the assembly of a catalytically competent prenicking complex. Here, we provide additional evidence for a slow assembly step in the nuclease reaction and demonstrate that the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubation of terminase with DNA also yields an observable fast phase of the reaction, but only when large (> or = 3 kb) DNA substrates are used. Finally, we present data which demonstrate that phage lambda terminase can efficiently utilize DNA from the closely related phage phi21 as an endonuclease substrate and that the enzyme binds efficiently to the cosB region of both phage genomes. The implications of these results with respect to the assembly of a catalytically competent nucleoprotein complex required to initiate genome packaging are discussed.

Bacterial Proteins↗