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PubMed1975

Sequence of the promoter-operator proximal region of the major leftward RNA of bacteriophage lambda.

The sequence of the first 149 nucleotides of the major leftward RNA of bacteriophage lambda has been determined. Preliminary sequence information was also obtained for a portion of the untranscribed area immediately upstream of the point on the template when RNA synthesis normally starts. Several restriction endonuclease sites, deletion endpoints, and single base changes have been localized within the sequence. The first potential translation initiation codon which is not followed by an in-phase termination codon is a GUG located 90 nucleotides from the transcription startpoint.

Base Sequence↗

PubMed1994

Synergistic activation of transcription by bacteriophage lambda cI protein and E. coli cAMP receptor protein.

Two heterologous prokaryotic activators, the bacteriophage lambda cI protein (lambda cI) and the Escherichia coli cyclic AMP receptor protein (CRP), were shown to activate transcription synergistically from an artificial promoter bearing binding sites for both proteins. The synergy depends on a functional activation (positive control) surface on each activator. These results imply that both proteins interact directly with RNA polymerase and thus suggest a precise mechanism for transcriptional synergy: the interaction of two activators with two distinct surfaces of RNA polymerase.

Base Sequence↗

PubMed1977

Physical structure of the replication origin of bacteriophage lambda.

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.

Base Sequence↗

PubMed1973

Genetic and biochemical investigation of the Escherichia coli mutant hfl-1 which is lysogenized at high frequency by bacteriophage lambda.

The Escherichia coli mutant hfl-1 is lysogenized at very high frequency by bacteriophage lambda. The normal requirement for the lambdacIII gene product in the establishment of repression is not observed in hfl-1 strains. These phenotypic characteristics are specified by a single locus at 82.5 min on the E. coli map in extremely close proximity to the purA gene, cotransduction frequencies ranging from 97 to 100% depending on the particular purA marker used. The lactose operon is shown to function normally in this strain, and there are also no demonstrable differences in ribonucleic acid polymerase activity or cyclic-adenosine monophosphate levels. Alterations in the cell envelope are indicated by a slight rifamycin resistance, which is reversible by pretreating the cells with ethylenediaminetetraacetic acid, and by a resistance to penicillin and a sensitivity to high concentrations of sodium dodecyl sulfate. It is not known whether this change in cell surface is the primary lesion, or a pleiotropic effect of some more basic metabolic shift.

Chromosome Mapping↗

PubMed1967

Protein components of bacteriophages lambda and lambda virulent.

Parallel studies have been made of the protein coats of the temperate bacteriophage lambda and of a deletion mutant, lambda virulent. A new method for preparing ghosts of both phages by the action of Cu(++) is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 +/- 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 +/- 1,500 and 16,000 +/- 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage lambda virulent is 0.016 g/ml less than that of lambda. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.

Amino Acids↗

PubMed1976

Structure of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.

Coliphages↗

PubMed1991

Expression of a human monoclonal anti-(rhesus D) Fab fragment in Escherichia coli with the use of bacteriophage lambda vectors.

A human anti-(rhesus D) antibody (IgG1 lambda) Fab fragment was cloned from an Epstein-Barr-virus-transformed cell line and expressed in Escherichia coli with the use of bacteriophage lambda vectors. The cloned protein is active in binding to human erythrocytes and permits the development of a recombinant reagent for the prevention of haemolytic disease of the newborn. The method offers a rapid and effective means of rescuing human Fabs from potentially unstable cell lines secreting human antibodies.

Antibodies, Monoclonal↗

PubMed1993

Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter. Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR.

We have deleted a single base-pair in the -35 region of the bacteriophage lambda PRM promoter. The deletion (PRM delta 34) creates a better match of PRM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo. Since the mutation also increases the overlap between OR2 and the -35 region of PRM, binding of repressor to OR2 no longer activates, but in fact represses PRM. Finally, the mutation decreases the distance between the PRM and PR transcription start sites from 82 to 81 base-pairs. As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other. Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion. Both promoters can be occupied by RNA polymerase at the same time. Determination of KB and kf revealed that inhibition of PRM delta 34 by PR was manifest in a 100-fold decrease in the value of kf, but at the same time KB was increased tenfold. These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes. In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone. These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter. Assays of the effects of the delta 34 mutation in vivo indicate that interference between PRM and PR does not limit the rate of open complex formation at PRM in the cell. Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.

Bacteriophage lambda↗

PubMed1992

Increase in susceptibility to EcoRII restriction of bacteriophage lambda produced by propagation on host cells growing in 5-azacytidine: a new in-vivo method for demonstration of DNA-methylation inhibition.

The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium. This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

Azacitidine↗

PubMed1965

Factors in lysis and lysis inhibition by lambda bacteriophage.

Groman, Neal B. (University of Washington, Seattle). Factors in lysis and lysis inhibition by lambda bacteriophage. J. Bacteriol. 90:1563-1568. 1965.-Induced Escherichia coli strain K-12(lambda112) exhibited lysis inhibition at 37 C but lysed at 44 C when incubated in LB medium lacking NaCl [LB - (NaCl)]. In LB medium containing NaCl, the temperatures for lysis and lysis inhibition were reversed. In contrast, induced K-12(lambda) lysed under all of these conditions. At 37 C, the addition of NaCl to LB - (NaCl) at various times after induction of K-12(lambda112) restored lysis. The degree of lysis decreased the longer the addition was delayed, but partial restoration occurred as late as 150 min postinduction. At 44 C, the addition of salt at various times after induction restored lysis inhibition even after lysis had begun. An attempt was made to correlate the conditions for lysis and lysis inhibition with the behavior of lambda112 endloysin. The enzymatic activities of lambda and mutant lambda112 endolysins were compared under various salt-temperature conditions. Both endolysins were progressively and equally inhibited by increasing concentrations of Na(+), K(+), and Li(+) salts, and exhibited similar relative activities at 24 and 37 C. Both were stable at 37 C in the presence and absence of NaCl, and were inactivated at comparable rates at 44 C. The results indicate that the effect of salt and temperature on lysis and lysis inhibition cannot be explained by their direct effect on lambda112 endolysin.

Bacteriolysis↗

PubMed1986

Expression of immunogenically reactive diphtheria toxin fusion proteins under the control of the pR promoter of bacteriophage lambda.

The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983). Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts. Deletion of 320 bp from the C-terminal region of the B-fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E. coli lon- strain in addition to beta Gal. The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT. Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT. Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT.

Amino Acid Sequence↗

PubMed1994

Single-site mutations in the C-terminal domain of bacteriophage lambda cI repressor alter cooperative interactions between dimers adjacently bound to OR.

Wild-type cI repressor dimers bind with 2.5-3 kcal/mol of cooperative free energy to the tripartite right operator region (OR) of bacteriophage lambda [Johnson, A. D., et al. (1981) Nature 294, 217-223; Brenowitz, M., et al. (1986) Methods Enzymol. 130, 132-181]. Quantitative modeling has suggested that cooperativity is required for maintenance of the lysogenic state and for the efficient switch from lysogenic to lytic growth [Ackers, G. K., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1129-1133; Shea, M. A., & Ackers, G. K. (1985) J. Mol. Biol. 181, 211-230]. Cooperativity and self-association are thought to involve protein-protein contacts between C-terminal domains of the repressor molecule [Pabo, C. O., et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1608-1612]. To address the importance of the C-terminal domain in mediating the cooperativity exhibited by lambda cI repressor, a number of single-site mutant candidates were screened for possible deficiencies in cooperative interactions [Beckett, D., et al. (1993) Biochemistry 32, 9073-9079; Burz, D. S., et al. (1994) Biochemistry 33, 8399-8405]. Since repressor dimerization and binding to operator sites are coupled processes, elucidation of the energetic basis of regulation in this system requires that the equilibrium dimerization constants and the intrinsic and cooperative free energies of binding be measured. In this work we evaluate the interaction of eight mutant repressors with OR DNA: Gly147-->Asp (GD147), Pro158-->Thr (PT158), Glu188-->Lys (EK188), Lys192-->Asn (KN192), Tyr210-->His (YH210), Ser228-->Arg (SR228), and Ser228-->Asn (SN228), each with an amino acid substitution in the C-terminal domain, and Glu102-->Lys (EK102) where the substitution lies in the "linker sequence" between domains. Self-assembly properties of six of these mutant repressors are presented in the preceding paper (Burz et al., 1994). In this work, the binding of mutant cI repressors to OR was examined using quantitative DNAse I footprinting. This technique monitors individual site occupancy concurrent with binding at the other sites within a multisite complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗

PubMed1990

Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli.

A new series of expression vectors that direct high-level overproduction of gene products in Escherichia coli is described. All contain strong bacteriophage lambda promoters, PR and PL, arranged in tandem so that both promote transcription into genes inserted into or between unique restriction sites. The vectors also direct expression of the lambda cI857 gene (from its natural promoter, PM), which enables their use in any E. coli host strain to effect controlled expression by shifting the temperature of cultures from 30 to 42 degrees C. The vectors pCE30, pND201, pPT150 and pMA200U are derivatives of the high-copy-number plasmid pUC9. Vector pCE33 is an analogous derivative of the heat-inducible runaway-replication plasmid, pMOB45, and directs overproduction of proteins by virtue of increase in both gene dosage and transcription following treatment at 42 degrees C. The vectors pND201 and pPT150 bear a ribosome-binding site (RBS) perfectly complementary to the 3' end of E. coli 16-S rRNA a few bp upstream from a unique HpaI site. Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described. The phagemid vector pMA200U is a direct analog of pCE30 designed to facilitate preparation of single-stranded DNA templates for use in oligodeoxyribonucleotide-directed mutagenesis of overexpressed genes.

Bacteriophage lambda↗

PubMed1984

Knowledge-based simulation of genetic regulation in bacteriophage lambda.

We have developed a general-purpose computer program for the functional simulation of regulatory genetics. This simulator is knowledge-based and was developed using the Unit System, a software tool for the acquisition, representation, and manipulation of hierarchically organized knowledge. The advantages of a knowledge-based design are presented, and the simulator's architecture is described. Its performance on the decision between lytic and lysogenic growth in Bacteriophage Lambda is reported.

Bacteriophage lambda↗

PubMed1995

Display of peptides and proteins on the surface of bacteriophage lambda.

The display of peptides or proteins on the surface of viruses is an important technology for studying peptides or proteins and their interaction with other molecules. Here we describe a display vehicle based on bacteriophage lambda that incorporates a number of features distinct from other currently used display systems. Fusions of peptides or protein domains have been made to the amino terminus of the 11-kDa D protein of the lambda capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand interactions, based on the ability of a monoclonal antibody to recognize an epitope fused to the D protein on phage heads. To produce large D fusion display libraries and yet avoid the cumbersome task of cloning many fragments into lambda DNA, we have used the Cre-loxP site-specific recombination system in vivo to incorporate plasmids encoding the D fusions into the phage genome. Finally, we show that D fusion proteins can be added in vitro to phage lacking D protein and be assembled onto the viral capsid.

Antigens, Surface↗

PubMed1979

Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda.

We have identified integrated proviral DNA sequences of m1 and HT-1 isolates of Moloney sarcoma virus (MuSV) in EcoRI digests of transformed mink cell genomic DNA and have cloned these fragments in bacteriophage lambda. Both the lambda-HT1 phage recombinant, containing a 12.3-kilobase MuSV pair (kb) fragment, and the lambda-m1 phage recombinant, containing a 7.0-kb fragment, possess full copies of the sarcoma viruses along with 5' and 3' host flanking sequences. The MuSV proviral DNA sequences, 6.7 kb for HT-1 and 5.2 kb for m1, are colinear by heteroduplex microscopy with the 1.5-kb difference in size accounted for by two approximately equal to 0.8-kb deleted regions in m1. Both integrated viral genomes are terminally redundant and have integrated at the same site in the provirus but at different sites on the host chromosome. The host sequence flanking integrated HT-1 MuSV have been identified as a single EcoRI restriction fragment of 5.6 kb in normal mink cells.

Animals↗

PubMed1985

A small mobilizable IncP group plasmid vector packageable into bacteriophage lambda capsids in vitro.

A mobilizable cosmid derivative of an IncP group plasmid was constructed by cloning the oriT region of RK2, a wide host-range plasmid, and the minimal DNA sequence of bacteriophage lambda required for efficient packaging in vitro. This cosmid is 13 kb in size and has unique restriction sites for EcoRI, XhoI, HindIII, and SalI. The XhoI and HindIII sites are within the kanamycin-resistance gene and the SalI site is in the tetracycline-resistance gene. This plasmid was mobilizable from an Escherichia coli donor to a number of diverse gram-negative bacteria at a frequency of 0.8 to 10 per 100 donors. This vector is one of the smallest of all wide host-range cosmids described in the literature. As part of this study, another mobilizable IncP group plasmid vector has also been constructed which, in addition to the sites listed above, has a unique BglII site, but which lacks the packager sequence.

Alcaligenes↗

PubMed1981

Structure of the baboon endogenous virus genome: cloning of circular virus DNA in bacteriophage lambda.

Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7. The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28. Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping. Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA. Eight other clones had the viral insert of 8.0 kb, i. e., the small circular form, and were deleted one of the repeated sequences. The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb. One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb. One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region. The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection. The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors.

Bacteriophage lambda↗