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A procedure for isolating and directly sequencing recombinant bacteriophage lambda gt11 DNA templates is described. Approximately 250-300 bases of sequence can be obtained directly from the lambda gt11 template, eliminating the need for subcloning prior to dideoxynucleotide sequencing of clones.
Previous experiments have indicated that the gam gene of bacteriophage lambda is responsible for an inhibition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene ("gamma-protein") and that characterize the inhibition reaction with highly purified preparations of gamma-protein and RecBC DNase. Genetic characterization was performed with partially purified fractions prepared from cells infected with various lambda mutants. An activity that inhibits RecBC DNase was absent in extracts prepared after infection by phage that carry nonsense or deletion mutations in the gam gene; this activity was highly thermolabile in an extract prepared after infection by phage that carry a temperature-sensitive mutation in the gam gene. For biochemical characterization, the gamma-protein has been purified more than 800-fold. This highly purified preparation inhibited all of the known catalytic activities associated with the RecBC enzyme, but exhibited no detectable DNase or ATPase activities by itself. These findings are discussed in terms of their implications for regulation of genetic recombination and bacteriophage lambda development.
Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.
When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.
Total intracellular DNA was isolated from replication-restricted bacteriophage lambda crosses in which the infecting parents were heteroallelic for wild-type and deletion mutant alleles. This DNA was examined for the presence of heteroduplex DNA molecules that contained wild-type sequences in one strand and deletion-mutant sequences in the other. Molecules hybrid for a 689-nucleotide deletion in the immunity region of lambda were detected at significant levels only in crosses in which both the red recombination system of lambda and the rec recombination system of Escherichia coli were active. Molecules hybrid for a 1300-nucleotide deletion in the central portion of the lambda genome were detected at significant levels in DNA isolated from both red+ and red- crosses in which recA function was present.
The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome. Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome. The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA. DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome. These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence. Bear et al. (J. Virol. 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor. Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites. Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.
The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.
The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with 0.1, 0.5 and 1 mM concentrations of sulfur mustard (SM) was investigated using agarose gel electrophoresis. Alkaline agarose gel electrophoresis also revealed single strand breaks at 0.5 and 0.1 mM concentrations of SM. The presence of magnesium ions in the reaction mixture prevented DNA degradation. It is proposed that the degradation of lambda DNA by its interaction with an excess of SM may be caused by the breakage of phosphodiester backbone of DNA via the formation of an intermediate phosphotriester bond.
The bacteriophage lambda O protein participates in the initiation of lambda DNA replication. The lambda O gene was cloned into plasmid pKC30 such that its expression was controlled by the lambda PL promoter. A lambda prophage-coded thermosensitive cI repressor was used to regulate transcription of the cloned O gene. Thermal inactivation of the lambda cI repressor resulted in overproduction of the O protein until it constituted approximately 20% of the total cellular protein of Escherichia coli. A simple three-step purification protocol was developed that yields several milligrams of homogeneous O protein per gram of cell paste. The precise position of the O gene in the known lambda DNA sequence was identified from the amino-terminal sequence of the isolated O protein. Purified O protein stimulated the replication of plasmid lambda dv DNA in vitro and specifically bound to duplex DNA fragments carrying the lambda replication origin.
The sequence of 18 nucleotides in the region preceding the initiation of transcription of the gene N of bacteriophage lambda has been determined to be as follows (see article). The basic approach used for the sequence determination involved Escherichia coli DNA polymerase I-catalyzed elongation of the octadecanucleotide primer, dT-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, possessing the appropriate polarity and nucleotide sequence corresponding to the 5' end of the gene N transcript. Following hybridization of the primer to the r-stand of bacteriophage lambda CI85657, sequences of the newly grown ollgonucleotide chains were determined by a) partial exonuclease digestion followed by two-dimensional fingerprinting; b) determination of pyrimidine tracts; and c) nearest neighbor analyses. Primer elongation was carried out in a controlled manner, the size of the newly grown chains being kept short by the following techniques: a) insertion of a ribonucleotide unit as the 3' terminus of the primer; b) use of a limited number of deoxynucleoside 5'-triphosphates in the elongation reaction; and c) enlongation of the primer using all the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.
The avian myeloblastosis virus provirus inserted in a lambda bacteriophage, recombinant clone 11A1-1 (Souza et al., Proc. Natl. Acad. Sci. U.S.A. 77:3004-3008, 1980), was transfected into chicken embryo fibroblasts which had been preinfected with either Rous-associated virus type 61 or the transformation-defective avian sarcoma virus tdB77. Within 4 to 5 h after transfection, the cells were injected into 16-day-old chicken embryos or 1-day-old chicks. Acute myeloblastic leukemia developed after a long latent period. Filtered (0.22-micrometer pores) supernatant of transformed buffy-coat cell cultures from one leukemic chicken of the lambda 11A1-1 (tdB77) group rapidly transformed yolk sac cells in vitro. Results from an infectivity interference assay and analysis of proviral DNA fragments generated with restriction endonucleases were consistent with the presence in leukemic cells of defective avian myeloblastosis virus and tdB77 as the helper virus.
The O protein of bacteriophage lambda is required for initiation of DNA replication at the lambda replicative origin designated ori lambda. The binding sites for O protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage lambda O protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage lambda O and P proteins and host DnaB protein interact at ori lambda to generate a larger structure than that formed by O protein alone; P and DnaB proteins fail to form any observable complex when O protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage lambda O protein and ori lambda provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.
Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.
Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.
Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+' strand as template) toward the M13 origin of replication (ori). Lethality can be relieved by mutation of PR, repression of the promoter by the lambda cl repressor, or by insertion of a strong transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR. This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively.
Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass. The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell. Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA. Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined. Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population. After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction. Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end. In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population. In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined. The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome. Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.
A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.
The tetrameric cII protein from bacteriophage lambda activates transcription from the phage promoters P(RE), P(I), and P(AQ) by binding to two direct repeats that flank the promoter -35 element. Here, we present the X-ray crystal structure of cII alone (2.8 A resolution) and in complex with its DNA operator from P(RE) (1.7 A resolution). The structures provide a basis for modeling of the activation complex with the RNA polymerase holoenzyme, and point to the key role for the RNA polymerase alpha subunit C-terminal domain (alphaCTD) in cII-dependent activation, which forms a bridge of protein/protein interactions between cII and the RNA polymerase sigma subunit. The model makes specific predictions for protein/protein interactions between cII and alphaCTD, and between alphaCTD and sigma, which are supported by previous genetic studies.