Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

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.     

Transfer of a bacterial gene using phage lambda transfecting DNA]

A new amber mutation of phage with the gene coding synthesis of beta-galactosidase was received by recombination. With the help of transfection DNA isolated from this phage the transfer of the gene coding the beta-galactosidase synthesis to the recipient phage-resistant E. coli cell was realized. The suggested model can be used for the gene transfer to the recipient phage-resistant cells or other species of bacteria with transfection DNA.     

Sequence of a secondary phage lambda attachment site located between the pentitol operons of Klebsiella aerogenes.

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.     

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.     

The arrangement of DNA in lambda phage heads. II. lambda DNA after exposure to micrococcal nuclease at the site of head-tail joining

DNA in lambda phage heads (lacking tails) is damaged by treatment with micrococcal nuclease. Alkaline sucrose gradient sedimentation of the DNA extracted from nuclease-treated and untreated heads reveals no hydrolysis of internal phosphodiester bonds. Transformation of helper-infected cells by whole or half molecules of treated DNA is as efficient as by control DNA. Thus, the site of nuclease damage must be near the ends and very limited.     

DNA base sequence changes induced by bromouracil mutagenesis of lambda phage

The base sequence changes induced by bromouracil mutagenesis in the cI gene of phage lambda have been determined by direct sequence analysis. Phage DNA mutagenized during prophage replication or during phage lytic growth showed predominantly A · T → G · C transitions. The frequency of this mutation was strongly sequence-dependent: 5′ A-C-G-C 3′ > A-C(A.C or T) > A(A.G or T). The difference in mutability of bases in the gene is not the result of specificity in mutL-dependent mismatch repair, since phage grown in mutL host cells showed the same distribution of bromouracil mutations. The observations made in phage mutagenized with bromouracil in the prophage state should be representative of bromouracil mutagenesis in the Escherichia coli chromosome.     

N-terminal sequence of phage lambda repressor

Summary Lambda repressor was purified from an E. coli strain which produces 150 times more lambda repressor than a single lysogen. The sequence of the fifty N-terminal residues was determined by automated Edman degradation. It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al. (1975) a substantial part of the operator-DNA-binding site of the repressor.     

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.     

The fate of phage lambda DNA in lambda-infected minicells.

The fate of phage lambda DNA in lambda-infected Escherichia coli minicells harboring the plasmid ColE1, and in plasmid-free minicells, were studied. Binding of lambda DNA to the minicell membrane, and formation of the supercoiled covalently-closed circular structure has been demonstrated. Phage infection abolishes plasmid DNA synthesis. Only a very slight, non-replicative lambda DNA synthesis occurs, soon after infection. This synthesis is associated with fragments of lambda DNA arising during, or soon after its penetration.     

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.     

Bending and supercoiling of DNA at the attachment site of bacteriophage lambda

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.     

Filter-supported preparation of lambda phage DNA

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.     

Rapid isolation of phage lambda DNA

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.