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Multiple repressor binding at the operators in bacteriophage lambda.

Short DNA duplexes are protected when lambda DNA is digested with nuclease in the presence of lambda repressor. As the ratio of repressor to operator is increased, six successively larger fragments are recovered, ranging in size from 35 to 100 base pairs, each of which binds repressor. Study of these fragments indicates that, at each of the two lambda operators (o(L) and o(R)), repressor first binds to a unique site (not necessarily terminal), and that five additional sites are then filled in linear right-ward or left-ward order. The nucleotide sequences and affinities for repressor of o(L) and o(R) are not identical, although six fragments of similar size are protected at each operator. Evidence is presented arguing against the existence of hairpin-like structures in the operator fragments, and, moreover, it is shown that the operator duplex does not unwind when repressor binds to it.

Autoradiography↗

Int-constitutive mutants of bacteriophage lambda.

The constitutive production of small amounts of trpB enzyme in an Escherichia coli strain carrying lambdacI857 prophage within the trpC gene has been examined in derivatives of this strain from which portions of the prophage have been deleted. Enzyme production requires a site (p(I)) within the prophage close to the left prophage end. Selection for mutants of this lysogen that grow on low concentrations of indole yielded two types of mutations within the prophage: (a) v2-type, in which all phage genes controlled by the major leftward operator are derepressed; and (b) int-c type, in which the only phage gene derepressed is int. The int-c mutations lie in the same part of the prophage as p(I). All int-c mutants appear deficient in xis gene function, even when derepressed.

Chromosome Mapping↗

Activity of empty, headlike particles for packaging of DNA of bacteriophage lambda in vitro.

A precursor head of phage lambda is synthesized after induction of cells lysogenic for lambdaD(-)F(-) and lambdaA(-) (head-defective) mutants. This precursor head can be assayed by complementation in vitro and can be purified by CsCl gradient centrifugation and sucrose gradient sedimentation. The precursor head contains no DNA and has the same dimensions as the petit lambda particle. It can be packed with lambda DNA in an extract from induced Escherichia coli lysogenic for a lambdaE(-) mutant.

Coliphages↗

Isolation of the bacteriophage lambda A-gene protein.

An in vitro assay for measuring the activity of the phage lambda A-gene product has been developed. The assay is based on the observation that A-donor extracts complement A minus extracts for packaging of exogenous immature lambda DNA into phage particles. A partial purification of the A-gene product activity using this assay is presented. A method is suggested by which this A protein-dependent in vitro system might be manipulated to analyze the mechanism of reforming the lambda cohesive termini during chromosome assimilation into phage precursors.

Centrifugation, Density Gradient↗

Repression and autogenous stimulation in vitro by bacteriophage lambda repressor.

Purified lambda repressor protein is shown to reduce the lambda DNA-directed synthesis of proteins in vitro as determined both by net amino-acid incorporation and by analysis of specific lambda-coded proteins resolved by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. By means of different lambda DNA templates carrying deletion and point mutations in the operators o-L or o-R, it has been possible to demonstrate repression of the synthesis of two classes of lambda proteins. The synthesis of one, class c, appears to be controlled from the operator o-L and is more efficiently repressed at low concentrations of the repressor than that of the other class of repressible lambda proteins, class d, which is controlled from the operator o-R. Several other proteins synthesized in vitro are not repressible. Some of these are coded by the J-att region. In addition, the repressor appears to have another activity, that of stimulating the synthesis of a protein identified as the repressor itself. Lambda repressor appears to stimulate its own synthesis by acting at prm, a site defined by the cis-acting mutation prm 116.

Chromosome Mapping↗

Proposed mechanism of bacteriophage lambda induction: acquisition of binding sites for lambda repressor by DNA of the host.

Interference with the in vitro binding of lambda phage repressor to lambda operator DNA was observed when Escherichia coli DNA containing the following lesions was present in the reaction mixture: (a) DNA with single-strand breaks from pancreatic DNase (nicked DNA); (B) DNA isolated from thymine-straved cells; (c) DNA from ultraviolet-treated cells; (d) DNA of mitomycin-treated cells; and (e) DNA from a temperature-sensitive ligase mutant after 1 hr at 42 degrees. Normal E. coli DNA did not interfere. Binding of lambda cIing-minus repressor to operator DNA was not affected by E. coli DNA with lesions. DNAs from cells treated with increasing doses of mitomycin were proportionately more effective in competition for repressor, suggesting increasing binding sites per unit of DNA. A general model of virus induction is proposed, based on binding affinity of ultraviolet-sensitive repressors for single-strand breaks in the host DNA. The model is extended also to the presumptive repressor of cell division.

Binding Sites↗

Visualization of a novel junction in bacteriophage lambda DNA.

At early times after infection of a recA derivative of Escherichia coli with lambdab221c126red270a42 phage, a low but significant proportion of intracellular lambda molecules show a novel junction. These junctions are also present, although in reduced numbers, in a lysate obtained at late times after infection of a recA+ host with lambdacIIcIII phage. Fine structure and denaturation mapping analyses showed that these junctions occur at homologous positions and that they are compatible with the occurrence of a cross-strand exchange between lambda DNA duplexes similar to the type proposed in most molecular models for genetic recombination. However, the results are also consistent with the structures expected if a replicating growing point undergoes branch migration.

Coliphages↗

Induction of sigma factor synthesis in Escherichia coli by the N gene product of bacteriophage lambda.

Thermoinduction of cells of E. coli carrying prophage lambdacI857 within the bfe gene brings about not only "escape synthesis" of core subunits of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2-7-7-6), but also a striking stimulation of sigma factor synthesis. The latter phenomenon, termed sigma induction, is generally observed after lambda phage infection or prophage induction. A series of experiments with various bacterial and phage strains led us to conclude that the N gene product of lambda is directly involved to the sigma induction. These and other results obtained with mutants defective in transcription termination factor rho suggest the involvement of a rho-sensitive site in the control of sigma gene expression in E. coli.

Chromosome Mapping↗

Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.

Cell Line↗

Effects of ribosomal mutations on the read-through of a chain termination signal: studies on the synthesis of bacteriophage lambda O gene protein in vitro.

In a DNA-dependent protein-synthesizing system that contains streptomycin-sensitive ribosomes, lambda DNA directs the synthesis of two proteins that are products of the O gene. The larger is produced as a result of read-through of a UGA termination codon. In the system containing streptomycin-resistant ribosomes this read-through protein is not synthesized, indicating that the mutational alteration in the ribosomal protein S12 restricts the read-through. The mutant ribosomes also fail to synthesize the read-through coat protein of RNA phage Qbeta. In addition, the mutant ribosomes restrict suppression of amber mutations in vitro, similar to their effect in vivo.

Coliphages↗

Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin.

A transducing phage lambdaasn was isolated. The late gene region of its genome was found to have been substituted by an Escherichia coli chromosomal segment containing the genes bgIR, bgIC, glmS, uncA, and asn. Restriction endonuclease cleavage mapping and electron microscopic analysis of the lambdaasn DNA revealed that the size of the bacterial segment is approximately 1.75 X 10(7) daltons, corresponding to about 26.4 kilobases. The circular DNA of lambdaasn was digested with restriction endonuclease EcoRI, diluted, and sealed with DNA ligase. When the reaction mixture was used to transform a recipient E. coli strain, a small plasmid of about 1 X 10(7) daltons (named pMCR115) was obtained. Restriction endonuclease cleavage mapping of pMCR115 and other evidence suggested that it contained the replication origin (oriC) of the E. coli chromosome.

Chromosomes, Bacterial↗

Interaction of bacteriophage lambda repressor with nonoperator DNA containing single-strand gaps.

In direct binding assays, purified lambdaind+ repressor displayed high affinity for nonoperator DNA containing single-strand gaps. Its affinity for this same DNA but completely double-stranded, nicked, or denatured was considerably lower. In contrast, purified lambdaind- repressor had 1/10th the affinity for the gapped DNA, a level comparable to that of purified lac repressor. In the presence of limiting amounts of ind+ repressor, nonoperator DNA containing gaps could be shown to compete effectively with lambda DNA for binding of repressor. A previous model of lambda induction [Sussman, R. & Ben-Zeev, H. (1975) Proc. Natl. Acad. Sci. USA 72, 1973--1976], based on the assumption that this phenomenon involves the binding of repressor to lesions in the host DNA, is reevaluated in the light of the data reported here.

Binding Sites↗

A single base-pair change creates a Chi recombinational hotspot in bacteriophage lambda.

X4+ mutations, responsible for the Chi phenotype in phage lambda, locally increase the rate of recombination promoted by the Escherichia coli recombination system (Rec). X+ mutations in the cII gene, one of a few sites in lambda at which such mutations arise, were located genetically and physically with overlapping deletions. DNA sequence analysis of the deletion segment containing the X+ C mutations showed that two independent X+ C mutations arose by the same A-T to T-A transversion. Presumably, this change creates a nucleotide sequence recognized by a protein involved in a rate-limiting step of recombination.

Base Sequence↗

Visualization of the intracellular development of bacteriophage lambda, with special reference to DNA packaging.

To reveal intermediates in lambda DNA packaging, infected cells were osmotically ruptured and the cell lysates were deposited on electron microscope grids by sedimentation through a sucrose/formalin cushion. A fixation procedure that crosslinks head-related structures to DNA allowed us to study successive stages in the process of head filling. Three types of head-related structures can be distinguished: (i) empty heads (petit lambda), less angular in outline than complete lambda heads; (ii) heads partially filled with DNA (partially filled heads), having a roundish outline; and (iii) particles tightly packed with DNA (full heads), having a hexagonal outline. DNA-head complexes were bound either at the terminal end of a DNA thread or at a point intermediate along the thread. The terminal complexes were more abundant. No head-related structures could be found in an induced lambda mutant lysogen blocked in the synthesis of petit lambda (amber in lambda gene E). One type of mutant blocked in DNA packaging (amber in gene A) produces empty heads and free tails, whereas another (amber in gene D) produces partially filled heads in addition. Our data suggest that a DNA-petit lambda complex may be an early intermediate in packaging and that the lambda DNA substrate can be a cohesive-ended concatemer or a concatemer with double-stranded cohesive site sequences.

Coliphages↗

Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads.

Evidence is presented that ColE1 hybrid plasmids carrying the cohesive-end site (cos) of lambda can be used as gene cloning vectors in conjunction with the lambda in vitro packaging system of Hohn and Murray [(1977) Proc. Natl. Acad. Sci. USA 74, 3259--3263]. Due to the requirement for a large DNA molecule for efficient packaging, there is a direct selection for hybrids carrying large sections of foreign DNA. The small vector plasmids do not contribute a large background in the transduced population, which is therefore markedly enriched for large hybrid plasmids (over 90%). The efficiency of the in vitro packaging system is on the order of 10(5) hybrid clones per microgram of foreign DNA for hybrids in the 20--30 million dalton range.

Coliphages↗

Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

5-Hydroxytryptophan↗

The structure and function of the DNA from bacteriophage lambda.

The position and orientation of genes in lambda and lambda dg DNA are described. The position of six genes located in the right half of isolated lambda DNA was found to be -(N, i(lambda))--O-P---Q-R-(right end of DNA), which is their order on the genetic map of the vegetative phage. The order of the three genes of the galactose operon (k, t, and e) located in the left half of lambda dg DNA was found to be (left end of DNA)----k-t-e-, consistent with Campbell's model (5) for the formation of this variant. Gene orientation, defined as the direction of transcription along the DNA, is inferred to be from right to left for the galactose operon in lambda dg DNA. The strand of lambda DNA which functions as template in transcription of N, an "early" gene required for normal replication of lambda DNA, was determined as a first step in ascertaining the orientation of this gene. The method includes isolation of each strand, formation of each of two heteroduplex molecules consisting of one strand from wild-type and one from an N mutant) and comparison of their N activities. The second step, which consists of ascertaining the 5'-to-3' direction of each strand, is discussed, as is a determination of the orientation of gene R.

Chemical Phenomena↗