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A pleiotropic regulatory mutation in lambda bacteriophage.

Lambda bacteriophage mutants, lambdasar, were isolated. These mutants can form plaques on a non lysogenic lawn and are unable to grow on nonimmune (imm-), cro constitutive hosts. Analysis of the restriction of lambdasar by a set of defective lysogens suggested that both the cro and cII gene products participate in the inhibition. The sar mutations were mapped in the ori region between the genes cII and O. Complementation experiments showed that under the restrictive conditions lamdasar is defective in the expression of both the N and the O genes. Transcription analyses support these findings, as lambdasar is unable to serve as a template for transcription after infecting cro constitutive hosts. In addition lambdasar does not replicate under the restrictive conditions, although its DNA can bind to the host membrane to some extent. The Sar phenotype can be relieved by removing sites of action of cro either by a V2 mutation or by substituting the lambda immunity region by imm434 or imm21. Similarly introducing a cy mutation, which interferes with the action of the cII gene product, also eliminates the Sar effect. The sar mutation can suppress cy mutations as manifested in plaque morphology, lysogenization frequency, cI repressor synthesis and the expression of rex function. Suppression takes place only when the sar mutation is present in cis to cy and it requires the action of the cII and cIII gene products. It is suggested that the sar mutation suppresses cy by activating a new promoter for repressor synthesis, pro. The results also suggest that the cII and cIII gene products may act at a site other than y.

Chromosome Mapping

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.

Coliphages

Explanations accounting for transduction by bacteriophage lambda in maltose negative bacteriophage lambda resistant mutants of Escherichia coli K-12.

Entry of DNA from lambda phages particles into lambdarMAl- mutants of Escherichia coli K-12 is shown to be due to two distinguishable processes. One, residual transduction, results from a low level expression of lamB. The other one, background transduction, is independent of gene lamB. Interpretations are presented for these results. It is propos that residual transduction is due to a weak promoter pB3 located within or near the distal part of the gene preceeding lamB in the same operon. It is proposed that background transduction is due to a secondary receptor structure for phage lambda. Finally a tentative hypothesis relatin pB3 to insertion sequences is presented.

Coliphages

[Antigenic structure of bacteriophage lambda. Identification of the chief antigens of bacteriophage lambda].

The antigenic composition of the bacteriophages lambdaC1857 and lambdagt-lambdaC was investigated by modified immunoelectrophoresis in a 1,2% agarose gel involving 1% Triton X-100 and 0,25% sodium desoxycholate. The phages lambdaC1857 and lambdagt-lambdaC were shown to have identical antigenic compositions and to comprise three basic antigens, such as a1, a2, a3. The main structural proteins of the phage such as pE, pV and pD were isolated by preparative electrophoresis in 13% polyacrilamide gel. The immunophoresis of individual proteins indicated the antigens a1, a2 and a3 to be proteins pV, pD and pE, respectively.

Antigens, Viral

Ribonucleic acid synthesis after adsorption of the bacteriophage lambda on Escherichia coli minicells.

Lambda bacteriophage adsorbs on the chromosome-less small bodies (minicells) produced by aberrant cell division of Escherichia coli P678-54. The plasmid-less minicells, as well as the minicells harbouring the colicinogenic factor E1, reveal a considerable inhibition of total RNA synthesis after phage adsorption. Both kinds of minicells synthesize RNA hybridizable to lambda DNA, but in the plasmid-harbouring minicells lambda RNA synthesis is much more intensive.

Adsorption

Charon phages: safer derivatives of bacteriophage lambda for DNA cloning.

The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.

Chromosome Mapping

[Interaction of lambda bacteriophages with mammalian cells. II. Elucidation of the role of interacting components].

The study of 3H-thymidine labelled bacteriophage lambda C185757 uptake by HeLa, RH and Chinese hamster cell revealed the lack of cells or phage specificity in the phage interaction with cells. The phage uptake is shown to be an active process depending on the cell state. The mechanism of "protective" action of calcium chloride is found to be as follows: the calcium phosphate precipitate formed in phosphate-containing media absorbs the phage, thus increasing its concentration on the cell surface, which makes the pinocytosis more effective.

Animals

Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES.

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.

Chromosome Mapping

Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration.

Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation.

Coliphages

Assembly of biologically active proheads of bacteriophage lambda in vitro.

Bacteriophage lambda DNA can be packaged in vitro into preformed proheads to generate plaque-forming units. This complex set of reactions is initiated when lambda DNA is mixed with the product of the phage A gene, and proheads. Because proheads are an essential early reactant, the system has potential as an assay for the formation of biologically active proheads. When extracts of cells infected with certain lambda head mutants (for example, B--, C--, Nu3--, and E--) are used as the prohead donor, plaque-forming units are not produced. However, when extracts of E- - and Nu3- - infected cells are first reacted together the combination provides prohead-donor activity to the in vitro packaging system. In vitro assembled, biologically active proheads have the same sedimentation properties and electron micrsocopic appearance as "wild-type" proheads isolated from lambdaA-D- -infected cells. Centrifugation analysis shows that the Nu3- extract contributes gpE, the major capsid protein, to the reaction in the form of monomers or small polymers.

Coliphages

Genetic heterozygosity in unreplicated bacteriophage lambda recombinants.

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely linked markers. Recombination between closely linked markers seems to be the consequence of the removal of base-pair mismatches that are present within the heteroduplex regions. This localized reduction of heterozygosity within the heteroduplex regions that join the parental components of recombinant DNA molecules can account for high negative interference.

Coliphages

Genetic structure of the replication origin of bacteriophage lambda.

A fragment of bacteriophage lambda DNA produced by the restriction endonuclease Eco RI and extending from the immunity region to a point inside gene O is found to have a fully functional origin of replication. Seven ori- mutations of lambda cluster in a small region just to the left of the Eco RI cleavage site which defines the right end of this fragment. These mutations lie within gene O.

Chromosome Mapping

The purification and properties of the scaffolding protein of bacteriophage lambda.

The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head. Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein. The Nu3 gene has been cloned and overexpressed, and its protein product has been purified. The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction. The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396. A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape.

Amino Acid Sequence

A map of the cleavage sites for endonuclease AvaI in the chromosome of bacteriophage lambda.

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5'-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.

Chromosome Mapping

Interaction between bacteriophage lambda and its Escherichia coli host.

Bacteriophage lambda relies to a large extent on processes requiring interactions between viral- and host-encoded proteins for its lytic growth, establishment of lysogeny, and release from the prophage state. Both biochemical and genetic studies of these interactions have yielded new information about important host and lambda functions. In particular, mutations in Escherichia coli that compromise lambda DNA replication, genome packaging, transcription elongation, and site-specific recombination have led to the identification of bacterial genes whose products are chaperones, transcription factors, or DNA-binding proteins.

Bacterial Proteins

Proteolytic cleavage of bacteriophage lambda repressor in induction.

The bacteriophage lambda repressor, a protein that maintains the lysogenic state of a bacterium containing a lambda prophage, is cleaved when the lysogen is induced by mitomycin C or ultraviolet light. This cleavage does not occur when induction is prevented by mutational alteration either of the phage repressor or of the host recA gene product. Proteolytic cleavage may be the primary mechanism of repressor inactivation in this induction pathway, or it may follow a different event which causes the initial inactivation.

Coliphages

Bacteriophage lambda PaPa: not the mother of all lambda phages.

The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

Adsorption

Nicking-closing activity associated with bacteriophage lambda int gene product.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.

Bacteriophage lambda