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PubMed1993

A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.

We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.

Bacteriophage lambda↗
PubMedDate not supplied

[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↗
PubMed1985

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes.

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.

Bacteriophage lambda↗
PubMed1995

Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda.

The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure. A turbidimetric assay utilizing chloroform-treated E. coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5. The lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-13C]methionine into the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5. Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme. The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.

Bacteriophage lambda↗
PubMed2004

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

Bacteriophage lambda↗
PubMed1993

Bacteriophage Lambda as a Delivery Vector for Tn10-Derived Transposons in Xenorhabdus bovienii.

Xenorhabdus bovienii wild-type strains lack a functional receptor protein (LamB) in the outer membrane and as a result are unable to adsorb coliphage lambda (lambda). Introduction of plasmids encoding lamB into X. bovienii T228 results in constitutive expression of LamB in the outer membrane of this organism. LamB-expressing strains of X. bovienii adsorb lambda bacteriophage particles and can be used as hosts for lambda::Tn constructs. A Tn10-derived transposon, element 9 (J. C. Way, D. Davis, D. Morisato, D. E. Roberts, and N. Kleckner, Gene 32:369-379, 1984) was used to construct a variety of insertion mutants of X. bovienii. Mutants that had altered expression of protease, lipase, DNase, dye-binding capability, and hemolytic activity, in addition to a series of auxotrophic mutants, were isolated.

Journal Article↗
PubMed1980

Morphogenetic genes C and Nu3 overlap in bacteriophage lambda.

In bacteriophage lambda, genes C and Nu3, two of the four cistrons which are essential for normal prohead formation, have overlapping nucleotide sequences. These genes are translated in the same reading frame so that the Nu3 protein is identical to the COOH-terminal one-third of the C protein. This structural relationship may provide for the functional interaction of the C and Nu3 proteins through their regions of structural homology during prohead assembly. The in-phase overlapping organisation of genes may constitute a general strategy to facilitate the mutual interaction of a pair of proteins through their common structural domains.

Bacteriophage lambda↗
PubMed1991

Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda.

The bacteriophage lambda cIII gene product is an early regulatory protein that participates in the lysis-lysogeny decision of the phage following infection. We have previously shown that the translation of the cIII gene is determined by two unique factors: (1) efficient expression is dependent upon the presence of RNaseIII in the cell; (2) alternative mRNA structures of the cIII coding region determine the rate of its translation initiation. In this study we demonstrate the presence of the alternative mRNA structures in vivo. The presence of minor RNaseIII cleavage sites within this region indicate that RNaseIII can differentiate between the two alternative structures. We localize by a deletion analysis the RNaseIII responsive element to the cIII coding region, and suggest that regulation of cIII translation by RNaseIII is achieved through binding to the alternative structures region of the mRNA.

Bacteriophage lambda↗
PubMed1987

RNase III stimulates the translation of the cIII gene of bacteriophage lambda.

The bacteriophage lambda cIII gene product regulates the lysogenic pathway by stabilizing the lambda cII regulatory protein. Our results show that the expression of the lambda cIII gene is subject to specific requirements. Tests of a set of cIII-lacZ gene and operon fusions reveal that a sequence upstream of the cIII ribosome binding site is needed for cIII translation. The sequence contains an inefficient RNase III processing site. Furthermore, expression of cIII is drastically reduced in cells lacking RNase III. We have isolated a phage carrying a mutation (r1), which lies in the upstream sequence, that leads to a reduction in cIII translation and inactivates the RNase III processing site. The r1 mutant is nevertheless still dependent on RNase III for cIII translation; r1 reduces cIII translation by a factor of 3 in wild-type cells and by a factor of approximately equal to 30 in an RNase III mutant host. We propose that RNase III stimulates cIII translation by binding to the upstream sequence and thereby exposing the cIII ribosome binding site. This stimulation does not involve RNA cleavage. Consistent with this hypothesis is our finding that, in vitro, unprocessed cIII mRNA is translated, whereas RNase III-cleaved cIII mRNA is not.

Bacteriophage lambda↗
PubMed1991

A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda.

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.

Attachment Sites, Microbiological↗
PubMed1988

Late stages in bacteriophage lambda head morphogenesis: in vitro studies on the action of the bacteriophage lambda D-gene and W-gene products.

The in vitro maturation of bacteriophage lambda can be divided into discrete steps. Concatemers of lambda DNA bind terminase to form complex I. This DNA-terminase complex then binds a prohead to form a ternary complex (II). Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII, as well as phage tails. By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied. gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients. This complex requires only the addition of gpW, gpFII, and phage tails to form mature phage particles. The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA. In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III. This latter reaction converts complex III to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV). After isolation of the complex IV only gpFII and tails are required for mature phage formation in vitro. gpW is a heat-stable protein of molecular weight approximately 10,000.

Bacteriophage lambda↗
PubMed1975

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↗
PubMed1974

The distribution of crossovers along unreplicated lambda bacteriophage chromosomes.

The distribution of crossovers along unreplicated chromosomes of bacteriophage lambda has been examined by determining the density distributions and genotypes of particles in the progenies of crosses of density-labeled by ordinary parents in the presence of genetic blocks to replication. The Red and Rec systems combined produce crossovers primarily near the ends (especially the right end) of the chromosome. Removal of the generalized lambda recombination functions by red and gam mutations results in loss of these terminal crossovers; coupled with this loss is a disappearance of the differential dependence of recombination frequencies in terminal and central intervals on DNA synthesis. Removal of the bacterial system by a recA mutation results in severe depression of crossing over among unreplicated phage, with the few recombinants produced by the lambda system occurring near the right end.

Chromosome Mapping↗
PubMed1977

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↗
PubMed1968

Evidence for a new endonuclease synthesized by lambda bacteriophage.

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.

Centrifugation, Zonal↗
PubMedDate not supplied

[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↗
PubMed1983

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda.

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.

Amino Acid Sequence↗
PubMed1979

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↗