Exchange of DNA in the recombination of bacteriophage lambda.
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Bacteriophage lambda↗
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Temperature-inducible amber suppressor: construction of plasmids containing the Escherichia coli serU- (supD-) gene under control of the bacteriophage lambda pL promoter.
An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppressor tRNASer2am was identified and purified by being cloned into a plasmid vector. Information obtained from DNA sequence analysis was used to select a serU132 fragment for insertion downstream from the bacteriophage lambda pL promoter in two pBR322-lambda derivatives. In nonsense mutant strains bearing the resulting serU132 hybrid plasmids, the presence of the lambda cI857 repressor gene carried on the same plasmid or in a prophage genome permits thermal regulation of suppressor synthesis.
Bacteriophage lambda↗
Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target.
To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage lambda vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda in vitro packaging. After infecting and plating phage on a hfl- bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on lambda transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-5) in testes, were comparable to ranges in lambda transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and 6. 7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of lambda transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models.
Animals↗
The effects of Escherichia coli and yeast DNA insertions on the growth of lambda bacteriophage.
The effects of Eco RI endonuclease-cleaved Escherichia coli and yeast (Saccharomyces cerevisiae) DNA fragments on the propagation of the lambda bacteriophage vectors containing them were determined on a nonmutanted and a PolA E. coli K12 host. Observable alterations in the growth of hybrids containing yeast DNA insertions were less frequent and less extreme than those seen in hybrids containing E. coli DNA. A lambda-E. coli hybrid was selected after extensive growth on the Pol A (deficient in polymerase I) host which also grew very well on the PolA+ host and may have resulted from some alteration in the hybrid. Hybrids selected on the PolA host gave no evidence for the expression of polymerase I activity. No lambda-yeast hybrid made from the lambdagt vector lacking lambda-specific recombination (red-) had a yield of viable bacteriophage on infection greater than two-thirds that of "wild-type" lambda.
Coliphages↗
Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda.
A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.
Bacteriophage lambda↗
Integrative recombination of bacteriophage lambda DNA in vitro.
An in vitro system for the production of integrative recombinant DNA of bacteriophage lambda is described. The in vitro recombination mimics the in vivo integration of viral DNA into host DNA in its requirement for int gene product, for the presence of a thermolabile component, and for the limitation of the recombination to a pair of specialized sites (attachment sites) on the DNA. The enzymes are extracted from Escherichia coli containing phage lambda gene products. The substrate is the DNA from lambda-attB-attP, a phage variant that contains two attachment sites on the same chromosome. The product is a recombinant phage chromosome that has lost the DNA between the attachment sites. The parental and recombinant DNA are distinguished following transfection to mature phage in spheroplasts. The reaction requires ATP, Mg++, spermidine, and a monovalent cation. Recombination occurs preferentially between attachment sites on the same molecule. The enzymatic activity is completely inhibited by extracts containing xis gene product.
Adenosine Triphosphate↗
EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system.
A derivative of bacteriophage lambda has been modified and tested together with an appropriate host system to meet the criteria of EK2 biologic containment for cloning DNA from higher organisms. In this report certain of the safety features are summarized and some of the tests carried out to confirm the containment properties of the vector are described. The cloning efficiency of this system, together with available gene purification and hybrid screening technology, indicate that it can be used to clone DNA fragments carrying specific, unique mammalian genes.
Animals↗
Application of paper chromatograms to the study of inducers of lambda bacteriophage in Escherichia coli.
A procedure has been developed whereby paper chromatograms of agents which induce lambda bacteriophage in Escherichia coli can be developed using bioautographs with a lysogenic test system. Well-defined plaque-forming zones are produced indicating the area on the paper chromatogram where the active inducing material can be located. A mixture of the bacteriophage-inducing antibiotic, mitomycin C, and the noninducing antibiotic, paromomycin, was resolved into its components on paper strips with an ethyl acetate-methanol solvent system. The location of both antibiotics could thus be readily observed. Antibacterial and inducing activities were found to be identical with a crude fermentation solid, NSC-B-158,791. The use of this procedure for resolution of multicomponent inducing activities in antibiotic beers and for characterization of active components which may be potential antitumor antibiotics is indicated.
Antineoplastic Agents↗
The leftward promoter of bacteriophage lambda. Isolation on a small restriction fragment and deletion of adjacent regions.
As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.
Bacteriophage lambda↗
[Measurement of antibodies to double-stranded and single-stranded DNA using bacteriophage lambda DNA as the antigen--avidity of antibodies to DNA in patients with lupus nephritis].
IgG-class antibodies to double-stranded (ds) and single-stranded (ss) DNA in patients with collagen disease were measured by enzyme-linked immunosorbent assay (ELISA) using bacteriophage lambda DNA as the antigen. Moreover, avidity of IgG-class antibodies to both dsDNA and ssDNA in patients with systemic lupus erythematosus (SLE) was evaluated by ELISA as salt-dependent binding activities. The results are as follows: 1) Elevated IgG-class antibodies to dsDNA were largely restricted to patients with active SLE. Furthermore, the levels of IgG-class antibodies to dsDNA correlated with the disease activity of SLE. 2) Lupus nephritis (LN) patients with nephrotic syndrome (NS) or diffuse proliferative LN as determined by renal biopsy had high levels of IgG-class antibodies to both dsDNA and ssDNA. 3) LN patients with NS showed the highest means of avidity index of IgG-class antibodies to both dsDNA and ssDNA. These findings suggest that the present method of measuring IgG-class antibodies to dsDNA is useful for the diagnosis and management of patients with active SLE and that IgG-class antibodies to both dsDNA and ssDNA with high avidity may be an important factor in the pathogenesis of LN with NS.
Adult↗
Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells.
Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.
Bacterial Proteins↗
Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals.
The positions of RNA processing events mediated by RNase III and of two ribosome binding sites have been defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL) of bacteriophage lambda. Purified RNase III makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PL start. The positions of these cuts suggest a secondary structure for the RNase III recognition site which is similar to other RNase III sites in which double-stranded cleavage occurs. Structure mapping experiments reveal a pattern of cleavages made in the PL transcript by nucleases specific for single- or double-stranded RNA that support the structure proposed for the RNase III stem and provide clear evidence for the stem-loop formed in the RNA at the position of the N recognition (nutL) site. Our finding that ribosomes bind efficiently in vitro to the region of the PL transcript which includes the AUG codon at position 223 supports other evidence that this triplet is the N protein start codon. The existence of an additional ribosome binding site in the N gene leader region just downstream from the nutL site identifies a second position possibly used for translational initiation or regulation. Its occurrence suggests potential roles for ribosome interaction and/or translation of the leader RNA in regulating phage development and N gene expression.
Bacteriophage lambda↗
Segments of bacteriophage lambda (orf 221) and phi 80 are homologous to genes coding for mammalian protein phosphatases.
The amino acid sequences of mammalian protein phosphatase 1 and 2A were compared pairwise with every sequence in the National Biomedical Research Foundation protein sequence database using an exhaustive searching programme [Coulson et al., Comp. J. 30 (1987) 420-424]. The N-terminal half of the protein encoded by an open reading frame, orf 221, in bacteriophage lambda (nt 43,224-43,886 in the map of Daniels et al. [in Hendrix et al. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 519-676] shows 35% identity to either protein phosphatase 1 or 2A in this region. If conservative replacements are included the overall homology rises to 49%. A gene in phi 80 also shows 35% identity with the mammalian protein phosphatases. The results indicate that orf 221 of phage lambda and the homologous phi 80 gene may encode protein phosphatases. The possible roles of protein phosphorylation in the propagation of bacteriophage are discussed.
Amino Acid Sequence↗
[DNA inside bacteriophage lambda forms a Z-form].
The site of the transition regions between the B- and Z-forms of the DNA duplex (B-Z junction) may serve as marker of the existence of Z-DNA in situ. The structure of (dC-dG)10 insert in the bacteriophage lambda gt10 has been studied in situ by modification of B-Z junction with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analogue of hydroxylamine possessing specificity with respect to unpaired cytidine. This modification inhibited the process of restriction at BamHI site adjacent to the Z-insert. Judging by the extent of the inhibition about 5% of all inserts has been converted in Z-form. The certain role of Z-form at process of the packaging DNA into bacteriophage's capsid suggest.
Bacteriophage lambda↗
The mechanism of regulation of bacteriophage lambda pR promoter activity by Escherichia coli DnaA protein.
Apart from its function as an initiator of DNA replication, the Escherichia coli DnaA protein is also a specific transcription factor. It activates and represses a number of promoters. However, mechanisms of transcription stimulation by DnaA remained unknown. Bacteriophage lambda pR promoter is one of the promoters activated by DnaA. It was reported previously that DnaA binds downstream of the pR promoter and perhaps interacts with the RNA polymerase beta subunit. Here we demonstrate that DnaA positively regulates transcription from pR by stimulation of two steps in transcription initiation: RNA polymerase binding to the promoter region and promoter escape. For this transcription activation, two weak DnaA boxes located downstream of pR are necessary and sufficient. Such a mechanism of transcription activation and location of the activator-binding sites relative to the transcription start point are unusual in prokaryotes. Changes in the distance between the transcription start point and the first DnaA box by 5 and 10 bp and alterations in the orientation of these boxes did not abolish the stimulation of transcription by DnaA, but the efficiency of the promoter activation was different for various mutations. It seems plausible that formation of higher order nucleoprotein structures, involving DNA looping, is necessary for effective stimulation of the pR promoter. At high concentrations, DnaA is a repressor of pR rather than an activator. This repression was found to be because of inhibition of RNA polymerase binding to the promoter region.
Bacterial Proteins↗
Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.
Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?
Adenoviridae↗
[C1 and cro repressors of lambda phages. I. Construction of vectors for expression of cro repressor of bacteriophage lambda imm434].
Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised. These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434. The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped. The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning.
Autoradiography↗
Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation.
Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.
Amino Acid Sequence↗