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PubMed1974

DNA polymerase I-dependent mutants of coliphage lambda.

Mutagenized lysates of bacteriophage lambda were screened for mutants unable to plate on DNA polymerase I-deficient (polA(-)) hosts. The mutants obtained were all recombination deficient (red(-)). These mutants, like red(-) and gam(-) mutants previously isolated by others, grow more poorly than wild-type lambda even on polA(+) hosts (burst size 14 to 30% of wild-type lambda.) In a polA(-) host, the burst size of red(-) and gam(-) mutants is reduced an additional five- to tenfold, and lysis is delayed. Wild-type lambda grows normally in polA(-) hosts. Neither lambdaN(-)nin (which doesn't express red or gam) nor lambdabio phages (from which all or part of the red-gam region is deleted) form plaques on polA(-) hosts. Apparent revertants, able to plate on polA(-) hosts, have been selected from both lambdaN(-)nin and lambdabio. Those derived from N(-)nin seem to be N(-)nin cro(-) mutants; whereas those coming from lambda bio have a new bypass mutation (pas) that lies between genes P and Q.

Chromosome Mapping↗
PubMed1997

UVB DNA dosimeters analyzed by polymerase chain reactions.

Purified bacteriophage lambda DNA was dried on a UV-transparent polymer film and served as a UVB dosimeter for personal and ecological applications. Bacteriophage lambda DNA was chosen because it is commercially available and inexpensive, and its entire sequence is known. Each dosimeter contained two sets of DNA sandwiched between UV-transparent polymer films, one exposed to solar radiation (experimental) and another protected from UV radiation by black paper (control). The DNA dosimeter was then analyzed by a polymerase chain reaction (PCR) that amplifies a 500 base pair specific region of lambda DNA. Photoinduced damage in DNA blocks polymerase from synthesizing a new strand; therefore, the amount of amplified product in UV-exposed DNA was reduced from that found in control DNA. The average lesion frequency per 500 base pair per strand at 16 PCR cycles was approximately 1.22, 1.00, 0.70 and 0.50 for 30 ng, 50 ng, 100 ng and 150 ng of dried DNA, respectively, after a total dose of 60 kJ m(-2) delivered with a solar UVB simulator. Although the average lesion frequency increases linearly with increasing doses for four different amounts of template DNA, the lesion frequency seems to be averaged by the amplified products from the protected lambda DNA molecules below the top few layers. The average daily dose, equivalent to the UV dose applied with the solar UVB simulator was 10.2 +/- 0.4 kJ m(-2) with the 50 ng containing DNA dosimeter in September 1995 in Melbourne, FL. Both 50 ng and 150 ng containing DNA dosimeters produced the same average daily dose within experimental error in January 1996, which was 5.2 +/- 0.3 kJ m(-2) at the same location. The dried lambda DNA dosimeter is compact, robust, safe and transportable, stable over long storage times and provides the total UVB dose integrated over the exposure time.

Australia↗
PubMed1998

Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector.

groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution. The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells. In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V. harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E. coli for bacteriophage lambda growth. Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E. coli groE region. Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E. coli. This confirmed that the lambda clone indeed carries the groE region of V. harveyi. The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria. The V. harveyi cloned groE genes did not suppress thermosensitivity of the E. coli groE mutants.

Bacteriophage lambda↗
PubMed1999

Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3.

Mapping of protein domains having a distinct function is essential to understanding the protein's structure-function relationship. We used a bacteriophage lambda surface expression vector, lambdafoo, in order to determine the minimal carbohydrate-binding domain of human galectin-3 (Gal-3). Gal-3 cDNA was randomly digested by DNase I and cloned into the phage vector. The library generated was screened by affinity selection using lactose immobilized on agarose beads. DNA sequence analysis of a set of isolated clones defined the minimal folding domain of Gal-3 required for lactose binding, which consisted of 136 amino-acid residues. Using the phage clones isolated, we also determined relative dissociation constants in solution between lactose and the minimal domain expressed on the phage surface. This technique does not require either purified or labeled proteins, and bacteriophage lambda surface display may, therefore, be useful for protein domain mapping and in vitro studies of various macromolecular interactions.

Antigens, Differentiation↗
PubMed1997

Chi-star, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain.

BACKGROUND: chi sequence (5'GCTGGTGG) of Escherichia coli was first identified as a site that increased the plaque size of bacteriophage lambda. Subsequent studies showed that this site is responsible for both the attenuation ofRecBCD exonuclease activity and the promotion of RecA, RecBCD-mediated recombination. It is known that bacteriophage lambda containing the chi site makes very small plaques on a recC* (recC1004) mutant because chi is not recognized by the RecBC*D mutant enzyme. RESULTS: We cloned E. coli chromosomal fragments in lambda which allowed lambda to form larger plaques on this recC1004 mutant. The fragments were found to share a chi-like 11-mer sequence, 5'GCTGGTGCTCG. Substitution of these fragments with a synthetic 11-mer of this sequence and single-base-pair substitution analysis of its last four nucleotides demonstrated that this sequence is both necessary and sufficient for the observed activity. The sequence, designated X* (chi-star), protected rolling-circle DNA replication in the recC1004 mutant and in the recBCD+ strain, most likely because it attenuated the exonuclease activity of the RecBC*D and RecBCD+ enzyme. chi-star, did not significantly stimulate lambda recombination in two assays. CONCLUSION: We have discovered that a mutant RecBCD enzyme responds, in vivo, to a longer chi variant.

Bacteriophage lambda↗
PubMed1987

Repair of a mismatch is influenced by the base composition of the surrounding nucleotide sequence.

Heteroduplexes with single base pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli. A series of transition (G:T and A:C) and transversion (G:A and C:T) mismatches located throughout most of the bacteriophage lambda cI gene has been examined. The results suggest that the transition mismatches are generally better repaired than the transversion mismatches and that, at least for the transversion mismatches studied, repair efficiency increases with increasing G:C content in the neighboring nucleotide sequence. This specificity of the E. coli mismatch repair system can account, in part, for the similar frequencies of base substitution mutations throughout the E. coli genome.

Bacteriophage lambda↗
PubMed1988

Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements.

Amino Acid Sequence↗
PubMed1990

Production of lambda-phi 29 phage chimeras.

Proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro. These hybrid proheads have a structure similar to that of normal lambda proheads. Furthermore, the chimeric proheads can package both lambda and phi 29 DNA. These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar. The DNA-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yield infective particles capable of DNA transfer.

Bacteriophage lambda↗
PubMed2005

On the role of Cro in lambda prophage induction.

The lysogenic state of bacteriophage lambda is exceptionally stable yet the prophage is readily induced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins; the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we confirm, in the native configuration, the previous observation that the DNA loop mediated by oligomerization of CI bound to two distinct operator regions (O(L) and O(R)), increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we show that the presence of the cro gene might be unimportant for the lysogenic to lytic switch during induction of the lambda prophage. We revisit the idea that Cro's primary role in induction is instead to mediate weak repression of the early lytic promoters.

Bacteriophage lambda↗
PubMed1999

Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences.

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage lambda and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated lambda DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage lambda DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-lambda-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after lambda DNA integration might have existed prior to lambda DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5'-CG-3' methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated lambda DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

Animals↗
PubMed1991

Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.

Bacteriophage lambda↗
PubMed1987

Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region.

New expression vectors were constructed for use in strains of Escherichia coli. Their most important feature is a polylinker system that facilitates the insertion of a gene in an optimal relationship to the highly efficient E. coli atpE translational initiation region (from nucleotide -50 to the start codon). Three ATG-containing restriction endonuclease sites can be used for the insertion of the 5' end of a gene at, or near to, its translational initiation codon. These sites may alternatively be used for the creation of a suitable translational start codon. Transcription is started by the bacteriophage lambda major promoters pR and pL in tandem and terminated by the bacteriophage fd terminator. Transcriptional initiation is very effectively repressed at 28-30 degrees C by the product of the bacteriophage lambda cIts857 gene, which is also present on the vectors. Full induction is achieved by shifting the incubation temperature to 42 degrees C. The combination of highly efficient transcriptional and translational signals on these vectors allowed high-level expression of sequences encoding human interferon beta and interleukin 2 and of the E. coli atpA, sucC and sucD genes.

DNA Restriction Enzymes↗
PubMed1998

Induction of mutations by 2-acetylaminofluorene in lacI transgenic B6C3F1 mouse liver.

Mutations induced in liver cells by the hepatocarcinogen 2-acetylaminofluorene (2-AAF) were characterized after i.p. administration on 4 consecutive days at 100 mg/kg per injection in male B6C3F1 Big Blue transgenic mice that harbored the Escherichia coli lacI reporter gene. Animals were sacrificed at 5, 10 or 60 weeks following the last injection, livers removed and DNA packaged in vitro into bacteriophage lambda particles. The bacteriophage were assayed for lacI function by plating on E. coli in the presence of X-gal. Approximately 3 x 10(5) plaques were assayed per animal. Solvent-treated control mice exhibited a slight increase in mutant frequency over time, from 3.93 x 10(-5) at 5 weeks to 5.02 x 10(-5) at 60 weeks. In contrast, treatment with 2-AAF yielded an approximately 2-fold increase in mutant frequency at 5 and 10 weeks after treatment relative to controls, with frequencies of 8.13 x 10(-5) and 7.43 x 10(-5) respectively. However, by 60 weeks post-treatment the mutant frequency was not significantly increased over concurrent controls. Similar to results in other systems, 2-AAF induced predominantly single base changes targeted to G:C base pairs, primarily G:C-->T:A transversions (27%). In contrast to results in other bacterial and eukaryotic systems, no deletions were observed among the 2-AAF-induced mutations and the 4 base hot spot deletion that is frequently observed in lacI in E. coli was not observed in this system, suggesting that the lacI transgene may be relatively refractory to frameshift mutations in vivo in the mouse.

2-Acetylaminofluorene↗
PubMed1986

Cooperative DNA binding by lambda integration protein--a key component of specificity.

Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies [(1982) J. Mol. Biol. 155, 447-466] has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (lambda attP) of bacteriophage lambda DNA. Optimal values of the equilibrium constant obtained were 3.0(+/- 1.0) X 10(10) M-1 for the P' site using a model of three sites with equal affinity and 1.9(+/- 0.4) X 10(10) M-1 for the P1 site on a two-site model. The value of the cooperativity parameter alpha is 172(+106)(-66) in all cases. The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential. The occurrence of binding site arrays distinguishes lambda attP very clearly from other DNA sequences containing single recognition sites by chance.

Allosteric Site↗
PubMed2005

Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function.

Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage-presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rac prophage. This type III restriction alleviation does not depend on lar on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.

Bacteriophage lambda↗
PubMed1993

Antigen-dependent stimulation by bone marrow-derived mast cells of MHC class II-restricted T cell hybridoma.

This paper describes a new role for mast cells as being able to present Ag to immune T cells. A mouse bone marrow-derived mast cell population obtained after 3 wk of culture in a conditioned medium has been shown to express a variety of membrane-associated Ag, including MHC class II and class I Ag, CD23, CD32, high affinity receptor for IgE, and CD4. Expression of MHC class II molecules was up-regulated upon stimulation with LPS but not with IFN-gamma and was down-regulated after exposure of mast cells to IL-3 treatment. We have demonstrated that mast cells were able to present native Ag as well as immunogenic peptides to MHC class II-restricted T cell hybridoma. The inhibition of Ag presentation after mast cells have been treated with ammonia suggests that Ag catabolism in intracytoplasmic compartment as a key step in Ag handling takes place in these cells. The MHC class II molecule is the restricting element for the presentation of OVA and the lambda repressor from bacteriophage lambda to a panel of specific T cell hybridomas, as demonstrated by the blocking effect of anti-MHC class II mAb on the Ag-presenting function. A characteristic feature of mast cells is the generation of a narrower immunogenic peptide repertoire as compared with A20 and LBB 3.4.16, a B lymphoma cell line, and a B cell hybridoma, respectively. This novel function of mast cells brings to a much closer connection inflammatory and immunologic processes and sheds new light on the biology of mast cells and particularly on the specific allergic responses.

Ammonia↗