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PubMed1991

A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

Amino Acid Sequence↗

PubMed2006

Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine.

Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.

Bacteriophage lambda↗

PubMed1984

Initiation by bacteriophage T1 of DNA packaging at a site between the P and Q genes of bacteriophage lambda.

The growth of phage T1 on cells tandemly lysogenic for heteroimmune lambdoid prophages leads to a nonrandom packaging of lambda DNA by T1. A site, called esp-lambda, is located between the P and Q genes of lambda and results in increased packaging to the left by T1. When cloned into pBR322, the esp-lambda site causes a significant increase in transduction of the plasmid by T1. The nin5 deletion inactivates esp-lambda.

Bacteriophage lambda↗

PubMed2005

Display libraries on bacteriophage lambda capsid.

Phage display is an established technology that has been successfully applied, in the last fifteen years, to projects aimed at deciphering biological processes and/or at the isolation of molecules of practical value in several diverse applications. Bacteriophage lambda, representing a molecular cloning and expression tool widely utilized since decades, has also been exploited to develop vectors for the display of libraries on its capsid. In the last few years, lambda display approach has been consistently offering new enthralling perspectives of technological application, such as domain mapping, antigen discovery, and protein interaction studies or, more generally, in functional genomics.

Animals↗

PubMed1991

Bacteriophage lambda promoters pL and pR: sequence determinants of in vivo activity and of sensitivity to the DNA gyrase inhibitor, coumermycin.

Sequence encompassing the region between bp -43 and +8 of the pL and pR promoters of bacteriophage lambda, as well as sequence variants of these promoters, were compared with respect to their ability to drive a promoterless cat gene in vivo. For both pL- and pR-based promoters, variants with one nonconsensus bp rather than the consensus promoter were found to be maximally active. Determination of promoter function in CSH26 and C600 revealed a marked strain dependence in the activity of some promoter variants. In response to the antibiotic coumermycin, which effects a reduction in DNA superhelical density in vivo, promoters were found to be activated, inhibited or unaffected, depending on their sequence. No simple correlation between a particular response and sequence features of a promoter has become apparent.

Aminocoumarins↗

PubMed1985

E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda.

The NusA protein of Escherichia coli is a factor which mediates termination of transcription. In this paper, we demonstrate that the NusA protein can bind in vitro to a specific site on the mRNA of bacteriophage lambda. Several RNAs were synthesized by in vitro transcription of truncated lambda DNA templates, and the activity of NusA binding to these RNAs was examined by a Millipore filter-binding assay. RNAs containing the sequence immediately upstream of the boxA site were trapped on the filter by association with the NusA protein, but those lacking the site were not. Anti-NusA antibody inhibits this binding. To determine the binding site precisely, we developed a new method which we have named 'reverse-transcriptase mapping'. The RNA transcribed from the pL promoter was incubated with 32P-labelled DNA primer and NusA, and the primer-extension reaction was started by adding the reverse transcriptase. In this way, the primer extension was blocked at the position G of the boxA RNA sequence (5'CGCUCUUA 3'), indicating that the NusA-protection site is immediately upstream of boxA and includes the 5'-end C. The NusA protein purified from a temperature-sensitive nusA mutant defective in transcription termination showed reduced and thermolabile RNA-binding activity, suggesting that the RNA-binding activity is related to the physiological function of NusA.

Bacterial Proteins↗

PubMed1998

Inactivation of bacteriophage lambda, Escherichia coli, and Candida albicans by ozone.

The effects of ozone (O3) on three types of microbes were studied. Test suspensions were exposed to 600 ppm O3 at room temperature. Control experiments were performed under identical conditions using oxygen gas. Bacteriophage lambda was completely inactivated at 10 min while Escherichia coli and Candida albicans were only inactivated by factors of 10(5) and 10(4) respectively at 40 min. Exposure of a mixed microbial suspension to O3 for 5 min resulted in 100% killing of bacteriophages while the viability of E. coli remained unchanged. Various body fluids containing phages were exposed to O3. Compared to buffered solution, the decrease in phage titers was significantly slower in whole blood, plasma, and albumin. Both E. coli and C. albicans had increased production of thiobarbituric-acid-reactive substances with increased O3 exposure. 3H-labelled amino acids were incorporated into E. coli. O3 treatment resulted in a loss of radioactivity, indicating leakage of cytoplasmic contents. The data indicate that microbes are inactivated by O3 at different rates, possibly related to differential membrane permeability. The milieu in which microbes are present determines the effectiveness and outcome of O3 treatment.

Bacteriophage lambda↗

PubMedDate not supplied

[Thermo-regulated expression of the htpR gene under the control of the PR-promotor of bacteriophage lambda induces supersynthesis of heat shock proteins].

The mechanisms of induction of heat shock protein synthesis in E. coli have been studied. For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed. An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.

Autoradiography↗

PubMed1977

Characterization of the integration protein of bacteriophage lambda as a site-specific DNA-binding protein.

The Int protein specified by bacteriophage lambda is required for the recombination event that integrates the viral DNA into the host genome at its specific attachment site. Using a DNA-binding assay, we have partially purified the Int protein and studied some of the features of its binding specificity and regulation. The DNA-binding activity is attributed to Int protein because the activity is eliminated by a nonsense mutation or a deletion in the int gene, and is rendered thermolabile by temperature-sensitive mutations in the int gene. The DNA-binding activity is specific for DNA carrying an appropriate attachment site, suggesting that Int protein directs the sequence-specific recognition essential for integrative recombination. The specific DNA-binding activity is also missing after infection by phage carrying mutations in the cII and cIII regulatory genes of lambda. This finding corroborates the conclusion from other types of experiments that regulation of the int and cI genes by cII/cIII provides for coordinate regulation of both major events of the lysogenic response, establishment of repression and insertion of viral DNA.

Carrier Proteins↗

PubMed1977

Cro regulatory protein specified by bacteriophage lambda. Structure, DNA-binding, and repression of RNA synthesis.

The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth. We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property. As purified, Cro protein is a dimer of identical subunits of molecular weight 8600. The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins. In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis. A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development.

Amino Acids↗

PubMed1992

Involvement of the Escherichia coli RNA polymerase alpha subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins.

Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.

Bacteriophage lambda↗

PubMed1995

Inhibition of transcription starting from bacteriophage lambda pR promoter during the stringent response in Escherichia coli: implications for lambda DNA replication.

Replication of lambda plasmid DNA is halted in amino acid-starved wild type (stringent) strains whereas it proceeds in relA (relaxed) mutants. The only transcription which could be important in lambda plasmid DNA replication in amino acid-starved Escherichia coli cells is that starting from the pR promoter. Using a fusion which consists of the lacZ gene under the control of bacteriophage lambda pR promoter we found that transcription starting from this promoter was inhibited during the stringent, but not the relaxed, response in E. coli. We confirmed our conclusion by estimating the relative level of the pR transcript by RNA-DNA hybridization. We propose that decreased transcription from the pR promoter which serves as transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid replication during the stringent response. The results presented in this paper, combined with our recent findings (published elsewhere), indicate that the transcriptional activation of ori lambda may be a main regulatory process controlling lambda DNA replication not only during the relaxed response but also in normal growth conditions.

Bacteriophage lambda↗

PubMed1988

The relationship between lipophilic-hydrophilic balance, uptake and anti-bacteriophage lambda activity of experimental anti-tumour bisquaternary salts.

The uptake by Escherichia coli of a series of bisquaternary experimental anti-tumour agents (quinolinium 4-[p-9(-pyridylamino)phenylcarbamoyl]-aniline-bisalkyl dibromides) has been measured both by association of radiolabelled compounds and their inhibition of the vegetative replication of bacteriophage lambda (after heat inactivation of the phage repressor) as a measure of biologically effective intracellular drug concentration. Uptake of these compounds was correlated with biological effect, and was a function of both incubation temperature and the lipophilic-hydrophilic balance of the compound. At 30 degrees C uptake was drug concentration-dependent and was not readily reversible. No saturation of uptake was apparent over the concentration range tested. Preliminary experiments indicated that time-dependent drug uptake was also related to growth inhibition in cultured L1210 murine leukaemia cells. These results are consistent with the hypothesis that uptake occurs by diffusion across the plasma membrane followed by strong binding to cell constituents such as DNA. The approximate range of uptake of the most active compounds, using an external drug concentration of 1 microM, are 100 and 2400 molecules/s respectively for bacteria and murine leukemia cells. For bacteria, the uptake of approx. 2 X 10(5) molecules of drug/cell inhibits the yield of phage lambda by 90%.

Animals↗

PubMed1994

Damage and mutagenesis of E. coli and bacteriophage lambda induced by oxathiolane and aziridinyl steroids.

lambda-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the radiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of lambda phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathiolane steroid treatment of lambda cI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32 degrees C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage lambda has been suggested.

Aziridines↗

PubMed1980

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.

Bacteriophage lambda↗

PubMedDate not supplied

[1H-NMR study of the OR1 operon in bacteriophage lambda. I. Assignment of signals from imino protons and adenine C2 protons].

An oligodeoxyribonucleotide composed of 17 residues, d(TATCACCGCCAGAGGTA), and a complementary chain were synthesized. Their duplex was identical with the operator OR1, the binding site for bacteriophage lambda cro and c1 repressors. The 1H NMR spectra (500 MHz) of the duplex imino and aromatic protons were studied at 10, 20 and 25 degrees C. Signals from the imino protons of complementary base pairs and from the C2 protons of adenine (with the exception of the duplex terminal nucleotides) were assigned using the NOE technique and the known characteristics of short DNA fragment melting. No signals from the imino protons of the terminal base pairs were detected even at 10 degrees C due to fraying which increased as the temperature was raised. The assignment of signals can be used to identify centers of interaction between the operator OR1 and repressors, as well as to study possible local changes in DNA geometry.

Adenine↗

PubMed2000

Transfer of conjugative plasmids and bacteriophage lambda occurs in the presence of antibiotics that prevent de novo gene expression.

Plasmids transferred between bacteria prevented from expressing genes by the presence of bacteriostatic antibiotics. Whereas it has long been known that de novo gene expression is not required in donor cells for conjugation, the observations reported here extend the autonomy of plasmid transfer to the early events of establishment in recipients. In addition, this phenomenon was extended to bacteriophage lambda.

Bacterial Proteins↗

PubMed1990

Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126.

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.

Bacteriophage lambda↗