PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Bacteriophage lambda”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources
PubMed1983

Differential binding of RNA polymerase to the pRM and pR promoters of bacteriophage lambda.

Escherichia coli RNA polymerase binding to the promoters pR and pRM of bacteriophage lambda was visualized and quantitated by electron microscopy. Although the two promoters are located close together in the phage genome, their proximity to the end of an 889-bp HaeIII DNA fragment made it possible to position binary complexes within 18 bp (2%) intervals. Thus, polymerase binding to pR and pRM could be distinguished by comparing the locations of binary complexes formed with wild-type and mutant (prm-) DNA at 37 degrees and 15 degrees C. We found that at 37 degrees C, RNA polymerase bound primarily to pR, while at 15 degrees C the efficiency of binding was the same at pRM as at pR. In addition, at 15 degrees C the overall efficiency of binding was significantly reduced relative to that at 37 degrees C. When the enzyme was incubated with prm- DNA, binding to pRM was reduced at both temperatures, as expected. Reduced binding to pRM was accompanied by an increase in binding to pR, apparently as a consequence of the low enzyme-to-DNA ratios used in these experiments.

Bacteriophage lambda↗
PubMed1989

Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication.

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.

Bacterial Proteins↗
PubMed1992

The Rex system of bacteriophage lambda: tolerance and altruistic cell death.

The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.

Adenosine Triphosphate↗
PubMed1993

Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-lambda is proposed for this unusual prokaryotic enzyme.

Amino Acid Sequence↗
PubMed1986

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.

Attachment Sites, Microbiological↗
PubMed1981

Beta protein of bacteriophage lambda promotes renaturation of DNA.

The protein encoded by the red beta gene of bacteriophage lambda was found to promote reannealing of complementary single strands of DNA. Reannealing activity was optimal at pH 6.0 and required a divalent cation. A threshold temperature of at least 15 degrees C was necessary in order to detect activity. The reaction was linear with time for about 20 min, but the extent of reaction was dependent upon the amount of beta protein added. Reannealing of complementary single strands was confirmed by measuring increased lability of DNA to lambda exonuclease. When protein preparations from red- lysogens were tested for ability to promote reannealing, high activity was observed in a mutant altered in the gene for exonuclease, but there was low activity in a mutant altered in the gene for beta protein.

Bacteriophage lambda↗
PubMed2001

The bacteriophage lambda attachment site in wild strains of Escherichia coli.

The attachment site (attlambda) of bacteriophage lambda was examined in wild strains of Escherichia coli. Although the att region is non-coding, the DNA sequence was invariant in the 13 strains examined. Two other non-coding regions showed nine changes, all associated with a single strain. In four of 33 strains, sequences were inserted in or near the attlambda site and in two of these the insert was related to lambda. Among strains that can be lysogenized by lambda, integration was via the attlambda site in all cases. Some resistant strains can be lysogenized, and these have been termed "lenient." Most of these fail to give normal phage yield after induction. In some cases rare lysogens have been formed in cells that belong to a mutant subpopulation.

Bacteriophage lambda↗
PubMed1980

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function.

Escherichia coli mutants, called groNB, which block the growth of bacteriophage lambda at the level of action of the gene N product, have been isolated as survivors at 42 degrees C of bacteria carrying a) the defective prophage lambda bio11 i lambda cI857 delta H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage lambda cI857. In addition, groNB bacterial mutants have been isolated at 37 degrees C, as large colony formers in the presence of lambda i lambda cI h434, lambda i lambda cI h lambda, and lambda i lambda cI h80 phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42 degrees C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of lambda growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro+ for lambda growth. Lambda transducing phages carrying the groNB+ bacterial gene have been isolated. GroNB mutant bacteria lysogenized by the transducing phage acquire the Gro+ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.

Bacteriophage lambda↗
PubMed1998

Guanosine tetraphosphate (ppGpp)-mediated inhibition of the activity of the bacteriophage lambda pR promoter in Escherichia coli.

It was previously demonstrated that the activity of bacteriophage lambda promoter pR is decreased in wild-type Escherichia coli cells starved for amino acids (during the stringent response). Since pR activity is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda. These results led to the proposal that the pR promoter susceptible to control by the stringent response. However, subsequent studies demonstrated that this promoter is activated by the host dnaA gene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of pR activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation. Here we present evidence that pR is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA function. We have checked that the level of ppGpp is increased during prolonged (up to 4 h) starvation for isoleucine in relA+ cells but not in the relA- mutant. At the same time we observed inhibition of lambda plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced. Finally, we found that the activity of a pR-lacZ fusion is inhibited after gratuitously induced overproduction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product. We conclude that the activity of the pR promoter is inhibited directly by ppGpp.

Bacterial Proteins↗
PubMed1986

Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.

Amino Acid Sequence↗
PubMed1987

Bacteriophage lambda cloning system for the construction of directional cDNA libraries.

We have developed a bacteriophage lambda cloning vector, lambda ORF8, that can be used for the construction of cDNA libraries. The wild-type lambda genome contains five BamHI, five EcoRI, and seven HindIII restriction sites that have all been removed from the genome of lambda ORF8. Sites for these endonucleases are present within the multiple cloning site of lambda ORF8. We report a method for preparing cDNAs that can be cloned in a single orientation in our phage vector. The method utilizes the synthesis of double-stranded cDNA, including priming of first-strand synthesis by oligo(dT). After completion of second-strand synthesis, a bifunctional oligodeoxynucleotide linker is ligated to the cDNA fragments. This linker, which contains a BamHI restriction site, will create a HindIII restriction site when ligated to the 3' end of cDNA fragments. Subsequent treatment of methylated cDNA with HindIII and BamHI endonucleases allows these fragments to be cloned directionally into lambda ORF8. To demonstrate the utility of this cloning system, we prepared a library from 5 micrograms of mRNA isolated from phytohemagglutinin-stimulated human peripheral blood lymphocytes. The primary library contained 2 X 10(8) plaque-forming phage, at least 80% of which contain inserts. A portion of the library was examined for the presence of gamma-interferon-related clones to verify the method had generated a library that was representative of phytohemagglutinin-stimulated peripheral blood lymphocytes. This simple and efficient cDNA cloning system significantly reduces the amount of RNA and effort required for the preparation of large directionally cloned libraries.

Bacteriophage lambda↗
PubMed2003

The optimal eukaryotic signal for translation initiation from non-AUG codons, present upstream of bacteriophage lambda P cistron, is inactive in Escherichia coli.

Expression of the replication genes of bacteriophage lambda, O and P, is believed to be translationally coupled. However, it was previously noted that, under conditions of amino acid starvation, when O is not synthesized, P continues to be expressed at a relatively high level. The results presented in this report, contrary to the previously presented hypothesis, suggest that an AGACUGGAU sequence (an optimal context for translation initiation from non-AUG codons in eukaryotes, and present upstream the P cistron) is inactive in Escherichia coli. Comparative sequence analysis confirms that such a signal is unlikely to be important for P synthesis. Instead, a weak Shine-Dalgarno sequence may be present upstream the P cistron, and be active in the absence of O gene expression.

Bacteriophage lambda↗
PubMed1989

The rex genes of bacteriophage lambda can inhibit cell function without phage superinfection.

The rexA and rexB genes of bacteriophage lambda are expressed from the prophage and cause the exclusion of many superinfecting mutant phages. We cloned the rexA and rexB genes into a multicopy plasmid so that they were overexpressed from the inducible tac promoter. No obvious phenotypes were associated with overexpressing both rexA and rexB or overexpressing rexA in the absence of rexB expression. However, induction of rexA in the presence of limiting rexB activity caused an immediate cessation of cell growth. All macromolecular synthesis abruptly ceased and amino acid transport was severely inhibited. Intracellular levels of adenosine 5'-triphosphate also dropped. These phenotypes are similar to those observed after phage superinfection, leading us to propose that at least some of the exclusion caused by the Rex proteins could be due to a change in their ratio following superinfection.

Bacteriophage lambda↗
PubMed1977

HindII, HindIII, and HpaI restriction fragment maps of the left arm of bacteriophage lambda DNA.

The sites on the left arm of bacteriophage lambda DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were localized on the lambda physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of lambda were mapped by analysis of the digestion profiles of deletion and substitution derivatives of lambda, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites of the lambda genome between the left vegetative end and the b2 region were mapped entirely by succesive digestion experiments. The restriction fragment map for the right arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).

Chromosome Mapping↗
PubMed1973

Regulation of the expression of the N gene of bacteriophage lambda.

A quantitative assay for the N protein of bacteriophage lambda has been used to study the in vivo regulation of N gene expression. The assay makes use of the observation that in a cell-free protein-synthesizing system from Escherichia coli programmed with lambdaN(-) DNA the lambda endolysin is made only if N protein is added to the reaction. The rate of synthesis of N protein in vivo is negatively controlled by the products of the CI and tof genes of the phage. Furthermore, N protein activity is extremely unstable in vivo. During normal cell growth at 35 degrees , the half-life of N protein is about 2 min.

Cell-Free System↗
PubMed1976

Single burst study of rec- and red-mediated recombination in bacteriophage lambda.

Single bursts from three-point crosses of bacteriophage lambda were analyzed for recombination of markers several thousand nucleotide pairs apart. Single recombination, mediated by the rec system of Escherichia coli, is usually reciprocal. Double recombinants are also significantly correlated in single bursts, although the correlation is weaker than for reciprocal singles. Reciprocity is not found in crosses mediated by the lambda red system. With respect to certain other paraments of recombination, the two systems appear to be alike. Double recombinants usually result from one event, not from two independent single recombinations, and their average clone size is about half that of single recombinants. The results are discussed in terms of current molecular models of recombination.

Base Sequence↗
PubMed1984

Long range base-pairing in the leftward transcription unit of bacteriophage lambda. Characterization by electron microscopy and computer-aided sequence analysis.

Restriction fragments of bacteriophage lambda DNA corresponding to the major leftward transcription unit were purified, denatured to form single-stranded DNA, self-annealed, and examined by electron microscopy. Three intrastrand stem and loop secondary structures were observed reproducibly and the locations of the paired regions were determined. A method for computer-aided sequence analysis of these regions is presented and used to identify sets of base-pairings likely to account for the observed structures. One loop observed within gene Ea47 is postulated to involve pairing of sequences which include the polypeptide initiation and termination codons. Another loop is postulated to involve pairing of sequences in gene int with sequences located in the gam-cIII region. A third loop appears to involve sequences in and to the right of gene Ea22 paired with sequences located in the bet-gam region. A general discussion of base-pairing which gives rise to long range interactions is presented along with possible effects of the postulated models on gene expression.

Bacteriophage lambda↗