Showing papers on "Lambda phage published in 1984"

Autodigestion of lexA and phage lambda repressors.

Abstract: Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli. In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction. I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein. This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reaction. Several lines of evidence argued that it was not due to a contaminating protease activity. Autodigestion was stimulated by alkaline pH. It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+. The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular. Purified phage lambda repressor also broke down under similar conditions to yield products like those resulting from recA protein action. Phage lambda repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo. This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway. These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease. The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene.

Abstract: We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.

Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus

Abstract: A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression of beta-galactosidase, the product of the lacZ gene, from Tn5 lac insertions in phage lambda depends both on insertion into a transcription unit in the correct orientation and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn5 lac can fuse lacZ expression to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was isolated and used to introduce Tn5 lac into Myxococcus xanthus, a bacterium that undergoes multicellular development. Stable kanamycin-resistant transductants are obtained that contain no P1 DNA sequences but have Tn5 lac inserted at different sites in the Myxococcus chromosome. Individual transductants express different levels of beta-galactosidase. A chromogenic substrate of beta-galactosidase, 5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus when cleaved in large amounts. In principle, Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured.

Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe.

Abstract: A single synthetic oligonucleotide was employed as hybridization probe to detect and enable isolation of the human insulin-like growth factor I (IGF-I) gene from a human genomic DNA library. The synthetic oligonucleotide probe coded for the B-chain of IGF-I and was designed for expression in Escherichia coli. Despite numerous interspersed mismatches, the synthetic probe hybridized specifically with seven recombinant lambda phage containing almost the entire B-chain region of the human IGF-I gene. The usefulness of this approach was further demonstrated by the detection of lambda phage containing human preproinsulin, using A and B chain synthetic oligonucleotides, 90 and 63 nucleotides in length, as hybridization probes. The nucleotide sequence of the human IGF-I exon suggests that IGF-I is synthesized as a larger precursor molecule.

Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor

Abstract: The thermal stabilities of mutant phage lambda repressors that have single amino acid replacements in the NH2-terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solvent-exposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.

Demonstration of two operator elements in gal: in vitro repressor binding studies.

Abstract: Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of two operator loci needed for gal repression. One (OE) is located immediately upstream to the two overlapping gal promoters and the other (OI) is inside the first structural gene. We have investigated the ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor. The repressor has been purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage lambda PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both O+E and O+I but not to OEc and OIc sequences. These results authenticate the proposed operator role of the two homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by greater than 90 base pairs.

Identification of the secY (prlA) gene product involved in protein export in Escherichia coli.

Abstract: The gene secY (or prlA) is essential for protein export across the cytoplasmic membrane of Escherichia coli The protein product of secY has been identified using the gene cloned under the control of the pL promoter of phage lambda in combination with the maxicell system The protein was found to have some unusual properties First, it is important not to heat the protein at 100 degrees C in the SDS sample buffer for its subsequent detection by gel electrophoresis Second, migration of the protein in SDS-polyacrylamide gel electrophoresis is variable depending on the gel compositions Gels with stronger sieving effect give higher apparent molecular weights These properties are similar to those of hydrophobic proteins of the cytoplasmic membrane, such as the lactose permease Finally, a major fraction of the protein synthesized from the overproducing plasmid is degraded rapidly in vivo The altered protein from the secY24 mutant gene is even more unstable These results provide information which is basic for the dissection of the protein export machinery of the bacterial cell

Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step.

Abstract: We isolated a plaque-forming derivative of phage lambda, lambda placMu1 , that contains sequences from bacteriophage Mu enabling it to integrate into the Escherichia coli chromosome by means of the Mu transposition system. The Mu DNA carried by this phage includes both attachment sites as well as the cI, ner (cII), and A genes. Lambda placMu1 also contains the lacZ gene, deleted for its transcription and translation initiation signals, and the lacY gene of E. coli, positioned next to the terminal 117 base pairs from the S end of Mu. Because this terminal Mu sequence is an open reading frame fused in frame to lacZ, the phage can create lacZ protein fusions in a single step when it integrates into a target gene in the proper orientation and reading frame. To demonstrate the use of this phage, we isolated lacZ fusions to the malB locus. These showed the phenotypes and regulation expected for malB fusions and could be used to isolate specialized transducing phages carrying the entire gene fusion as well as an adjacent gene (malE). They were found to be genetically stable and rarely (less than 10(-7] gave rise to secondary Lac+ insertions. We also isolated insertions into high-copy-number plasmids. The physical structure of these phage-plasmid hybrids was that expected from a Mu-dependent insertion event, with the lambda placMu prophage flanked by the Mu attachment sites. Lac+ insertions into a cloned recA gene were found at numerous positions and produced hybrid proteins whose sizes were correlated with the position of the fusions in recA.

Enzymes and sites of genetic recombination: studies with gene-3 endonuclease of phage T7 and with site-affinity mutants of phage lambda.

Abstract: The ability of bacteriophage T7 endonuclease I to cleave Holliday structures was invesigated. Holliday structures are an intermediate in genetic recombination, and their cleavage is essential for bateriophage growth. 37 references, 11 figures.

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r.

Abstract: The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.

Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

Abstract: A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta-galactosidase gene of phage lambda Charon 16 so as to obtain expression of random protein fragments as fusion proteins. The monoclonal antibody is used to screen the phage library to isolate phage expressing the specific antigenic determinant. DNA of immunoreactive phage can be analyzed rapidly and subcloned to allow DNA sequence determination. The method is generally applicable and permits antigenic determinants of functionally interesting monoclonal antibodies to be mapped and related to specific protein sequences. We have used this procedure to determine the region of the feline leukemia virus envelope protein gp70 recognized by a virus-neutralizing monoclonal antibody, cl.25. Antibody binding was mapped to a 14-amino acid region in the amino-terminal half of gp70. This region may be directly involved in an essential function of the gp70 protein, perhaps in gp70-mediated host recognition functions. Synthetic peptides derived from this region may provide useful vaccine antigens for the prevention of feline leukemia virus-associated disease in cats.

DNA methylation pattern is determined by the intracellular level of the methylase.

Abstract: Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage lambda DNA was observed after thermal induction of a lambda c1857 lysogen while the integrated lambda phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and lambda phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.

Cloning of terminal transferase cDNA by antibody screening.

Abstract: A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.

Regulation of outer membrane protein synthesis in Escherichia coli K-12: deletion of ompC affects expression of the OmpF protein.

Abstract: A chromosomal deletion beginning at a Tn10 located ca. 8 kilobases upstream from the ompC structural gene and extending through the 2.6-kilobase HindIII fragment carrying the ompC was isolated. The 2.6-kilobase ompC fragment was cloned into lambda 540 to obtain phage lambda 540C1. When the deletion mutant was lysogenized with lambda 540C1, the resulting strain produced normal levels of OmpC protein, and expression of this protein was regulated by osmolarity, carbon source, and the lc gene of phage PA-2, indicating that the cloned fragment contained all of the information required for regulated expression of ompC. The strain carrying the deletion was partially constitutive for expression of OmpF protein, whereas the lambda 540C1 lysogen of this strain and other strains with mutations in ompC repressed OmpF synthesis under conditions which lead to high-level expression of OmpC protein. Strains which are diploid or triploid for ompC show strong inhibition of synthesis of OmpF protein. We conclude that a regulatory element located upstream from the ompC coding sequence inhibits translation of OmpF protein under conditions which favor OmpC expression. Since ompF is known to repress transcription of ompC, we propose that these two genes constitute a closed regulatory loop which acts to amplify regulatory signals which control expression of these proteins.

Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene

Abstract: A set of plasmids has been constructed that carry a constitutive lamB gene (LamBc phenotype) from Escherichia coli and that confer functional phage lambda receptors to bacteria other than E. coli. This E. coli LamBc strain has been selected to escape both maltose-inducible and glucose-repressible control. Constitutivity results from an IS-3 insertion, carrying a mobile promoter, proximal to lamB. The LamBc DNA has been cloned into both broad and narrow host-range plasmids, and the resulting pTROY plasmids have been transferred to diverse bacteria. Both Salmonella typhimurium/pTROY and Klebsiella pneumoniae/pTROY strains efficiently adsorb phage lambda; Pseudomonas aeruginosa/pTROY strains do not. Introduction of a functional E. coli LamB protein into foreign bacterial will allow these bacteria carrying pTROY plasmids to be infected by phage lambda recombinant DNA libraries, phage lambda::Tn insertion mutagenesis vectors, and in vivo lambda-packaged cosmids.

Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA.

Abstract: Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C. This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature.

Isolation and bacterial expression of a murine alpha leukocyte interferon gene.

Abstract: A murine alpha interferon gene (MuIFN-αA) was isolated from a murine genomic DNA library in phage lambda. The gene was cloned and its sequence determined. It was expressed in Escherichia coli under...

Overproduction and purification of the connector protein of Bacillus subtilis phage ø29

Abstract: A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E coli protein after 4 hours of induction These proteins represent less than 1% of the B subtilis protein in phi 29-infected cells Protein p10 has been highly purified from the E coli cells carrying the recombinant plasmid Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B subtilis

Regulated protein production using site-specific recombination

Abstract: In vivo regulation of protein production is achieved by rearranging DNA segments comprising a protein-producing gene (i.e., protein-encoding DNA as well as regulatory DNA to effect the expression of the protein-encoding DNA in the host), in response to a change in an environmental condition such as temperature. The rearrangement is synchronized and directional (irreversible) in members of the cell population, because it is catalyzed by a lambda phage site-specific recombination enzyme system that operates on a pair of lambda phage attachment sites to rapidly drive the rearrangement and to avoid the reverse reaction. The cells include means to produce the lambda enzyme system in response to the change in environmental condition. By engineering one of the attachment sites within the gene that produces the protein whose production is to be regulated (yielding two gene segments), the synchronized rearrangement operates to change the gene from one configuration to another. In only one of these configurations are the gene segments positioned and oriented for protein production. Specifically, the protein-producing configuration is: gene segment one-attachment site-gene segment two. The attachment site is exogeneous to the gene, i.e, it does not occur in that location naturally and is positioned there by engineering techniques. The regulated protein production is useful, e.g., in fermenting the desired product, by allowing cell growth to proceed in the absence of product formation. When the desired cell mass is achieved, product production is enabled by the rearrangement.

Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance.

Abstract: We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him-. These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature.