Showing papers on "Lambda phage published in 1977"

Lambdoid phages that simplify the recovery of in vitro recombinants.

Abstract: Derivatives of phage lambda are described for use as vectors for fragments of DNA generated with the HindIII and EcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the lambda gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of the lacZ gene of E. coli able to complement a lacZ host. The appropriate lacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.

A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways

Abstract: A mutant of E. coli (designated the STS mutant) has been isolated in which the phage induction and error-prone DNA repair pathways appear to be expressed constitutively without the cells having received an inducing signal. Phage lambda was not able to lysogenize this mutant, whereas a noninducible mutant of lambda, lambdacIind-, known to synthesize a repressor that is insensitive to the induction mechanism, lysogenized it normally. This result suggested that normal phage repressor was synthesized in the STS mutant but was then inactivated by the induction mechanism. The STS strain also had mutator characteristics, and showed spontaneous, error-prone repair of UV-damaged phage lambda. Derived from a lexA tif sfiA parent strain, the STS mutant carried an additional mutation spr at the lexA locus that resulted in a high level of expression of the induction pathways. The properties of this and related strains provide additional evidence that induction of phage and induction of error-prone DNA repair occur by a similar mechanism, and further suggest a model for the regulation of these pathways.

UV-induced alleviation of K-specific restriction of bacteriophage lambda.

Abstract: A partial release of K-specific restriction of phage lambda grown in Escherichia coli C was observed when E. coli K strains AB1157 (having wild-type repair of UV-produced DNA damage) and AB1886 (uvrA) were irradiated with UV light before infection. The effect occurred in AB1886 at lower UV fluences than it did in AB1157. Little or no release of restriction was observed when AB2463 (recA) or AB2494 (lex-1) was used. Such release of restriction appears to be another of the UV-induced phenomena associated with "SOS" repair.

Isolation and characterization of conditional-lethal rho mutants of Escherichia coli

Abstract: Temperature-sensitive nitA (rho) mutants of E. coli were isolated; one of them was characterized as an amber mutant. These strains show the Nit phenotype (transcription of phage lambda DNA independent of the N gene) at low temperatures and are inviable at high temperatures. The mutated sites appear to be between cya and metE on the chromosome. Temperature-sensitive nitA bacteria not only permit leftward transcription of the lambda genome at a high rate in the absence of the lambda N protein, but also allow lambda growth at low temperatures. At high temperatures, phages lambda and T4 are incapable of normal development in these cells, while growth of T7 is not affected. The production of thermally unstable rho by the nitA temperature-sensitive mutant suggests that nitA is the structural gene for rho.

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

Abstract: A λ phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the α and β subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperaturesensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the α and β subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.

uv-induced alleviation of K-specific restriction of bacteriophage lambda. [Escherichia coli]

Abstract: A partial release of K-specific restriction of phage lambda grown in Escherichia coli C was observed when E. coli K strains AB1157 (having wild-type repair of uv-produced DNA damage) and AB1886 (uvrA) were irradiated with uv light before infection. The effect occurred in AB1886 at lower uv fluences than it did in AB1157. Little or no release of restriction was observed when AB2463 (recA) or AB2494 (lex-1) was used. Such release of restriction appears to be another of the uv-induced phenomena associated with ''SOS'' repair.

Characterization of lambda Escherichia coli hybrids carrying chemotaxis genes.

Abstract: Molecular cloning techniques were used to construct hybrid Escherichia coli lambda phage and isolate Col E1 factors that carried the cheB region of the E coli genome The products of these genes were examined by using a series of deletions in the phage to stimulate specific polypeptide synthesis in ultraviolet-irradiated cells and by using Col factor to program protein synthesis in minicells Seven flagellar related polypeptides were synthesized Three of these with apparent molecular weights of 38,000, 28,000, and 8,000 were associated with the cheB region; three polypeptides 63,000, 61,000, and 60,000 were associated with the region that maps between cheB and cheA These bands were referred to as the triplet group We suggest that these polypeptides are the same as the methyl-accepting chemotaxis protein described by Kort et al (Proc Natl Acad Sci USA 72:3939-3943, 1975) Another polypeptide with a molecular weight of 12,000 is associated with the cheA region which also produces at least three gene products We conclude that the cheA-cheB region in E coli is complex Further genetic and biochemical analyses are required to describe all of these products

Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII.

Abstract: A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.

Formation of recombinant DNA of bacteriophage lambda by recA function of Escherichia coli without duplication, transcription, translation, and maturation

Abstract: Genetic recombination of phage lambda DNA mediated by Rec function of Escherichia coli was studied in the absence of duplication, transcription, translation, and maturation. Cells were jointly infected with double amber mutants, λ D - F 1 - and λ S - R - , and incubated in the presence of chloramphenicol and rifampin. The am + recombinant DNA molecules formed within the cell were detected by in vitro packaging as viable recombinant phages. This system was used to measure the recombination activity of rec - bacteria. In recA or recA recB bacteria, the number of recombinant DNA molecules was about 1% of the rec + level. In contrast, almost normal numbers of recombinant DNA molecules were formed in recB or recC cells.

Mutagenesis of lambda phage: 5-bromouracil and hydroxylamine.

Abstract: Mutagenesis by 5-bromouracil of lambda phage to clear plaque formers does not depend on the recA function of the host E. coli cell or on the red function of the phage. Pretreatment of the host cells with ultraviolet light does not affect bromouracil mutagenesis of the adsorbed phage. Mutagenesis by hydroxlamine to clear plaque formers takes place at a high level in recA - host cells, and is not changed by preirradiation of rec + (wild type) hosts with ultraviolet light. Thus, bromouracil and hydroxylamine appear to mutate λ phage by a process which differs from that responsible for ultraviolet mutagenesis. Two characteristics of bromouracil mutagenesis —the nonlinear dependence of the number of mutants on bromouracil incorporation, and a high frequency of heterozygotes — fit in with Rydberg's (1977) picture of bromouracil mutagenesis as a consequence of base mispairing, with mismatch repair removing the mutations at low incorporation of the analog.

Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

Abstract: Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.

Genetic and physiological control of host cell lysis by bacteriophage lambda.

Abstract: The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.

Transduction of bacteriophage lambda by bacteriophage T1.

Abstract: When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.

Isolation and characterization of lambda pleu bacteriophages.

Abstract: In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage lambda into cistron leuA. The orientation of lambda in the chromosome is ara leuDCB lambda JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., lambda pleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., lambda pleu 13) transduces leuA strains to prototrophy. lambda pleu 13 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 lambda pleu13). A phage carrying the entire leucine operon (lambda K2) was constructed by a cross between lambda pleu9 and lambda pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with lambdaK2 indicated that leuO and leuP are present and functional in lambda K2. leu-specific messenger ribonucleic acid from E. coli hybridizes to the heavy (r) strand of lambdaK2. The leucine operon of lambda G4 pleuABCD (an S7 derivative of lambda K2) exists intact on a 7.3 x 10(6)-dalton fragment (lambdaG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between lambda G4 and lambda show a 5.4 x 10(6)-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 10(6)-dalton piece of lambda DNA starting at 0.46 fractional unit on the map of lambda. Fragment lambda G4EcoRI-B has about 0.6 x 10(6) daltons of lambda DNA from the b2 region at one end and about 1.4 x 10(6) daltons of lambda DNA from the int region at the other end.

DNA sequence at the end of the cI gene in bacteriophage λ

Abstract: The nucleotide sequence of 57 base pairs near the end of the cI gene in phage lambda is presented. This sequence was determined by direct sequencing techniques and includes the codons for 11 carboxyterminal aminoacids of the cI product, the lambda repressor. The sequence reveals that the cI gene, which has recently been shown to have a unique initiation region, is terminated by a UGA codon. A GUG triplet, which could act as a translation start signal for the rex gene occurs 8 base pairs beyond the cI termination codon. This GUG triplet is preceded by a sequence that could serve as a strong ribosome binding site for the rex message.

Isolation and characterization of plaque-forming lambdadnaZ+ transducing bacteriophages.

Abstract: The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with lamdacI857 integrated at a secondary attachment site near purE, transducing phages (lambdadnaS+) that transduced a dnaZts (lambda+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the lambda DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i,e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of lambda DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed lambda DNA between the points 39.3 to 66.5% of lambda length (measured in percent length from the left and of lambda phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard lambda transducing phages are deleted for one prophage end only. In lambdagal and lambdabio strains, the deletions of lambda DNA begin at the union of prophage ends (i.e., position 57.3% of lambda length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A, D. Hershey [ed.], The Bacteriophage Lambda, p. 45-82, 1971). Models for formation of the lambdadnaZ+ phages are discussed.

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Abstract: Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.

Transcription of a mammalian sequence under phage lambda control.

Abstract: RECOMBINANT DNA techniques have recently enabled bacterial plasmids carrying eukaryotic sequences syn-thesised in vitro from purified mRNAs to be constructed and cloned in Escherichia coli1–10. We show here that a rabbit globin sequence is not transcribed in E. coli when carried by the pCR1 plasmid. However, after transfer into phage λ, the globin sequence is transcribed under λ control. Thus, there is no basic hindrance to the transcription of a specific mammalian sequence in E. coli.

Restoration of Immunity in Lysogens Carrying Prophage P2, Derepressed at High Temperature

Abstract: The possibility that a strain lysogenic for phage P2 could be brought into the so-called “antiimmune” state in which the synthesis of phage repressor is permanently turned off, was tested in the following way. Two lysogenic strains that could be derepressed at 42°C were prepared. In one, the prophage had, in addition to a temperature-sensitive repressor mutation (c5), amber mutations in the two early genes A and B. In the other, the prophage had an unknown defect that blocked expression of the A and B genes. Both strains could multiply at 42° C as well as or almost as well as a non-lysogen. After the strains had grown for several generations at 42° C, they were returned to 30° and the resynthesis of repressor was followed by measuring the restoration of immunity to super-infection. In both cases, the immunity returned slowly over a period of 2 to 3 h. In a strain made doubly lysogenic for two amA amB c5 prophages, immunity was restored at a more rapid rate, suggesting that the rate of restoration depended mainly on the number of copies of repressor gene present. Attempts to demonstrate “channeling” towards the lytic pathway in the derepressed lysogens was also negative. The temperature treatment tended instead to increase the frequency of lysogenization of superinfecting P2. Thus, the presence of a system, similar to the cro system described for phage lambda, to regulate repressor synthesis in phage P2 could not be demonstrated.

The development of a lambda phage vector for recombination cloning of mammalian genes

Abstract: Top of pageAbstract The successful cloning of genes of higher organisms depends on the development of a simple and safe organism for recombination. In the Edinburgh Laboratory of Ken and Noreen Murray the Lambda phage was altered to permit the insertion of eucaryotic DNA. This phage has been modified to prevent lysogeny and recombination with the host E. coli. In addition, we have added three amber mutations to the phage (in genes for lysis, head formation, and capsid protein). This prevents growth of the phage in any wild-type, non-suppressor host. This phage is useful for cloning DNA because of the limited number of endonuclease restriction sites and the presence of a tryptophan gene. It has two targets for the endonuclease Hind III which are on each side of the tryptophan gene. The Hind III nuclease cuts the phage DNA into 3 separate fragments which can be rejoined with DNA ligase. The presence of the trp gene can be detected on a trp deficient E. coli host. The trp gene can also be replaced with a mammalian DNA of a molecular weight up to 15 × 106. In exchanging a new DNA for the trp DNA, the phage phenotype changes to permit easy selection of recombinant phage. We have recombined this phage with Xenopus ribosomal genes. This cloned DNA was labeled and used for mapping the human ribosomal genes and for in situ hybridization with human chromosomes.

Rolling circle replicative structure of bacteriophage lambda DNA in a recombination deficient system.

Abstract: Rolling-circle replicating structures which represent late stage lambda DNA replication can be detected among intracellular phage lambda DNA molecules under recombination deficient conditions as well as in wild-type infections. Furthermore, if initiation of lambda replication is delayed until the late stage of lambda infection, then nearly all replicating molecules are rolling-circle, even in the first round. Thus neither genetic recombination nor termination of a round of replication are required for generation of rolling-circle replicating molecules of lambda DNA.

Inhibition of beta-galactosidase synthesis in Escherichia coli after infection with different DNA and RNA phages.

Abstract: Infection ofEscherichia coli with T1, T2r+, T3 and T4 phages leads to an immediate inhibition of β-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of β-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.

Transcription of insertion elements IS1 and IS2 in vitro.

Abstract: Insertion elements IS1 and IS2 integrated within the gal operator-promoter region, an IS1 element in gene galT and insertions IS1 and IS2 integrated in the xycIIOP region of phage lambda were transcribed in vitro with E. coli RNA-polymerase. The insertion elements are transcribed exclusively by polymerase molecules started at the gal promoter and the lambdaPR promoter respectively. No promoter exists on IS1 or IS2 which can be recognized by RNA-polymerase in the pure in vitro transcription system used. Both insertions apparently are transcribed with a lower elongation rate than gal operon DNA or lambdaDNA. RNAs transcribed from the termini of IS1 and IS2 respectively were analysed by hybridization experiments. They are different in sequence.

[Mapping of the c-region of phage phi80].

Abstract: A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.