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PubMed1980

DNA rearrangements in a hybrid plasmid carrying the redB imm region of coliphage lambda.

The hybrid plasmid consisting of pSC101 and the redB--N--imm region of phage lambda cI857 persists in cells grown at 30 degrees C but not in cells grown at 37 degrees C. In the latter case the plasmid was found to undergo several modifications. Restriction maps of these new plasmids indicate the following modifications: (1) the insertion of an IS1 element into gene N carried by the lambda fragment; (2) a mutation in the pL oL site of the same fragment, and (3) four large deletions (30 to 50% of the hybrid plasmid) which remove almost the entire lambda fragment. For the latter deletions, one endpoint seems to be fixed in the same restriction fragment of pSC101 while the other endpoint assumes four different positions on the lambda fragment; this might suggest a site-specific recombination event.

Bacteriophage lambda↗
PubMed1980

Packaging of plasmid DNA containing the cohesive ends of coliphage lambda.

High yields of ColE1::Tn3-cos lambda plasmid genomes packaged in phage lambda virions (2.10(9) per ml) are produced by thermal induction of E. coli W3350 (lambda cI1857S7) lysogens carrying the plasmid DNA. The plasmid DNA is packaged in the linear form, with the right m' terminus of lambda being associated with the lambda tail.

Bacteriophage lambda↗
PubMed1981

Antitermination and termination functions of the cloned nutL, N, and tL1 modules of coliphage lambda.

A plasmid containing a pp-galK operon was constructed to assay galK expression as a measure of transcriptional regulation by the cloned nutL, N and tL1 modules in a Rho+, Nus+ Escherichia coli host. Insertion of tL1 and rut, both carried on a lambda fragment located between the bamHI site and gene N, between the promoter and galK reduced expression of galK by 80--90%. Gene N alone, when controlled by pp, stimulated galK expression by about two-fold. Cloning of both gene N and nutL in the proper orientation resulted in about a 60% decrease in the tL1 termination. Additional tandem nutL sites increased efficiency of antitermination. A shortened nutL segment, containing only 25 bp of the original genomic nutL sequence, was found to have nearly equal ability to bring about antitermination. If the orientation of the nutL fragment is reversed, antitermination is abolished and the insert now displays a termination function. Termination efficiency is 60 to 73% for one to four tandem nutL modules in reverse orientation. Similarly, the inverted N module acts as a terminator, with 67% efficiency for one and 90% for two tandem inserts. Termination by inverted nutL or N fragments is probably unrelated to their normal functions, but indicates a fortuitous presence of a terminator sequence in the inverted orientation.

Bacteriophage lambda↗
PubMedDate not supplied

Sequence organization of the origins of DNA replication in lambdoid coliphages.

We have determined the sequences of the ori region DNA of several phage lambda mutants and hybrids, which shed light on the mechanism of DNA replication in the lambdoid phages. These include the heterologous substitution hybrids lambda rep82:lambda and lambda rep80:lambda, a pseudorevertant of the ori-r93 mutant lambda r93hot5, and the insertion mutant lambda pk35. The ori regions of the three lambdoid phages, lambda, phi 80 and 82, all have repeated sequences, termed iterons, and A . T-rich zones. We note that a similar arrangement of DNA is also found in several other prokaryotic origins of replication. lambda and phi 80 have four iterons, and 82 has five. The origin of lambda r93hot5 is unusual in that contains only three iterons, yet the phage grows normally. Analysis of this mutant indicates that the spacing of iterons is crucial to ori function, whereas their number is not. This argues against the cloverleaf model for lambda ori structure (Hobom et al., 1979). In lambda pk35 the drug resistance element Tn903 is inserted into the "inceptor" (ice) site, proposed to be crucial for lambda replication initiation (Hobom et al., 1979); yet this phage grows normally.

Bacteriophage lambda↗
PubMed1981

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles.

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Bacteriophage lambda↗
PubMed1982

Synthesis of the nutL DNA segments and analysis of antitermination and termination functions in coliphage lambda.

The nut antiterminator sequence, when present between a promoter and a terminator, permits the N-mediated antitermination of transcription in phage lambda. The efficiency of nutL was determined by assaying the activity of gene galK placed on a plasmid downstream from the promoter, nutL and terminator modules. As a reference and an estimate of the plasmid copy number, we have used an improved and very reproducible assay for bla activity. Sequences consisting of the 17-bp nutL core flanked by two HindIII cohesive sites were synthesized by the phosphite coupling method, and cloned in proper orientation between the Pp promoter of pBR322 and lambda gene N followed by the tL1 terminator on a galK-expression plasmid. The antitermination efficiencies for two synthetic 17-bp nutL sequences, one wild type and one point mutant at the base of the nutL stem, are similar but substantially reduced in comparison with the native 25-bp nutL sequence cloned at the same site in the otherwise identical galK-expression plasmid. Multiple tandem insertions of the synthetic 17-bp nutL segment successively increase antitermination efficiency, but also to levels below those of comparable plasmids carrying multiple copies of the native 25-bp nutL sequence. Thus, several specific base pairs in the flanking sequences appear to be important for the efficient nut function. In an inverted orientation the 17-bp nutL sequence has lost its antitermination function. It also lost the termination activity exhibited by inversion of the longer 25-bp and 74-bp native nutL sequences.

Bacteriophage lambda↗
PubMed1983

A cluster of leftward, rho-dependent t'J terminators in the J gene of coliphage lambda.

While searching for the N-unresponsive terminator described by Honigman (Gene 13 (1981) 299-309), a 1680-bp DNA fragment from within gene J of bacteriophage lambda was cloned into plasmid pD12 between promoter p'R and the galK gene. In vitro transcription of this plasmid and S1 mapping assays, together with nucleotide sequencing, demonstrated that this DNA fragment contains a cluster of at least four in rho+ Escherichia coli hosts and only 30% in rho- hosts at at 30 degrees C. At the elevated temperature of 42 degrees C, the rho-dependent termination component of the t'J cluster becomes somewhat leaky, with the readthrough increasing by about twofold. The t'J terminators appear to be less responsive to nutR- and N-mediated antitermination (efficiency of 84-87%) than tL3, which responds to the same antitermination function with an efficiency of 97%. The relationship between the t'J cluster and the same antitermination function with an efficiency of 97%. The relationship between the t'J cluster and the highly N-unresponsive leftward termination signal tJ, which is also located within the J gene region (Gottesman et al., J. Mol. Biol. 140 (1980) 57-75; Honigman, Gene 13 (1981) 299-309), is unknown.

Bacteriophage lambda↗
PubMed1983

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum.

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.

Bacteriophage lambda↗
PubMed1984

The maturation of coliphage lambda DNA in the absence of its packaging.

In vivo, lambda DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize lambda terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of lambda gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos. Since the only lambda genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe lambda maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another lambda gene that maps to the right of gene B.

Bacteriophage lambda↗
PubMed1984

Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL.

The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular lambda pL. Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30 degrees C and readily amplified by thermal induction of strains carrying a thermolabile lambda repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.

Bacteriocin Plasmids↗
PubMed1985

Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage.

In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.

Bacteriophage lambda↗
PubMed1985

Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements.

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR, was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.

Bacterial Proteins↗
PubMed1985

EcoK restriction during in vitro packaging of coliphage lambda DNA.

The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.

Bacteriophage lambda↗
PubMed1986

Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements.

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.

Bacteriophage lambda↗
PubMed1986

Boundaries of the nutL antiterminator of coliphage lambda and effects of mutations in the spacer region between boxA and boxB.

To study the relationship of sequence to function for the phage lambda nutL transcriptional antiterminator, we cloned the 354-bp HincII lambda DNA fragment (coordinates 35261-35615; Daniels et al., in Lambda II, 1983, pp. 485-486 and 618-619) between the plac promoter and Rho-dependent or Rho-independent terminators (t) in the plac-t-galK plasmid derived from vector pKO3, and assayed the galK expression in Escherichia coli hosts in the presence or absence of the N gene product. The 354-bp fragment displayed the complete antitermination activity, as did several shorter fragments obtained by restriction cutting, exonucleolytic deletions and ligations. The minimal length of cloned and fully active nutL comprises 43 bp and consists (in the 5'-to-3' order) of a 10-bp sequence upstream from boxA, the 8-bp boxA (5'-CGCTCTTA-3'), the 7-bp A-B spacer between boxA and boxB; the 17-bp boxB (nutL core; 5'-AGCCCTGAAGAAGGGCA-3') and 1-bp downstream from the nutL core. Deletion of the 10-bp sequence upstream from boxA reduces anti-termination by 40%. Deletion of both that sequence and boxA reduces antitermination by 90% at both 30 degrees C and 42 degrees C. Most of the deletions entering boxB abolish antitermination. Also, some of the small internal deletions within the 7-bp A-B spacer region have a strong negative effect on the nutL function, when transcription is from the plac promoter.

Bacteriophage lambda↗
PubMed1987

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins.

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.

Antibodies, Monoclonal↗