Protein synthesis induced by infection with packaged lambda dv plasmid

Plasmid λdv or imm²¹dv DNA was joined to a λ arm having a cos site. This recombinant plasmid can be packaged in a λ head, and used to infect Escherichia coli K12 cells. The injected DNA molecules become plasmids in cells. By adding these particles to uv-irradiated uvrA cells, the packageable λdv or imm²¹dv plasmids can be induced to synthesize proteins coded by genes on the plasmid genome. The packageable plasmid system is thus suitable for studying on synthesis and regulation of plasmid-coded biopolymers. Analyses of the dv-coded proteins in gel electrophoreses revealed that among several genes carried on the dv plasmid genome, only those genes that are members of the pRoR-tof-cII-O-P operon can be expressed. Evidence has been presented to show that expression of this operon, which is directly correlated with replication of the genome, is only partially allowed in cells perpetuating the dv plasmid. These observations are discussed in connection with the autorepressor model (D. E. Berg, 1974, Virology62, 224–233; K. Matsubara, 1976, J. Mol. Biol.102, 427–439) that genetically accounts for the control mechanism of plasmid replication.     

Inactivation of bacteriophage λ and λ DNA by nitrogen mustard

Bacteriophage λ and λ DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage λ were more sensitive to nitrogen mustard (Cl(CH₂)₂NMe(CH₂)₂Cl; HN2) than was λ DNA. The inactivation of λ DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by λ DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and intact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in λ particles. From these data we conclude that the inactivation of λ by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.     

A selective λ phage cloning vector with automatic excision of the insert in a plasmid

A bacteriophage λ cloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (1) It has a unique BamHI site engineered into the λ gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi⁻ phenotype). (2) When the recombinant phage infects a Cre-producing E. coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids. The vector, λMGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.     

A new chromatographic procedure for separation of typical λ-type molecular species from commercial λ-carrageenan

A commercial λ-carrageenan preparation was dissolved in 3·0 ammonium sulphate and loaded onto a Sepharose CL-4B column equilibrated with the same solvent. Fractionation was carried out by stepwise elution with decreasing concentrations of ammonium sulphate at a low temperature. Each fraction obtained was analysed for sulphur content, 3,6-anhydro- -galactose content, and molecular weight (Mw). The Mw of all the fractions was lower than the Mw of the starting material. The species with the highest sulphur content and lowest 3,6-anhydro- -galactose content, a composition closest to that of an ideal structure, was recovered in the 1·5 fraction.     

Protein synthesis shut-off induced by influenza virus infection is independent of PKR activity

The role of PKR activity in influenza virus-induced cell shut-off was studied by infection of PKR(+) or PKR(-) cell cultures and metabolic labeling in vivo. No differences in the synthesis of viral proteins or the decay of cellular protein synthesis were observed. To investigate the relevance of the inhibition of cellular pre-mRNA polyadenylation and nucleocytoplasmic transport in virus-induced shut-off, we carried out similar experiments with mutant viruses lacking C-terminal sequences of NS1 protein. No differences in the shut-off induced by mutant versus wild-type viruses were observed, indicating that these nuclear events are not relevant for shut-off. The analysis of cytoplasmic mRNA stability indicated that the accumulation of viral mRNA during the infection correlated with the progressive decay of cellular mRNA, in both the wild type and an NS1 deletion mutant.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.     

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

λ-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 irradiation 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 λ 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 oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°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 λ has been suggested.     

Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic–lytic switch

The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well‐known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin‐antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic–lytic switch.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage λ carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Successful restoration of Metrosideros polymorpha (ʻōhiʻa) is possible in forest sites with active Rapid ‘Ōhiʻa Death infections

Rapid ?ōhi?a Death (ROD), caused by the fungal pathogen Ceratocystis, is killing large numbers of ?ōhi?a trees (Metrosideros polymorpha) in Hawai?i. ?ōhi?a are a dominant tree in Hawaiian forests, have a range that goes from arid to wet forest climates, and are important for endangered species habitat and ecosystem function. To test whether actively planting ?ōhi?a seedlings is a viable restoration strategy in areas with high ROD mortality, we planted ?ōhi?a in a ROD‐affected forest and crossed this with weeding and fencing treatments to compare ROD mortality to other stressors. We also tested for viable Ceratocystis spores in soils around planting areas. We found that seedlings were more likely to die in unweeded and unfenced treatments than controls. Although viable Ceratocystis spores were found in soil, none of the 41 dead seedlings tested positive for Ceratocystis. This indicates that competition from exotic plants and exotic feral ungulate damage are more likely to kill seedlings than ROD within the first year after planting.     

Fluorescence enhancement of warfarin induced by interaction with β‐cyclodextrin

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism (blood clots). In aqueous media, warfarin forms inclusion complexes with a family of cyclic oligosaccharides, α, β, γ‐cyclodextrins (CD). The formation of these complexes results in enhancement of the fluorescence of warfarin. Such spectroscopic changes offer a venue for the development of bioanalytical methodologies for warfarin quantification in biological liquids. We characterized the photophysical properties of warfarin in solvents with varying polarity and viscosity. The fluorescence quantum yield of warfarin correlated: (1) strongly with the solvent viscosity (R = 0.979) and (2) weakly with the solvent polarity (R = 0.118). These findings indicate that it is the change of the viscosity, rather than polarity, of the microenvironment that causes the fluorescence enhancement of warfarin upon binding to β‐CD. Utilizing the observed fluorescence enhancement in fluorescence titration measurements, the binding constants of warfarin to β‐CD were obtained (2.6 × 10² M^?1–3.7 × 10² M^?1). Using multivariable linear analysis, we extracted the stoichiometry of warfarin‐β‐CD interaction (1:1). © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Protein synthesis induced by heat in an Ixodes tick

We determined whether ambient temperature influences the proteins produced by Ixodes dammini ticks. Nonfed adult females were subjected to temperature pulses, and ³⁵S-labeled methionine was injected into the hemocoel or used as an incubation medium for excised salivary glands. Heat-induced protein synthesis was observed in both whole-body and excised salivary gland preparations from nonattached ticks solely when ambient temperature was raised to 42°C. The approximate M_r of each protein was 88 kilodaltons (kDa), 75 and 74 kDa, and between 21 and 27 kDa for a group of lower molecular weight proteins. In another experimental series, ticks were allowed to attach to rabbits and then were subjected to temperature pulses. The 88 and 74 kDa proteins were present in preparations from nonheated ticks that had attached for 1 h. Only small amounts of these proteins were evident in tissues prepared from ticks attached for 2 days or more. Heat-induced proteins became apparent in ticks that were incubated at 42°C, regardless of time of attachment.     

Preparation of plasmid DNA by γ-irradiation of recA cells

If recA bacteria are exposed to appropriate doses of gamma-irradiation, nondividing cells which can sustain the multiplication of "small" plasmids are produced. The gamma-irradiation technique has a number of advantages over other methods for preparing pure plasmid DNA: (1) there is little, if any, contamination of DNA preparations by chromosomal DNA owing to extensive degradation of the irradiated DNA by endogenous nucleases, (2) there is no need to introduce a uvr mutation to the host bacteria (there is when UV is used to inactivate the chromosome), (3) the method is extremely simple to work with since operations are not limited by considerations of volume and cell density, and (4) there is no need to transfer material from container to container. Yields of plasmid DNA obtained by the gamma-irradiation technique compare favorably with those obtained by other methods.     

Initiation of Infection by Deoxyribonucleic Acid From Bacteriophage λ

Kinetic studies conducted on the early stages of infection of Escherichia coli K-12 by deoxyribonucleic acid (DNA) isolated from bacteriophage lambda indicate a rapid adsorption of the phage DNA to receptor sites at the bacterial surface prior to deoxyribonuclease-insensitive incorporation. A direct relationship found between the number of DNA molecules adsorbed per bacterium and the multiplicity of helper phage infection indicates a requirement for helper function during the attachment process. An apparent lack of attachment specificity with regard to the source of the DNA preparation, to the size of the inhibiting fragment, to the base ratio of the inhibiting DNA molecule, and to "cohesive" ends suggests a nonspecific interaction between the infectious DNA and the sites of helper phage attachment.     

Protein composition of 6K2‐induced membrane structures formed during Potato virus A infection

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein‐induced membranous structures from Potato virus A (PVA)‐infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N‐terminal Twin‐Strep‐tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non‐tagged Cerulean‐6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep‐tag‐based affinity chromatography was developed. Both (+)‐ and (–)‐strand PVA RNA and viral protein VPg were co‐purified specifically with the affinity tagged PVA‐SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA‐SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2‐induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co‐purified with PVA‐derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.