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PubMed1985

Ion etching bacteriophage T4: support for a spiral-fold model of packaged DNA.

Ion etching of bacteriophage T4 erodes virus components progressively from the outside to the inside while preserving the overall structure. The terminal portion of the T4 DNA molecule packaged can be specifically radiolabeled and was found to be eroded more rapidly than the remainder of the DNA. This strongly suggests that the first DNA to enter the prohead is condensed in the center of the capsid and is therefore shielded from the ion beam by the surrounding last packaged DNA. The results support a "spiral-fold" model for the arrangement of DNA within the icosahedral bacteriophage head. According to this model, phage T4 DNA strands run parallel to the long axis of the phage, with sharp (180 degrees) bends at the top and bottom of the capsid. The folds themselves are arranged radially about the long axis of the head in spirally organized shells.

DNA, Viral↗

PubMed1990

Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation.

The packaging of bacteriophage PI DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full. We have previously shown that pac is a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of pac is methylation sensitive. Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not. Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase. Comparison of that uncut pac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated. In contrast, virion DNA containing a cut pac site was not undermethylated. We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle.

Adenine↗

PubMed1990

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology.

We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

Animals↗

PubMed1991

Inhibition of Moloney murine leukemia virus-induced leukemia in transgenic mice expressing antisense RNA complementary to the retroviral packaging sequences.

Recombinant plasmids pLP psi as and pCP psi as were constructed by positioning the Moloney murine leukemia virus (M-MuLV) proviral packaging (psi) sequences in reverse orientation under the transcriptional regulation of lymphotropic promoter/regulatory elements from the M-MuLV long terminal repeat or the cytomegalovirus immediate-early region. Linear fragments containing the antisense psi and the appropriate transcriptional regulatory sequences from these plasmids were introduced into the mouse germ line by zygote microinjection. The chromosomal integration, germ-line transmission, and lymphocyte-directed expression of the antisense psi RNA were confirmed. Control (nontransgenic) and transgenic mice containing either the pLP psi as or the pCP psi as sequences were infected with M-MuLV on the day of birth and assayed for signs of leukemia between 12 and 14 weeks of age with standard assay procedures. While 31% (11 of 36) of the control, nontransgenic, mice developed leukemia, none of the antisense psi transgenic mice developed any symptoms of leukemia. The pCP psi as sequences were also introduced into mouse NIH 3T3 cells and stably transformed cell lines were isolated. When infected with M-MuLV these cells were shown to produce virus devoid of packaged viral RNA.

Animals↗

PubMed1992

Evidence that the packaging signal for nodaviral RNA2 is a bulged stem-loop.

Flock house virus is an insect virus belonging to the family Nodaviridae; members of this family are characterized by a small bipartite positive-stranded RNA genome. The larger genomic segment, RNA1, encodes viral replication proteins, whereas the smaller one, RNA2, encodes coat protein. Both RNAs are packaged in a single particle. A defective-interfering RNA (DI-634), isolated from a line of Drosophila cells persistently infected with Flock house virus, was used to show that a 32-base region of RNA2 (bases 186-217) is required for packaging into virions. RNA folding analysis predicted that this region forms a stem-loop structure with a 5-base loop and a 13-base-pair bulged stem.

Base Sequence↗

PubMed1996

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.

3T3 Cells↗

PubMed1998

Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors.

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to produce retrovirus vectors that can transduce and integrate genes with introns. However, this has proven difficult because the viral RNA is spliced when it is synthesized in the nucleus of a producer cell. We describe a novel approach to avoid this problem. In our system the retroviral RNA is synthesized in the cytoplasm of the cell, not in the nucleus, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leukemia virus genome containing the chloramphenicol acetyltransferase (CAT) gene in association with an intron. This was inserted into a SFV transcription plasmid and the corresponding SFV vector RNA was transcribed in vitro. BHK-21 cells were then transfected with this vector RNA together with two additional SFV vectors that encode the Moloney murine leukemia virus packaging proteins. Retrovirus vectors containing intron-CAT sequences were produced at titers up to 1.3 x 10(6) infectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into NIH 3T3 cells, where the intron-CAT RNA was subjected to efficient splicing and used for high level enzyme expression. Thus, the results show that intron containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.

3T3 Cells↗

PubMed1998

Transcriptionally active Xenopus laevis somatic 5 S ribosomal RNA genes are packaged with hyperacetylated histone H4, whereas transcriptionally silent oocyte genes are not.

The relationship between histone acetylation and transcription of the Xenopus laevis oocyte and somatic 5 S ribosomal RNA genes was investigated. Chromatin fragments from a X. laevis kidney cell line were immunoprecipitated with an antibody specific for hyperacetylated histone H4. The DNA from the hyperacetylated chromatin was probed with both oocyte- and somatic gene-specific sequences, and the results showed that the upstream, nontranscribed region of the transcriptionally active somatic genes is packaged with acetylated histone H4. In contrast, the corresponding region of the transcriptionally silent oocyte genes is packaged with hypoacetylated histone H4 in this cells line. Further study also showed that this region of the oocyte genes was less sensitive to digestion with the enzyme, micrococcal nuclease. Together these results suggest that, as described for both RNA polymerase I and II transcribed genes, there is a correlation between histone acetylation and transcription of the RNA polymerase III transcribed 5 S ribosomal RNA genes in X. laevis.

Acetylation↗

PubMed2001

Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein.

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.

Binding Sites↗

PubMed2003

Bacillus subtilis bacteriophage SPP1 DNA packaging motor requires terminase and portal proteins.

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P.G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.

Bacillus subtilis↗

PubMed2003

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2.

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (HØHØHØØ where Ø is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Goal-directed fantasy does not explain the training effect of the Carleton Skills Training Package.

Two treatments to enhance the hypnotic responsiveness of subjects who pretested as low in hypnotic susceptibility were compared. Subjects in one modification treatment (n = 20) received the Carleton Skills Training Package (CSTP). Those in a second modification treatment (n = 20) were administered a partial training that did not include information about imaginal strategies. The authors of the CSTP assume that goal-directed fantasies underlie subjects' feelings of involuntariness, so teaching imaginal strategies is the primary agent of subjective changes after the training. Controls (n = 20) received no treatment. Both training packages enhanced behavioral and subjective response to an equivalent degree. Control subjects' performances were stable across tests. Modest increases in hypnotizability scores following training were related neither to enhancing goal-directed fantasies nor to use of imagery. Other mechanisms, possibly compliance, may underlie the CSTP effect.

Adult↗

PubMedDate not supplied

Migration from packaging materials into foodstuffs--a need for more fundamental information.

During recent years considerable legislative attention has been focused on food packaging materials, especially plastics, and in a number of countries extensive legislation has been formulated. Such legislation often takes the form of a positive list of permitted substances accompanied either by compositional limits or migration limits in foods or stimulant liquids. In the UK this approach has not been followed owing to the general lack of fundamental information on which to base such a system. Instead, the UK has opted for a basic legislative framework supported by informal controls which rely to a large extent on close co-operation between Industry and Government. It is recognized, however, that in future years there is likely to be a move towards more extensive legislation on packaging materials in the UK, particularly as a result of the EEC's harmonization programme. It is imperative that any future legislation is placed on a sound scientific footing and affords adequate protection for the consumer. The Government of the UK is, therefore, currently sponsoring a number of studies aimed at providing this necessary basic information.

Chemical Phenomena↗

PubMedDate not supplied

Migration of epoxidised soya bean oil into foods from retail packaging materials and from plasticised PVC film used in the home.

Epoxidised soya bean oil (ESBO) is used as a plasticiser and heat stabiliser in a number of feed contact materials, in particular in poly(vinyl chloride) (PVC) films and gaskets. The level of ESBO migration into foods has been determined using a combined gas chromatographic/mass spectrometric (GC/MS) analytical procedure. The study has included both the use of ESBO-containing materials for retail packaged foods and the domestic use of plasticised PVC films for applications such as wrapping food and covering food for re-heating in a microwave oven. Levels of ESBO in fresh retail meat samples wrapped in film ranged from less than 1 to 4 mg/kg, but were higher (max. 22 mg/kg) in retail cooked meat. Migration into sandwiches and rolls from 'take-away' outlets ranged from less than 1 to 27 mg/kg depending on factors such as the type of filling and the length of the contact time prior to analysis. The levels of migration of ESBO into cheese and cakes were consistent with previous experience with plasticiser migration--direct contact with fatty surfaces leading to the highest levels. When the film was used for microwave cooking in direct contact with food, levels of ESBO from 5 to 85 mg/kg were observed, whereas when the film was employed only as a splash cover for re-heating foods, ESBO levels ranged from 0.1 to 16 mg/kg. For a variety of baby foods there was no significant difference in ESBO levels between foods packaged in glass jars with PVC gaskets and foods in cans containing ESBO in the can lacquer. In both cases ESBO levels were low, ranging from less than 0.1 to 7.6 mg/kg. It is not clear for these retail samples, if the low levels observed (average 1.9 mg/kg) result solely from migration or contain some contribution from naturally occurring epoxides.

Epoxy Compounds↗

PubMedDate not supplied

Sensory analysis of polystyrene packaging material taint in cocoa powder for drinks and chocolate flakes.

Polystyrene packaging material taint was sensorily evaluated in cocoa powder for drinks and chocolate flakes using short-cut signal detection measures on differences between control and test samples and on recognition of styrene. No differences were observed in cocoa powder for drinks and plain chocolate flakes treated with 0.5 dm2 polystyrene of 1 mm thickness. However, differences were detected in milk chocolate flakes and plain chocolate flakes, which were in contact with a larger area or thicker polystyrene packaging material. The latter results were confirmed by the styrene recognition test, so polystyrene is a potential source of off-flavour for chocolate products. The amount of residual styrene in the polystyrene used was about 320 ppm, while the amounts of styrene ranged from 7 to 132 ppb in cocoa drinks and from 414 to 1447 ppb in chocolate flakes.

Cacao↗

PubMedDate not supplied

Leaching of aluminium from aluminium dishes and packages.

A study was carried out of the leaching of aluminium from aluminium cooking vessels and packages. Very small or undetectable levels of aluminium leached from packaging materials into foodstuffs. In boiling tests with neutral porridge no migration of aluminium into the test matrix was observed from the pan. When boiling milk, the leaching of aluminium was 0.2-0.8 mg/kg. The aluminium content of tap water in aluminium pans when reaching boiling point was 0.54-4.3 mg/l and increased with increasing boiling time to 6.3-17 mg/l. Aluminium dissolved in foods based on acidic fruit juice rose to levels of 2.9-35 mg/kg when the foods were boiled in aluminium pans. Steaming of currant berries in an aluminium vessel gave aluminium concentrations of 19-77 mg/kg in the resulting juice. The highest aluminium concentration of 170 mg/kg was measured in rhubarb juice prepared in the steaming vessel. Aluminium dissolved in water to levels of 0.81-1.4 mg/l when heated for the first time in new coffee percolators. The aluminium concentration of water heated in older percolators was 0.09-0.78 mg/l.

Aluminum↗

PubMedDate not supplied

Plasticizers from printing inks in a selection of food packagings and their migration to food.

A survey of plasticizers in printing inks present in a selection of food packaging including confectionery, snacks, crisps, potatoes, chocolate bars and biscuits, has been carried out both in England and Spain. The identification of the polymer used as packaging showed that almost all of them were oriented polypropylene. Printing inks that are always used on the outer surface contain phthalates as major plasticizers, N-ethyl- and N-methyl-toluenesulphonamides and tris(2-ethylhexyl)trimellitate were found in some of the samples studied. Several food samples were also analysed to check the level of plasticizers migrating from printing inks.

Data Collection↗

PubMedDate not supplied

Testing and evaluation of recycled plastics for food packaging use--possible migration through a functional barrier.

A coextruded three-layered polypropylene (PP) cup with recycled PP material (R-PP) in the middle layer was investigated with respect to its possible use for a specific food packaging application. From the results of this investigation a general approach to testing and evaluating the use of recycled plastic materials in food packaging is presented. The method which focuses on the direct comparison of the R-PP containing cup with a food grade PP cup manufactured from virgin material involves essentially three investigation levels: 1, compositional analysis of the raw materials, i.e. PP and R-PP granules; 2, compositional analysis of the PP and R-PP cup materials and 3, migration testing of both types of cups applying practical as well as more severe test conditions. The aim of this approach is to test the PP food contact layer for its functional barrier behaviour. One key step is the estimation of migration under practice-like test conditions in concentrations below the analytical detection limits. This can only be achieved by selecting appropriate R-PP specific guide compounds whose migration can be monitored under more severe conditions than occur in practice. From the results of this investigation general conclusions on functional barrier testing are drawn.

Alkanes↗