Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Packaging”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.

Bacteriophage P22↗

The packaging signal in hepatitis B virus pregenome functions only at the 5' end.

When the hepatitis B virus (HBV) replicates, a full-size transcript of the viral genome, called pregenome RNA, is made and is selectively packaged into virus core particles. This selective encapsidation is dependent upon the presence of a specific cis-acting sequence called the packaging signal that is found at the 5' end of the pregenome RNA. Deletion analysis revealed that this packaging signal was located in a 70 bp stretch of nucleotides. Packaging defective virus genome containing the 70 bp packaging signal sequence at several locations, showed that the packaging occurs effectively only when the signal is located near the 5' end of the pregenome RNA. Packaging also occurred with a 2.4 kb viral RNA whose 5' region sequence was different from that of the pregenome RNA, but which has the packaging signal sequence inserted at the 5' region.

Base Sequence↗

A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors.

Lentiviral vector safety has been the impetus underlying the progress in packaging cell line development. The prospects of generating replication-competent lentiviruses (RCLs) and the potential for vector mobilization continue to be the driving force for the advancement of packaging cell lines. We have exploited the trans-lentiviral packaging system to develop the SODk3 packaging cell line for the generation of conditional self-inactivating (cSIN) vectors. Separating the gag-pol genome into two distinct expression cassettes (gag-pro and vpr-RT-IN) may reduce the potential for RCL formation, while concurrently employing cSIN vectors supports retention of the SIN phenotype in target cells and alleviates technical constraints associated with generating producer cell lines. Through development of the SODk3 packaging cell line we determined that the ratio of Gag/Pol in vector particles may be used as an indicator for packaging cell clones that yield high vector titers. Conditional SIN vector titers (1 x 10(7) TU/ml) were augmented through clonal selection. Distinct producer cell clones revealed a parallel between vector titer and transgene expression levels. We exploited this observation to demonstrate that incorporation of an internal ribosome entry site between the GFP marker and a relevant transgene affords efficient selection of high-titer producer cell lines. Furthermore, cSIN vectors generated from SODk3 packaging cells imparted efficient transduction of primary human fibroblasts, an indication of the future applicability of the SODk3 packaging cell line.

Cell Line↗

Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.

Bacteriophage T7↗

[Packages of care in the departments of mental health in Lombardy].

OBJECTIVE: The analysis aims to study the packages of care in the public Departments of Mental Health by diagnosis and service utilisation intensity. DESIGN: Data on community, hospital and residential contacts were provided by the Regional Psychiatric Information System. The sample has been composed by 55,518 patients residents in Lombardy and treated in public Departments of Mental Health. SETTING: The public Departments of Mental Health in Lombardy. MAIN OUTCOME MEASURES: Fifteen packages of care were defined according to researchers' experience; the package "community care only" has been divided in five sub-packages; for every package the care weight has been attached. RESULTS: Four packages of care ("community care only", "hospital care plus community care", "hospital care only" "community care plus day centre care") represented 95% of the patients. Three quarter of the patients were treated only in the community setting, without hospital, residential and day centre contacts in the year. Heavier patients (patients with more than 5000 care weight) represent only 4%. Residential care is the heavier setting (36% of the total weight), while schizophrenia is the diagnosis with mayor impact on the community services (59% of the total weight). Of the patients treated only in the community setting one third receives only psychological and psychiatric visits, while two thirds integrated community care. Conclusions in community care the mixed packages represent the exception not the rule. More complex or heavier packages are addressed to severe mental illness patients.

Catchment Area, Health↗

Secondary structure and interactions of the packaged dsDNA genome of bacteriophage P22 investigated by Raman difference spectroscopy.

Vibrational spectra of the double-stranded DNA genome of bacteriophage P22 in packaged and unpackaged states are compared by digital difference Raman spectroscopy. The difference Raman spectrum, which is sensitive to structural changes at the level of < 2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking, and the electrostatic environment of DNA phosphate groups. For both packaged and unpackaged states, the experiments were performed on aqueous solutions at 25 degrees C containing effective P22 DNA concentrations of 30-50 mg/mL in 200 mM NaCl + 10 mM MgCl2 + 10 mM Tris at pH 7.5. At the experimental conditions employed, the B-form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid. However, the electrostatic environment of DNA phosphates is dramatically altered with packaging. Specifically, we find the following: (1) C2'-endo sugar pucker and anti glycosyl orientations are conserved for all nucleosides. (2) Watson-Crick base pairing is essentially completely retained. (3) Alternative secondary structures, whether right- (A or C form) or left-handed (Z form), are not evident in either the packaged or unpackaged viral genome. (4) Small Raman hyperchromic effects (< 10%) observed for certain marker bands of dG, dA, and dT in the packaged state of P22 DNA suggest slightly reduced base-stacking interactions with packaging. These are consistent with previously reported UV hyperchromic effects, but the Raman spectrum shows that they are not associated with either base unpairing or strand separation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗

Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors.

To date, only adherent cell lines have been used for the generation of packaging cells for the production of type C retrovirus vectors. The large-scale production of high titre retrovirus vectors could benefit from the development of packaging cells growing in suspension. Here, we describe the ability of two different lymphoid cell lines, one B- and one T-lymphoblastoid cell line (Namalwa and CEM, respectively), to produce MLV-based vectors. Upon transfection with a third generation packaging construct, the virus particle production by Namalwa cells was characterised by low RT-activity, and by CEM cells as high RT activity as previously established adherent packaging cells. An amphotropic packaging cell line (CEMFLYA) was therefore established from CEM cells. Upon introduction of a lacZ vector genome, the novel packaging cell line produced vector particles routinely in the region of 10(7) infectious units/ml. The vectors were helper-free and highly stable in fresh human serum. The potential for scaled up vector production was demonstrated by continuous culture of the new packaging cells for 14 days in a 250 ml spinner flask. These suspension packaging cells should be applicable to large bioreactor systems to bulk produce high titre, complement-resistant retrovirus vectors for gene therapy.

B-Lymphocytes↗

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line.

Using retroviral supernatants derived from the amphotropic murine packaging cell line PA317 and the amphotropic canine packaging cell line (DA), cord blood and mobilized peripheral blood CD34+ cells were transduced with the vector LN (neomycin resistance) and the vector L-TR/TAT neo (neomycin resistance in conjunction with a double-hammerhead ribozyme conferring anti-HIV activity). Different multiplicities of infection (MOI) were applied in the setup according to vector titrations on NIH-3T3 cells. PA317-based supernatants were tested at MOI of 10 and 30. Purified concentrated DA-derived vector preparations were tested at MOI of 10, 30, 100, and 300. Immediately after transduction, CD34+ cells were plated into colony assays in the presence and absence of G418 to evaluate the amount of gene transfer and potential toxic effects of the vectors on colony growth. The remaining cells were subjected to G418 selection in liquid culture for 12 days and subsequently challenged with HIV-1JR-FL to test for efficacy of the anti-HIV gene in macrophages derived from transduced CD34+ cells. Transduction by the PA317-packaged vectors was maximal at the lowest MOI used and did not increase with increasing MOI. In contrast, transduction by the DA-packaged vectors could be progressively increased using increased MOI. The net transduction efficiency per unit of reverse transcriptase activity in the DA vector preparations was 8.7-fold higher than in the PA317 vector supernatants. HIV-1 challenge of the cells transduced by the ribozyme vector derived from the PA317 packaging cells resulted in a 1.5 log inhibition of p24 output compared with the control cells containing neomycin resistance only. A 2.5 log inhibition of p24 output could be observed in the cell population transduced with DA-packaged vector supernatants. Compared with retroviral supernatants from PA317 packaging cell lines, DA packaging line-derived vector preparations demonstrated higher transduction efficiency into CD34+ cells, particularly at higher MOI, and increased efficacy of the transferred anti-HIV gene when challenged with HIV-1JR-FL. The increase in transduction efficiency may be due to a higher ratio of intact vs. defective vector particles in the DA-derived vector preparations.

Animals↗

[Influence of the drug package information paper on compliance of neurological and psychiatric outpatients].

The influence of demographic parameter, the number of different drugs or the frequency of the single doses on the reliability of compliance was subject in different studies. The goal of this investigation was to examine the influence of the drug package information paper on the compliance of neurological-psychiatric patients. 951 patients of one quarter of a neurological-psychiatric practice were given a questionnaire, which examined the income behavior and the estimate of the meaning of the drug package information paper. 352 patients answered the questionnaires. Only 15.6 % refused it to answer the questionnaire. The remaining were not able to answer because of different reasons (dementia, aphasia, acute psychiatric disorders, foreign language origin, immobility, etc.). 98.1 % considered the drug package information paper to be important. Older patients and patients with lower education degree judged the drug package information paper to be too extensive. Only few patients (11.5 %) let the physician explain the drug package information paper. 86.3 % of the patients assumed the medication prescribed by the physician to be correct. 58.1 % of the patients however were not satisfied with the information by the physician. Patients with neuroses were particularly dissatisfied over the clearing-up by the physician (77.3 %). 73.3 % of the patients were deterred from taking in their medicines occasionally or frequently by the side effects described in the drug package information paper. 59.9 % of the patients would take the medicine, if the physician insisted on it. Independently of the influence of the drug package information paper 57.5 % of the patients forgot to take their medicines occasionally or frequently. Patients with epilepsies and M. Parkinson were most compliant. In summary the investigation showed that the clearing-up only by the drug package information paper in contrast to the clearing-up by the physician leads to more non-compliance.

Adult↗

DNA sequences necessary for packaging of bacteriophage lambda DNA.

The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay. From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs). A 60-base-pair stretch of the left arm leaves an efficiency of only 1%. The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host. By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA. The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required.

Bacteriophage lambda↗

Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4.

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.

Adenosine Triphosphatases↗

Coupling with packaging explains apparent nonreciprocality of Chi-stimulated recombination of bacteriophage lambda by RecA and RecBC functions.

Chi (chi, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli. In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a+ chi +b- X a- chi 0b+), the chi-containing recombinant (a- chi +b-) is less abundant than the non-chi-containing recombinant (a+ chi 0b+). Previously this was taken was evidence for nonreciprocality of chi-stimulated exchange. This inequality, however, is now seen to result from an event at cos (lambda's packaging origin) that both activates Chi and initiates DNA packaging. An event at rightward cos leads to activation of leftward chi on the same chromosome for an exchange to its left. From the resulting circulating dimer (--cos-a+- chi 0-b+-cos-a-- chi +-b- --), the cos that activated chi is more likely to be used for rightward packaging initiation than is the cos from the other parent. Consistent with this coupling model is "biased packaging" in lambda carrying two cos sites per monomer genome. When their maturation is dependent on dimerization by chi-stimulated exchange, the phage particles result more often from packaging from the cos that activates chi than from packaging from the other cos. Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates chi. A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase.

Bacterial Proteins↗

Non-verbal communication: evaluation of a computer-assisted learning package.

A computer-assisted learning (CAL) package was developed on non-verbal communication. Its effectiveness was evaluated by comparing learning based on use of the package with that based on a didactic lecture covering the same topic. A class of 151 first-year medical students was divided into two groups, balanced for gender and home/overseas students. One group was asked to use the CAL package, the other group attended the lecture. Knowledge was assessed one week later by a written test, and reactions to using the CAL package were obtained via a questionnaire. Each group was then allowed and encouraged to use the other resource and then asked about their preferences for type of resource at the end of term. Mean score on the knowledge test was reliably better in the CAL group. In addition, scores increased as the time spent using the CAL package increased: this relationship was highly significant. Use of the CAL package varied from 15 to 120 minutes (median 45). Users reported that it was easy to operate, was an adequate or good resource for learning about the subject, and was a good or reasonable use of their time. After using both types of learning resource half the students judged the CAL package more useful for learning about the subject, and half preferred it to the lecture (the other half had the opposite judgement and preference). This study provides evidence that a CAL package can effectively substitute for traditional didactic teaching in a medical school. Good quality CAL, however, requires substantial resources and high calibre staff to develop and maintain it.

Computer-Assisted Instruction↗

Retroviral gene transfer using safe and efficient packaging cell lines.

One of the requirements for the use of retroviral vectors in human gene therapy is a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. Wild-type virus may be produced through recombinational events between the helper virus and a retroviral vector. We have constructed an ecotropic packaging cell line, GP + E-86, and an amphotropic packaging cell line, GP + envAm12, in which the viral gag and pol genes are on one plasmid and the viral env gene is on another plasmid. Both plasmids contain deletions of the packaging sequence and the 3' LTR. The fragmented helper virus genomes, when introduced into 3T3 cells, produce titers of retrovirus which are comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid. We have found no evidence for the generation of wild-type retrovirus using the GP + E-86 and GP + envAm12 packaging lines, either alone or in combination with the N2 retroviral vector. We also show that these packaging cell lines can be used to transfer the neoR gene of the N2 vector into mouse hematopoietic cells, followed by successful (48-52%), long-term (up to 200 days) transplantation into irradiated recipients. These results indicate that these packaging lines are safe and efficient for use in experiments designed for murine (using GP + E-86) and human (using GP + envAm12) gene therapy.

Animals↗

Effect of vacuum packaging on growth of Clostridium botulinum and Staphylococcus aureus in cured meats.

Incrimination of vacuum-packaged smoked fish in outbreaks of botulism has raised questions about the safety of this process in comparison with other methods of packaging foods. It has been suggested, for example, that Clostridium botulinum may grow better in a vacuum-packaged product than in one that is packaged without vacuum. To evaluate this possibility, sliced bologna was inoculated with spores of C. botulinum type A, packaged in transparent plastic film with and without vacuum, and stored at temperatures within the growth range of the organism. There was no detectable difference in the rate of toxin development in the two types of packages. In contrast, vacuum packaging markedly inhibited the growth of Staphylococcus aureus on sliced ham. The results indicate that vacuum packaging has little if any effect on the ability of C. botulinum to grow in cured meats, but it may reduce the likelihood of staphylococcal food poisoning.

Clostridium botulinum↗

Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein.

In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4(Trans) and CP(Trans)) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4(Rep) and CP(Rep)), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).

Bromovirus↗

Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids.

Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However, sedoheptulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min.

Bacteriophages↗

Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions.

A region near the 5' end of Moloney murine leukemia virus (MoMLV) is required for packaging of viral RNA into virions. Retroviral vectors based on MoMLV have been constructed that are also efficiently packaged into virions despite removal of most of the interior region of the parental virus. To further localize sequences which are sufficient for packaging, we inserted various fragments from an MoMLV-based retroviral vector into a nonretroviral transcription unit, transfected these constructs into retrovirus-packaging cells, and measured packaging of RNA transcribed from these constructs into virions. Transcripts from some of these constructs were packaged at least as well as those from the parental vector or MoMLV itself. Sequences extending into the gag region, but not the long terminal repeat or tRNA-binding sequences, were required for efficient RNA packaging. RNAs transcribed from constructs which did not contain an insert, or in which the orientation of the insert was reversed, were not packaged at detectable levels. These studies define sequences which are necessary and sufficient for encapsidation of murine leukemia virus RNA into virions.

Animals↗