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At least 487 records · Page 27Linked to original sources
PubMed1997

The conformation of packaged bacteriophage T7 DNA: informative images of negatively stained T7.

Within the icosahedral protein outer shell of bacteriophage T7, a 40-kbp DNA genome occupies a cavity also occupied by a protein cylinder that projects into the DNA from the outer shell. However, neither the internal cylinder nor separately resolved DNA segments are revealed in the conventional negatively stained specimens of intact bacteriophage T7. In the present study, a procedure of negative staining is used that reveals both internal proteins and separately resolved segments of packaged DNA during electron microscopy of intact particles of a hybrid T7 bacteriophage; the hybrid is genetically T7, except for a tail fiber gene that has a segment from the T7-related bacteriophage, T3. The negatively stained packaged DNA segments of this hybrid bacteriophage are found to be wrapped around the axis of the internal cylinder. To obtain additional information about the conformation of packaged T7 DNA, electron microscopy is performed of negatively stained capsids that are incompletely filled with DNA (ipDNA-capsids); a procedure is described for improved isolation of ipDNA-capsids from lysates of hybrid bacteriophage T7-infected cells. The packaged DNA segments of ipDNA-capsids are found not to be wrapped around any axis. Images of ipDNA-capsids are explained by the hypothesis that DNA does not achieve its wrapped condition until the capsid is more than 40% full of DNA. Wrapping via folding is, therefore, proposed to explain the images of DNA packaged in bacteriophage T7.

Bacteriophage T3↗
PubMed1993

DNA packaging ATPase of bacteriophage T3.

A defined in vitro DNA packaging system of phage T3, which is composed of purified proheads and two packaging proteins, the products of genes 18 and 19 (gp18 and gp19, respectively), displayed a DNA-dependent ATPase activity. ATP was hydrolyzed to ADP and Pi. The ATPase activity was stimulated by nonpackageable DNA, such as single-stranded or circular DNA, or RNA (nonpac-ATPase). Among the inhibitors of DNA packaging, actinomycin D specifically inhibited the ATPase activity that was tightly coupled to DNA packaging (pac-ATPase), but did not inhibit the nonpac-ATPase activity. Both activities depended upon a functional packaging complex, but the nonpac-ATPase, once activated, did not require DNA. Unpackageable pUC18 DNA inhibited the pac-ATPase and the phage yield in parallel. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.

Adenosine Triphosphatases↗
PubMed1995

Analysis of ALV-based packaging cell lines for production of contaminant defective viruses.

We have previously described avian leukosis virus-based packaging cell lines that express gag, pol, and env proteins from two transcomplementing genomes and produce helper-free stocks of retroviral vectors with different host ranges. In this report, we demonstrated that (i) despite the deletion of the psi packaging sequence, the packaging-defective transcomplementing retroviral transcripts were packaged into virions at a level that could reach 2.3% of a wild-type virus packaging level and (ii) despite deletion of the 3' LTR, these genomes were transferred along with the vector to target cells. As these genomes were also bearing a selectable gene, titers of the resulting contaminant particles could be estimated, depending on the cell line to be between 0 and 6 infectious particles/ml of supernatant.

Animals↗
PubMed1998

Assembly of hepatitis delta virus particles: package of multimeric hepatitis delta virus genomic RNA and role of phosphorylation.

We previously demonstrated that both casein kinase II (CKII) and protein kinase C (PKC) positively modulate the hepatitis delta virus (HDV) RNA replication but not the assembly of the empty hepatitis delta antigen (HDAg) particle. In this study, we investigated whether phosphorylation of HDAg by these two kinases plays a role in assembly of the HDV virion. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation level of large HDAg but not small HDAg in HDAg-expressing HuH-7 cells was diminished by CKII inhibitor (DRB), whereas no effect was observed for the phosphorylation level of two HDAgs when treated with protein kinase A (PKA) inhibitor (HA1004) or PKC inhibitor (H7). Cotransfection experiment also demonstrated that packaging of HDV genomic RNA was not affected by the kinase inhibitor DRB or H7 and mutation at the putative CKII phosphorylation sites (serine-2, serine-123, or both), and the putative PKC site (serine-210) of HDAg did not elicit any significant effect on the HDV virion assembly. Therefore, based on the previous work and the present study, it seems that the status and biological significance of phosphorylation of HDAg vary depending on the HDV life cycle. Although in the HDV RNA replication cycle, phosphorylation of small HDAg by CKII or PKC plays important role in HDV replication, phosphorylation of the same HDAg by these two kinases does not occur during the HDV RNA virion assembly, and phosphorylation of the large HDAg by CKII does not confer any regulatory role in the assembly of HDV virion and empty viral particles. Our study also showed that the large HDAg without the small HDAg could efficiently assemble both monomeric and dimeric HDV genomic RNAs into secreted HBV-enveloped virus-like particles. Increasing the transfected small HDAg-expressing plasmid led to an enhancement of the packaging efficiency for the monomeric HDV genomic RNA with little effect on the packaging of dimeric HDV RNA. Similarly, HDAgs could package the trimeric HDV genomic RNA, albeit less efficiently. CsCl density gradient centrifugation confirmed that HDAgs and the monomeric and multimeric (dimer and trimer) HDV genomic RNAs formed an HBV-enveloped virus-like particle at a density of 1.23-1.25 g/ml. Thus, the assembly of the HDV virion seems to not impose much restriction on the size of HDV RNA for packaging.

Genome, Viral↗
PubMed2001

Chemical modification patterns of active and inactive as well as procapsid-bound and unbound DNA-packaging RNAof bacterial virus Phi29.

During replication, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into the limited space within the preformed procapsid. We previously found that a viral-encoded RNA (pRNA) played a key role in bacterial virus phi29 DNA translocation. Design of mutant pRNA sets containing two and three inactive mutant pRNAs, respectively, led to the conclusion that the stoichiometry of pRNA in DNA packaging is the common multiple of 2 and 3. Together with studies using binomial distribution of mutant and wild-type pRNA, it has been confirmed that six pRNAs of phi29 form a hexagonal complex to drive the DNA translocating machine. These findings have brought about commonality between viral DNA packaging and other universal DNA/RNA-riding processes including DNA replication and RNA transcription. Chemical modification was used to compare the structures of active and inactive as well as free and procapsid-bound pRNA. Our results explain why certain pRNA mutants are inactive in DNA packaging while remaining competent in procapsid binding, since the mutations were located in a domain involved in DNA translocation that is dispensable for procapsid binding. A mutant pRNA that had reduced procapsid binding was revealed to have a structural alteration within the procapsid-binding region that may account for the binding deficiency. Chemical probing of procapsid-bound pRNA revealed a large area of protection, while a 3-base bulge, C(18)C(19)A(20), was accessible to chemicals. A pRNA with a deletion of this 3-base bulge was fully competent to form dimers, bind procapsids, and inhibit phi29 virion assembly in vitro; however, its activity in DNA packaging and virion assembly was completely lost. The results suggest that this bulge is not involved in procapsid binding but may interact with other DNA-packaging components. A computer model showing the location of the CCA bulge was presented.

Bacillus Phages↗
PubMed2001

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Amino Acid Sequence↗
PubMed1988

Construction of a safe and efficient retrovirus packaging cell line.

Ecotropic and amphotropic retrovirus packaging cell lines have been constructed in which the helper virus genome have been separated onto two plasmids, and the psi packaging signal and 3' LTR have been removed. The gag and pol genes on one plasmid and the env gene on another plasmid were transfected into NIH 3T3 cells. Packaging cell lines produced by these transfected genes released titers of replication-defective retroviral vectors which were comparable to titers produced by packaging cell lines containing the helper virus genome on one plasmid. There has been no evidence of recombination events between the ecotropic helper virus plasmids and the vector virus plasmid that would result in the generation of intact replication-competent virus. These results suggest that a packaging cell line containing gag, pol and env on different plasmids is efficient and safe for use in retroviral gene gransfer.

Cell Line↗
PubMed2000

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.

This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'

Adenosine Triphosphatases↗
PubMed1999

Low efficacy of gene therapy for rat BT4C malignant glioma using intra-tumoural transduction with thymidine kinase retrovirus packaging cell injections and ganciclovir treatment.

BACKGROUND: The purpose of this study was to test the use of Herpes Simplex virus thymidine kinase (HSVtk) retrovirus packaging cell injections in the treatment of malignant brain tumours. METHODS: Therapeutic effect and tissue responses were examined in vivo in a syngeneic BT4C rat glioma model after HSVtk-producing PA317 packaging cell injections and intraperitoneal ganciclovir (GCV) medication. MRI was used to visualise the tumours before and after the treatment. Immunohistochemical stainings were performed to study astroglia and microglia responses and apoptosis-mediated cell death. RESULTS: The results suggest that only a limited treatment effect can be achieved with HSVtk packaging cell injections with no prolonged survival rates. Histological examination showed a strong astroglia response but only a modest microglia response after the treatment. HSVtk and GCV-induced cell death was at least partially mediated by apoptosis. It is concluded that HSVtk packaging cell injections and GCV treatment do not lead to eradication of malignant cells in a syngeneic BT4C rat glioma model. The lack of efficacy is most likely due to low gene transfer efficiency and limited life span of the injected packaging cell inside the tumours. CONCLUSIONS: Improvements in gene transfer efficiency, and stimulation of immunoresponse against tumour cells might lead to a more effective therapeutic response in vivo.

Animals↗
PubMed1992

Formation of the right before the left mature DNA end during packaging-cleavage of bacteriophage T7 DNA concatemers.

During bacteriophage T7 morphogenesis in a T7-infected cell, mature length T7 DNA molecules join end-to-end to form concatemers that are subsequently both packaged in the T7 capsid and cut to mature size. In the present study, the kinetics of the appearance in vivo of the mature right and left T7 DNA ends have been analyzed. To perform this analysis, the intercalating dye proflavine is used to interrupt DNA packaging. When used at 0.5 to 8.0 micrograms/ml, proflavine progressively inhibits events in the T7 DNA packaging pathway, without either altering protein synthesis or degrading intracellular T7 DNA. Restriction endonuclease kinetic analysis reveals that proflavine (8 micrograms/ml) completely blocks formation of the mature T7 DNA left end, but only partially blocks formation of the mature T7 DNA right end. Both these and other observations are explained by the hypothesis that, in the T7 DNA packaging pathway, events occur in the following sequence: (1) formation of a mature right end; (2) packaging of at least some of the genome; (3) formation of the mature left end.

Capsid↗
PubMed1983

Identification of a sequence likely to be required for avian retroviral packaging.

Two assays have been utilized to assess the ability of avian retroviral molecules to be packaged into virus particles. Cloned viral genomic molecules were microinjected into the nuclei of chick cells infected by either a lymphoid leukosis virus or an envelope glycoprotein-deficient sarcoma virus. The titer of focus-forming virus released by injected cells, or the ratio of these to helper virus, is then used to determine packaging efficiency, although biological properties other than packaging might also effect these assays. With either assay, deletions up to 3.0 kbp introduced in the viral gag or pol genes did not affect packaging unless sequences near the SstII restriction site (approximately 150 bp 3' of the splice donor site) were deleted. Deletions differing by 2 bp at the SstII site were found to express radically different packaging efficiencies.

Animals↗
PubMed1986

A defined in vitro system for packaging of bacteriophage T3 DNA.

Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg2+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM. A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.

Adenosine Triphosphate↗
PubMed1987

Early events in DNA packaging in a defined in vitro system of bacteriophage T3.

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19. The reaction requires Mg2+, ATP, and polyethylene glycol and is inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). About 30% of added mature T3 DNA was packaged into heads in the defined system. A complex with a sedimentation coefficient of about 50 S (50 S complex) accumulated in the reaction mixture containing ATP-gamma-S. The 50 S complex was DNase sensitive and was converted to filled heads by a second reaction in the presence of ATP without addition of DNA, proheads, gp18, and gp19. These results indicate that during early stages of DNA packaging, formation of precursor complexes proceeds by an allosteric mechanism with ATP acting as effector. The movement of DNA into the head is driven by the energy released by hydrolysis of ATP. gp18 formed a complex with DNA without addition of ATP-gamma-S and gp19. gp18-DNA complex was DNase sensitive and did not bind gp19; it was converted to filled heads by way of a second reaction after addition of ATP, gp19, and proheads. gp19 formed a functional complex with prohead in the presence of ATP-gamma-S or ATP. The complex did not bind gp18 but was converted to filled heads by incubation with ATP, gp18, and DNA. In the absence of ATP-gamma-S, gp19 formed complexes with prohead that were abortive in DNA packaging. Formation of the 50 S complex occurred in a reaction mixture containing gp18-DNA and gp19-prohead complexes in the presence of ATP-gamma-S. From these results, we propose details of the molecular mechanism of DNA packaging in the defined in vitro system.

Adenosine Triphosphate↗
PubMedDate not supplied

Evaluation of statistical packages for suitability for use by clinical investigators in medicine.

With the increased availability of personal computers and statistical software packages, it is inevitable that there will be increasing attempts by clinical investigators to perform data management and statistical analysis. Reviews of statistical packages are abundant in computer and statistical journals. However the majority of them were not written for clinical investigators in medicine. This paper presents an analytic approach to evaluate the suitability of statistical packages for use by clinical investigators for data-management and preliminary statistical-analysis purposes. The evaluation scheme addresses five areas of concern: availability of data-management features; availability of basic statistical-analysis features; ease of use; documentation; and quality of programs. Among six statistical packages reviewed by this process, CRISP is recommended as the most suitable package for clinical investigators to use for data-management and preliminary statistical-analysis purposes.

Evaluation Studies as Topic↗
PubMed1982

Identification of sequences necessary for packaging DNA into lambda phage heads.

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.

Bacteriophage lambda↗
PubMed1983

Lambda phage DNA sequences affecting the packaging process.

Our previous work identified a minimal region of bacteriophage lambda DNA that is necessary for packaging into phage particles. It consists of 40 bp of the right arm and 45 bp of the left arm [Miwa and Matsubara, Gene 20 (1982)267-279]. A part of this region, 22 bp of the right arm and 38 bp of the left arm, is sufficient for cutting at cos lambda (the minimal sequence for cos lambda cutting). An 84-bp region to its right contains a binding site for lambda terminase, a complex of Nu1 and A gene products. This second region, which we called the enhancing region for packaging, stimulates cutting at cos lambda as well as packaging. This region is not active if it is physically separated from the minimal packaging region. The enhancing region has 15-bp inverted repeats. These sequences are conserved in the corresponding region of bacteriophage phi 80, which has the same packaging specificity as lambda.

Bacteriophage lambda↗
PubMed2004

An evaluation of a supplementary road safety package.

A Supplementary Road Safety Package (SRSP) was developed in New Zealand in 1995/1996 to supplement the compulsory breath test (CBT) and speed camera programmes introduced in 1993. A major feature of the package was the use of emotion and shock advertising campaigns not only to affect high risk driving attitudes and behaviours towards speeding and drink-driving but also to encourage the use of safety belts. Furthermore, the SRSP also emphasised targeting enforcement to these three areas. This package continued for 5 years. This paper estimates the effect of the package on road trauma. The analysis shows that the Package made substantial impact on road safety and saved over 285 lives over the 5-year period.

Accidents, Traffic↗
PubMed2006

Geographic variability in alcohol-related crashes in response to legalized Sunday packaged alcohol sales in New Mexico.

On July 1, 1995 the state of New Mexico lifted its ban on Sunday packaged alcohol sales. Legislation lifting the ban included a local option allowing individual communities within the state to hold an election to reinstitute the ban on Sunday packaged alcohol sales. Previous research has shown a clear statewide increase in alcohol-related crash and crash fatality rates after the ban was lifted. The goal of this study is to measure county-level variability in changes in alcohol-related crash rates while adjusting for county socio-demographic characteristics, spatial patterns in crash rates and temporal trends in alcohol-related crash rates. Bayesian hierarchical binomial regression models were fit to the observed quarterly crash counts for all counties between July 1, 1990 and June 30, 2000. Results show marked variability in the impact of legalized Sunday packaged alcohol sales on alcohol-related crash rates. Relative risks of an alcohol-related crash for the post-repeal versus pre-repeal period vary across counties from 1.04 to 1.90. Counties with older population suffered a greater negative impact of legalized Sunday packaged alcohol sales. Counties with communities that quickly passed the local option to re-ban packaged sales on Sundays were able to mitigate most of the deleterious impact of increased alcohol availability that was observed across the state.

Accidents, Traffic↗