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PubMed2003

Use of PEG to acquire highly soluble DNA-packaging enzyme gp16 of bacterial virus phi29 for stoichiometry quantification.

All linear dsDNA viruses package their genome into a preformed procapsid via a ATP-driving motor involving two nonstructural enzymes or ATPase. This essential viral replication step has been investigated in the quest for new antiviral drugs. These DNA-packaging motors could be potential parts in nanotechnology. But both the low solubility and self-aggregation of all nonstructural enzymes have seriously hampered studies on these motors. Bacterial virus phi29 DNA-packaging motor has been well characterized. But the role of the nonstructural ATPase gp16 has not been well defined due to its hydrophobicity, low solubility, and self-aggregation. Here we report a novel approach to obtain affinity-purified, soluble, and highly active native gp16 with the aid of polyethylene glycol (PEG) or acetone. With several thousand-fold increase in specific activity in comparison to the traditional method, this unique approach has made the quantification of gp16 feasible. The basic functional unit of gp16 in solution was found to be a monomer, as determined by sedimentation and size exclusion chromatography. This result leads to a subsequent finding that the stoichiometry of gp16 for phi29 DNA-packaging was about 11+/-2. These findings will facilitate the study on this novel motor that involves three pRNA dimers and a 12-subunit connector.

Adenosine Triphosphatases↗
PubMed2000

HIV RNA packaging and lentivirus-based vectors.

Since the mid-1990s, the number of publications on lentivirus-based vectors has expanded dramatically as people have realized the opportunity that they represent. High-titer helper-virus free transfer of genes to nondividing cells is a reality and it can only be a short time before clinical trials are initiated. The most efficient vector to date appears to be HIV-1 and it is no coincidence that this is the virus in which there is the greatest theoretical understanding of the encapsidation process and viral assembly. Basic studies in the other viruses are at an earlier stage and this is reflected to some extent in their relative inefficiency. Emphasis is placed in some publications on non-HIV-based vector systems having the additional safety feature of a viral vector not based on a human pathogen. As yet, this is largely a cosmetic advantage in that no system would be used which was capable of regenerating a full-length wild-type HIV and the vectors all have single round replication kinetics. More important will be elucidation of the mechanism of packaging in the different lentiviruses. Cis and trans packaging preferences may influence efficiency. Accurate delineation of packaging signals will be important. Most influential, however, will be a deeper understanding of all the viral and cellular factors involved in the packaging pathway.

Base Sequence↗
PubMed2000

RNA secondary structures of the bacteriophage phi6 packaging regions.

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Bacteriophage phi 6↗
PubMed1992

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.

Circular Dichroism↗
PubMed1988

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging.

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.

Capsid↗
PubMed1997

Formation and cleavage of a DNA network during in vitro bacteriophage T7 DNA packaging: light microscopy of DNA metabolism.

To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer-containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.

Adenosine Triphosphate↗
PubMed2002

Using cost-effectiveness analysis to define a breast cancer benefits package for the uninsured.

OBJECTIVES: In 1999, California was considering legislation to fund breast cancer treatment for its uninsured. We sought to define the most cost-effective breast cancer benefits package in order to inform this debate. METHODS: We use cost-effectiveness analysis to calculate the additional costs and benefits of various adjuvant therapy strategies, radiation after breast conserving surgery, and reconstruction compared to those of surgery alone in order to define the most cost-effective breast cancer benefits package for uninsured women. RESULTS: Using cost-effectiveness analysis, we define a Minimum Breast Cancer Benefits Package that includes only the most cost-effective life-saving breast cancer treatments. To provide these benefits for an estimated 550 breast cancer patients will cost $10,200,000. We present two options that each cost an additional $1,700,000 - to expand the benefits to these patients to include post-mastectomy radiation and breast reconstruction; or to provide the Minimum Package to an additional 93 uninsured women. CONCLUSIONS: California legislators must decide whether to offer comprehensive benefits to a limited number of breast cancer patients or to provide only the most life-saving treatments to a greater number of women.

Breast Neoplasms↗
PubMed1983

Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophage T1 DNA packaging.

The packaging of intracellular DNA into heads is a key feature in the morphogenesis of bacteriophage particles. In many phages a performed empty head precursor, the prohead, is filled with DNA from a concatemeric substrate consisting of tandemly repeated genome lengths. The addition of outer shell proteins completes head formation. The DNA molecules released from particles of the coliphage T1 exist as three major permutations of nucleotide sequence. Such limited permutation can be explained by the modification of Streisinger's 'headful' mechanism proposed for phage P22. DNA packaging is initiated at a specific site (the pac site) on the concatemeric precursor. While this site is cleaved, subsequent cleavages (headful cleavages) are dependent only on head-filling and are not defined in terms of nucleotide sequence. Headfuls of DNA, consisting of slightly more than a genome length, are packaged in three successive cycles of head-filling to produce the permuted and terminally redundant molecules characteristic of T1 DNA. To elucidate the regulation of this process, we have studied the DNA metabolism of T1 head mutants. We describe here the properties of a mutant in gene 13.3 which is defective for headful cleavage but remains proficient in pac site cleavage. The observation in this mutant that concatemers are degraded to unit-length molecules by repeated pac site cleavage suggests a model of headful packaging in which pac site initiation and processive head-filling compete for the DNA substrate.

Bacterial Proteins↗
PubMed1995

Pilot study of an orthodontic treatment need learning package for general dental practitioners.

This pilot study evaluated an Index of Orthodontic Treatment Need Learning Package which was developed for use by general dental practitioners. Fifty-seven dentists participated in a randomised controlled trial which involved assessing the need for orthodontic treatment among two representative samples of 16 study casts. All the dentists assessed the first set of study casts without any aids or assistance. During the assessment of the second set of study casts one group of dentists used an IOTN Learning Package in the form of a poster, another group used an IOTN Learning Package in the form of a booklet, and the remaining group acted as a control and did not use any Learning Package. The results reveal that compared with the control dentists, the dentists using the IOTN poster and the IOTN booklet had, respectively, 1.8 and 2.6 fewer errors when assessing aesthetic need and 2.0 and 2.1 fewer errors when assessing dental health need.

Female↗
PubMed1997

A field trial of an orthodontic treatment need learning package for general dental practitioners.

OBJECTIVE: To evaluate whether GDPs using an index of Orthodontic Treatment Need (IOTN) learning package in a practice setting referred (or treated) more patients with a definite need for orthodontic treatment. DESIGN: A randomised controlled trial. SUBJECTS AND METHODS: 20 GDPs (test group) used the package when selecting patients for orthodontic care during normal dental practice. A control group of 20 GDPs did not use the package. Over a 12-month period impressions were taken by the participating GDPs of all patients selected for orthodontic treatment, either referred to another practitioner or to be treated by the GDP. MAIN OUTCOME MEASURE: IOTN used by two expert examiners. RESULTS: Among the patients selected for orthodontic treatment by the test group there were more patients with a definite objective need for orthodontic treatment (78.1% versus 62.5% in the control group). After adjustment for potential confounding variables, the odds on a patient selected by a test group dentist being in definite need were 2.24 (95% CI 1.25, 4.01) more than the odds on a patient selected by a control group dentist (P = 0.007). CONCLUSIONS: Use of the IOTN learning package by GDPs would reduce the proportion of patients selected for orthodontic treatment with low or moderate objective need.

Adolescent↗
PubMed1997

Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer.

We report increased transduction of human hematopoietic progenitor cells through a combination of novel retroviral vector packaging cell lines, and improved vector supernatant production. The new ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic particles (ProPak-A cells), and ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing. Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells. All vector types transduced primary human CD34-positive or CD4-positive cells, regardless of tropism. However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector. The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines. This ping-pong amplification yielded supernatant containing vector targeted to two distinct receptors present on human cells, and did not result in detectable RCR formation. In addition, we describe conditions for improved vector supernatant production in a packed-bed bioreactor.

3T3 Cells↗
PubMed1997

Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene.

The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells. Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity. To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter. Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells. Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines. This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production. Similar results using the third generation psi CRIP packaging cell line confirmed these findings. Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line. These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.

Animals↗
PubMed1997

In vitro packaging of an infectious recombinant adeno-associated virus 2.

Adeno-associated virus 2 (AAV), a human parvovirus, has properties such as stable chromosomal integration, high infectivity and lack of known human pathogenicity, making it a potentially useful vector for human gene therapy. AAV requires a helper virus, such as an adenovirus, for optimal replication and packaging in mammalian cells. Although replication of the wild-type AAV genome has been demonstrated in vitro, packaging of infectious viral particles was not documented until now. In this study, we produced in vitro infectious recombinant AAV virions containing the neomycin resistance (NeoR) and the human CD-16 (FC gamma RIIIa) gene. Our in vitro packaging of AAV has the same characteristics as AAV produced in vivo and demonstrates the feasibility of developing a safe packaging method for AAV to be used in gene therapy.

DNA, Recombinant↗
PubMed2000

Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle.

Antisense RNA has proven a potent inhibitor of gene expression and has the potential to inhibit retroviral replication at a number of stages in the virus life cycle by targeting both viral and cellular RNA sequences. Antisense RNA complementary to three target regions in the 5' leader/LTR of human immunodeficiency virus type-1 (HIV-1), the TAR region, the primer binding site and the splice donor (SD)-packaging signal (psi) region were stably expressed from the CMV IE promoter in Jurkat cells, and expression confirmed by RT-PCR. When challenged with HIV-1, cell lines expressing antisense RNA targeting the SD/psi region showed significant inhibition of replication (at up to 10(6) TCID 50/ml). These sequences were also expressed in lymphocytes after transduction using recombinant retroviruses and one sequence complementary to the SD/psi region inhibited replication of HIV-1. A co-transfection assay using COS-1 cells was also developed both to confirm the antiviral potential of these sequences, and to determine the predominant site of action of these molecules. Antisense RNAs targeting the psi region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore, analyses of relative levels of cellular and virion RNA from these assays suggest each of these antisense molecules exerts its effect at an early stage in the transcription-translation pathway, while the longer of the sequences also inhibited packaging of virion RNA. These results suggest that the packaging signal (psi) of HIV-1 represents an attractive target for antisense RNA-based gene therapy, although the main mode of action of such molecules may well be through antisense effects at an earlier stage of replication than packaging.

3T3 Cells↗
PubMed1997

Phage DNA packaging.

Phage DNA packaging occurs by DNA translocation into a preformed protein shell--a prohead--with the aid of a packaging enzyme or a terminase. The packaging enzyme is composed of two subunits: the large subunit has ATP-binding, prohead binding, and DNA cleavage activities, and the small subunit is a DNA binding protein. DNA translocation is driven by ATP hydrolysis. In general, phage DNA replication mechanisms lead to the accumulation of concatemers. Concatemers are processed to mature DNA during and depending upon DNA packaging. This review will focus on the molecular mechanism of concatemer processing and the coupling of ATP hydrolysis to DNA translocation.

Adenosine Triphosphate↗
PubMed1998

Gynaecological examination: a teaching package integrating assessment with learning (693).

A self-directed learning package which includes assessment was developed to allow students to acquire the psychosocial and motor skills needed to conduct a gynaecological examination with sensitivity. The package integrates assessment with learning and uses the Assessment Form as an educational instrument. The introduction, including a video of a vaginal examination and pap smear, is followed by five learning stations for groups of 3 or 4 students to visit. At each station a series of tasks are given which enable the objectives of the station to be fulfilled. The materials necessary to complete the tasks are provided. Gynaecological assistants, themselves trained by participation in the learning sessions, guide the students throughout the session and act as adviser 'patients' to help students acquire the necessary motor skills. In the assessment, students are required to integrate what they have learnt in the learning stations. The students assess themselves and the 'patient' and an observer assesses the student using the same Assessment Form. The form assesses and gives examples of the psychosocial, including legal and motor skills required. The assistants-observers give feedback to the students about their performance and make suggestions for future improvement. Of the 232 medical students completing their clinical terms in Obstetrics and Gynaecology in 1995, those students receiving the learning package rated their practical experience as significantly better. They rate the learning method, content, enjoyment and package overall very highly (median 4, 5 point scale 1-5). Open-ended comments by students confirmed these findings.

Education, Medical, Undergraduate↗
PubMed1999

Evaluation of a self-paced education package on violence against women for rural community-based health workers.

There are no reported education programs specifically focusing on the needs of rural health workers in the area of violence against women. The most commonly reported contact sought by women experiencing injuries and health problems associated with violence and abuse is with health workers. Women report a failure by health workers to make direct enquires, which may be due to their lack of education and confidence in responding to these issues. A convenience sample of 60 community-based rural health workers from a range of occupations and settings within the Wide Bay Health Region, Queensland, participated in the evaluation of a self-paced, distance education package on violence against women. The package contained seven modules. These included written and audio tape material, and activities that together formed a community development approach to addressing the needs in the participants' local community. Participants were given a mentor and teleconference support during the 8 weeks allocated to complete the package. A pre- and post-course evaluation, containing quantitative and qualitative data, was completed. Analysis of the quantitative data identified significant changes in participants' knowledge, and the qualitative data highlighted an increased sense of confidence in assisting women, forming support networks and using resources more effectively. Participants reported the most useful aspects of the package were: (i) modular- and user-friendly format; (ii) flexible, practical, health-focused content; and (iii) real world examples.

Adult↗
PubMed1977

DNA packaging and the pathway of bacteriophage T4 head assembly.

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction.

Coliphages↗