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At least 739 records · Page 41Linked to original sources
PubMed1989

Bovine leukaemia virus packaging cell line for retrovirus-mediated gene transfer.

Retroviral packaging cell lines were constructed by using the gag-pol gene of spleen necrosis virus, the gag-pol gene of Moloney murine leukaemia virus and the env gene of bovine leukaemia virus. The plasmids containing the gag-pol genes and the plasmid containing the env gene were cotransfected into NIH/3T3 and D17 cells. The cells containing the helper virus constructs were tested for their ability to package replication-defective murine leukaemia and avian reticuloendotheliosis retrovirus vectors. The titre of vector virus produced by each of the retroviral packaging cell lines was about 10(2) colony-forming units per ml of medium. Tests for events that might result in intact replication-competent retroviruses showed no evidence for the generation of such viruses. The vector viruses were able to infect dog and rat cells. Bovine cells were infected only after their cocultivation with the retroviral packaging cell lines producing murine leukaemia virus vectors, perhaps as a result of a low concentration of receptors.

Animals↗
PubMed1998

Subgenomic RNAs of bamboo mosaic potexvirus-V isolate are packaged into virions.

Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6.4 kb genomic RNA and two RNAs of 2.0 and 1.0 kb, in addition to associated satellite RNA (0.85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2.0 and 1.0 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for the 2.0 and 1.0 kb subgenomic RNAs of BaMV-V were located 1 1 and 16 nt upstream of the initiation codon of open reading frames (ORFs) 2 and 5, respectively. The 2.0 and 1.0 kb subgenomic RNAs functioned as messengers for the ORF2 protein and capsid protein, respectively. Packaging of the 1.0 kb subgenomic RNAs resulted in the formation of rod-shaped particles about 70 nm in length. Our results indicate that BaMV isolates have evolved distinctly for packaging of subgenomic RNAs.

Capsid↗
PubMed2003

Delineation of sequences important for efficient packaging of feline immunodeficiency virus RNA.

We have used systematic deletion analysis of the 5' untranslated region (UTR) of the feline immunodeficiency virus (FIV) genome, both in the presence and absence of various amounts of gag, to define the cis-acting sequences responsible for efficient RNA packaging. Our analyses revealed that the primary FIV packaging signal consists of two essential core elements located within the first 90-120 bp of the 5'UTR and the first 90 bp of the gag gene. Interestingly, the region between the major splice donor (SD) and gag, including approximately 130-160 bp upstream of the SD, is dispensable for encapsidation. Finally, other determinants of packaging were found to be present in the viral LTR and/or within the 3' end of the viral genome. Taken together, our results suggest that the primary packaging determinants of FIV are multipartite and discontinuous, composed of two elements within the 5'UTR and gag gene.

5' Untranslated Regions↗
PubMed2004

Design of miniaturized RF SAW duplexer package.

This paper provides a comprehensive methodology for accurate analysis and design of miniaturized radio frequency (RF) surface acoustic wave (SAW) duplexer package. Full-wave analysis based on the three dimensional (3-D) finite element method (FEM) is applied to get the package model. The die model is obtained by combining the parasitics and acoustics models. The modeling of bonding wire is also discussed. The models of package, die, and bonding wires are assembled together to get the total response. Based on this methodology, several novel ideas are proposed to significantly improve the isolation. Simulation and measurement results are compared, and excellent agreement is found. The technique developed in this paper reduces the design cycle time greatly and can be applied to various RF SAW device packages.

Journal Article↗
PubMed2004

Waste package performance evaluations for the proposed high-level nuclear waste repository at Yucca Mountain.

The evaluation studies of the proposed repository for long-term storage of spent nuclear fuel and high-level nuclear waste at Yucca Mountain, Nevada, are underway. Fulfillment of the requirements for limiting dose to the public, which includes containment of the radioactive waste emplaced in the proposed repository and subsequent slow release of radionuclides from the Engineered Barrier System (EBS) into the geosphere, will rely on a robust waste container design, among other EBS components. Part of the evaluation process involves sensitivity studies aimed at elucidating which model parameters contribute most to the waste package and overlying drip shield degradation characteristics. The model parameters identified for this study include (1) general corrosion rate parameters and (2) stress corrosion cracking (SCC) parameters. Temperature dependence and parameter uncertainty are evaluated for the general corrosion rate model parameters while for the SCC model parameters, uncertainty treatment of stress intensity factor, crack initiation threshold, and manufacturing flaw orientations are evaluated. Based on these evaluations new uncertainty distributions are generated and recommended for future analyses. Also, early waste package failures due to improper heat treatment were added to the waste package degradation model. The results of these investigations indicate that the waste package failure profiles are governed by the manufacturing flaw orientation model parameters.

Journal Article↗
PubMed1979

Independent learning packages (audio tape/pamphlet) for a course in cell excitation: evidence that they can be at least as effective as conventional lectures.

Three tape/pamphlet independent learning packages have been constructed to serve the same learning objectives as three conventional lectures on the physiology of the nerve axon. The learning efficacy of each package was compared with that of its companion lecture with the co-operation of fifty first-year medical students in a controlled experiment. The packages were at least as effective as the lectures in generating both short- and long-term attainment of their intrinsic learning objectives. The findings are discussed in relation to learning efficacy, student study time, student acceptability, package preparation time and costing.

Audiovisual Aids↗
PubMed1985

The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging.

The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in phi X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of phi X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of phi X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the phi X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the phi X174 and G4 systems respectively). Previous studies [Fluit, A.C., Baas, P.D., van Boom, J.H., Veeneman, G.H. and Jansz, H.S. (1984) Nucleic Acids Res. 12, 6443--6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by phi X174 gene A protein. Moreover, Brown et al. [Brown, D.R., Schmidt-Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402--8412] have shown that omission of the last 2 bp of the origin region does not interfere with phi X174 rolling-circle DNA replication in vitro. Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for phi X174 phage DNA replication and morphogenesis.

Bacteriophage phi X 174↗
PubMed1987

Factors controlling packaging of peptide hormones into secretory granules.

Endocrine, exocrine, and neuronal cells package only a subset of their secretory products into the electron-dense secretory granules. To investigate the factors controlling selective packaging of proteins into these granules, we utilized the mouse pituitary tumor cell line, AtT-20, which retained the capability to sort adrenocorticotropic hormone (ACTH) into secretory granules in vitro. Packaging of ACTH was blocked by treatment with weak bases, but was unaffected when N-linked glycosylation or sulfation was inhibited. To test whether the targeting information is specified by sorting domains present on peptide hormone sequences, we determined if a protein could be diverted to the dense secretory granules by attachment to a peptide hormone sequence. A plasmid DNA was constructed that encoded a hybrid protein in which a fragment of a viral membrane protein was fused to the carboxy terminus of human growth hormone. AtT-20 cells transfected with the hybrid were found to target it to dense secretory vesicles efficiently. These results support the hypothesis that sorting domains on peptide hormones direct their packaging into dense secretory vesicles.

Adrenocorticotropic Hormone↗
PubMed1993

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.

Amino Acid Sequence↗
PubMed1997

Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.

Adenosine Triphosphatases↗
PubMed2003

Isolation and analysis of mutants of double-stranded-RNA bacteriophage phi6 with altered packaging specificity.

The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented double-stranded RNA genomes into preformed polyhedral structures called procapsids or inner cores. This packaging requires hydrolysis of nucleoside triphosphates and takes place in the order S-M-L. Packaging is dependent on unique sequences of about 200 nucleotides near the 5' ends of plus strand transcripts of the three genomic segments. Changes in the pac sequences lead to loss of packaging ability but can be suppressed by second-site changes in RNA or amino acid changes in protein P1, the major structural protein of the procapsid. It appears that P1 is the determinant of the RNA binding sites, and it is suggested that the binding sites overlap or are conformational changes of the same domains.

Bacteriophage phi 6↗
PubMed2003

Analysis of specific binding involved in genomic packaging of the double-stranded-RNA bacteriophage phi6.

The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus-strand transcripts of the segmented double-stranded-RNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires the hydrolysis of nucleoside triphosphates and takes place in the order segment S-segment M, segment L. Packaging is dependent upon unique sequences of about 200 nucleotides near the 5' ends of plus-strand transcripts of the three genomic segments. It appears that P1 is the determinant of the RNA binding sites. Directed mutation of P1 was used to locate regions that are important for genomic packaging. Specific binding of RNA to the exterior of the procapsid was dependent upon ATP, and a region that showed a high level of cross-linking to phage-specific RNA was located. Antibodies to peptide sequences were prepared, and their abilities to bind to the exterior of procapsids were determined. Sites sensitive to trypsin and to factor Xa were determined as well.

Adenosine Triphosphate↗
PubMed2006

Assembly of two independent populations of flock house virus particles with distinct RNA packaging characteristics in the same cell.

Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.

Animals↗
PubMed2006

Ebola virus VP35-VP40 interaction is sufficient for packaging 3E-5E minigenome RNA into virus-like particles.

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minigenome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.

Animals↗
PubMed1977

In vitro packaging of UV radiation-damaged DNA from bacteriophage T7.

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.

Cell-Free System↗
PubMed1978

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.

Coliphages↗
PubMed1982

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging.

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.

Bacterial Proteins↗
PubMed1983

On the sequential packaging of bacteriophage P22 DNA.

Bacteriophage P22 is thought to package daughter chromosomes serially along concatemeric DNA. We present experiments which show that the average DNA packaging series length increases with time after infection, which supports this model. In addition, we have analyzed the effect on average series length of lowering the amount of the various individual proteins involved in DNA packaging. These results support the notion that the protein products of gene 2 and gene 3 are both more stringently required for initiation of sequential DNA packaging series than for their extension, and they are compatible with a model for the control of series length in which that length is determined, at least in part, by a competition between series initiation events and extension events.

Coliphages↗