Packaging Specificity Research Articles

Multiple view environment supporting VDM and Ada

The paper presents a multiple view, software development environment that supports transformation between VDM and Ada. The environment allows a software system to be represented in a canonical, source language independent form. This representation may then be viewed, and modified, via editors which support different notation. Two specific editors have been developed; one supports the Vienna Development Method (VDM) notation, and the other supports the Ada programming language package specification notation. Automatic transformation between the two notations is achieved via the canonical representation. The representation is stored in a repository implemented using the Portable Common Tool Environment (PCTE), and the editors are implemented using Tcl/Tk.

Isolation of a Mutant That Changes Genomic Packaging Specificity in φ6

Bacteriophage φ6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Plus strand transcripts of the segments are packaged in a serially dependent fashion in which S can package alone, M depends on S, and L depends on S and M. We have isolated a mutant form of the virus in the carrier state that has lost segment S. This finding presented an apparent anomaly with respect to the packaging program. Sequencing of gene 1 of segment L in this virus showed a translational change of arginine to glycine at the 14th position. Procapsids prepared from cDNA containing this mutation show behavior inin vitropackaging that is consistent with the phenotype of the mutant virus. The procapsids are able to package segment S alone, but this RNA is present in reduced amounts when the other segments are present. Segments M and L package without dependence on segment S. The mutant virus appears to produce procapsids that are at the second stage of the packaging program.

Nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector RNA.

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Psi) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Psi, but MLV can recognize only MLV Psi. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

MODIFIED-ATMOSPHERE PACKAGING OF FRUITS AND VEGETABLES: REDUCING CONDENSATION OF WATER IN BELL PEPPERS AND MANGOES

A dynamic model of gas and water vapor exchange in a package containing fresh fruit and vegetables was based on the Michaelis-Menten equation combined with Fick's diffusion law. A computer simulation evaluated package specifications which would provide best conditions for storage of a given commodity. Model predictions were confirmed by measured observations. Perforating the film greatly affected O 2 and CO 2 concentrations and water condensation, but not the in-package relative humidity. Bell peppers packaged in perforated film lost less weight and maintained higher quality during storage than fruit stored in open carton boxes and, at the same time, had lower decay levels than peppers kept in non-perforated packages. Perforating the film negated inhibition of ripening that prevailed for mango in non-perforated packages. Perforated packages retained most advantages of the modified atmosphere. Non-perforated film of the polyamide type had similar effects to perforated polyolefin film. Water condensation on the fruit and container walls was prevented by use of absorbers.

ILLIADS-T: an electrothermal timing simulator for temperature-sensitive reliability diagnosis of CMOS VLSI chips

In this paper, we present a new chip-level electrothermal timing simulator for CMOS VLSI circuits. Given the chip layout, the packaging specification, and the periodic input signal pattern, it finds the on-chip steady-state temperature profile and the resulting circuit performance. A tester chip has been designed for verification of ILLIADS-T, and very good agreement between simulation and experiment was found. Using this electrothermal simulator, temperature-dependent reliability and timing problems of VLSI circuits can be accurately identified.

Structured pluralism: towards an innovative model for health system reform in Latin America

Health systems throughout the world are searching for better ways of responding to present and future challenges. Latin America is no exception in this innovative process. Health systems in this region have to face a dual challenge: on the one hand, they must deal with a backlog of accumulated problems characteristic of underdeveloped societies; on the other hand, they are already facing a set of emerging problems characteristic of industrialized countries. This paper aims at analyzing the performance of current health systems in Latin America, while proposing an innovative model to promote equity, quality, and efficiency. We first develop a conceptualization of health systems in terms of the relationships between populations and institutions. In order to meet population needs, health systems must perform four basic functions. Two of these-financing and delivery-are conventional functions performed by every health system. The other two have often been carried out only in an implicit way or not at all. These neglected functions are `modulation' (a broader concept than regulation, which involves setting transparent and fair rules of the game) and `articulation' (which makes it possible to organize and manage a series of transactions among members of the population, financing agencies, and providers so that resources can flow into the production and consumption of services). Based on this conceptual framework, the paper offers a classification of current health system models in Latin America. The most frequent one, the segmented model, is criticized because it segregates the different social groups into three segments: the ministry of health, the social security institute(s), and the private sector. Each of these is vertically integrated, so that it performs all functions but only for a particular group. As an alternative, we propose a model of `structured pluralism', which would turn the current system around by organizing it according to functions rather than social groups. In this model, modulation would become the central mission of the ministry of health, which would move out of the direct provision of personal health services. Financing would be the main function of social security institutes, which would be gradually extended to protect the entire population. The articulation function would be made explicit by fostering the establishment of `organizations for health services articulation', which would perform a series of crucial activities, including the competitive enrollment of populations into health plans in exchange for a risk-adjusted capitation, the specification of explicit packages of benefits or interventions, the organization of networks of providers so as to structure consumer choices, the design and implementation of incentives to providers through payment mechanisms, and the management of quality of care. Finally, the delivery function would be open to pluralism that would be adapted to differential needs of urban and rural populations. After examining the convergence of various reform initiatives towards elements of the structured pluralism model, the paper reviews both the technical instruments and the political strategies for implementing changes. The worldwide health reform movement needs to sustain a systematic sharing of the unique learning opportunity that each reform experience represents.

A subgenomic mRNA transcript of the coronavirus mouse hepatitis virus strain A59 defective interfering (DI) RNA is packaged when it contains the DI packaging signal.

In infected cells, only the genomic RNA of the coronavirus mouse hepatitis virus strain A59 (MHV-A59) is packaged into the virions. In this study, we show that a subgenomic (sg) defective interfering (DI) RNA can be packaged into virions when it contains the DI RNA packaging signal (DI RNA-Ps). However, the sg DI RNA is packaged less efficiently than the DI genomic RNA. Thus, while specificity of packaging of RNAs into MHV-A59 virions is determined by the DI RNA-Ps, efficiency of packaging is determined by additional factors.

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6.

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

DNA Sequences Responsible for Specificity of DNA Packaging and Phage Growth Interference of Bacteriophages T3 and T7

T3 and T7 phages package recombinant plasmids carrying DNA necessary for DNA packaging (thepacsequences) of T3 and T7, respectively. Packaging is specific between T3 and T7. Thepacsequence has a bipartite structure, consisting of target sequences for processing of concatemeric DNA (pacC) and its left side flanking sequence containing a promoter for phage RNA polymerase (pacB). To determine the sequences responsible for the specificity of plasmid DNA packaging, plasmids chimeric for thepacB andpacC sequences of T3 and T7 were constructed. Analysis of packaging of the chimeric plasmid DNAs showed thatpacB is responsible for the packaging specificity of T3 and T7 DNAs. Plasmids carrying the genetic right end of T3 and T7 DNA interfered with the growth of T3 and T7 phages, respectively. Interference was specific between T3 and T7.pacB and sequences betweenpacB andpacC, but notpacC, were responsible for the interference. The specificity of interference was determined bypacB and sequences responsible for interference were partially defined.

Packaging and Transport of Waste to the Nirex (UK) Repository

United Kingdom Nirex Limited (Nirex) is developing a deep repository for the disposal of ILW and some LLW from UK waste producers. Two key parts of the programme are the specification of waste packages—containers and their contents—and the development of a transport system to convey packaged wastes to the repository. Nirex and waste producers have established a range of standard waste packages and transport packages. Each standard waste package meets an identified need and is defined by a Waste Package Specification which provides the essential link to the repository design. Re-usable shielded transport containers are being developed to transport certain waste packages to the repository, the transport container and contents together forming a Type B package. A Probabilistic Safety Assessment for transport associated with a repository at Sellafield predicts that ‘conventional’ transport risks will be simply related to the volumes of rail and road traffic. The additional radiological risks are expected to be extremely low.

Hepadnaviruses: Current Models of RNA Encapsidation and Reverse Transcription

Publisher Summary Progress in the field of hepadnaviral research has advanced considerably through the use of tissue culture systems for viral expression and the development of systems for the in vitro expression of the polymerases. Although the essential similarities between hepadnaviruses and the classically studied retroviruses have been confirmed, differences between the two groups of viruses have become strikingly apparent. Synthesis of the hepadnaviral polymerase as a distinct translation product and the central role played by this protein in directing RNA encapsidation may be contrasted with the retroviral model in which polymerase is expressed as a gag-pol fusion protein and the major determinants of RNA packaging specificity reside on the gag protein. Whereas retroviruses typically carry out both proteolytic processing and RNA splicing, there is no evidence for either protein processing or splicing in hepadnaviruses. The use of a protein primer for the synthesis of minus strand DNA is without precedent among the retroviruses, which employ a host cell tRNA for this function. In both cases, strand transfer is mediated through the use of terminal sequence similarities. However, the plus and minus strand primer translocations intrinsic to hepadnaviral reverse transcription are very different from the strongstop DNA translocations that occur in retroviruses. A characteristic feature of hepadnaviral replication is transcription from the unintegrated CCC DNA and the maintenance of this DNA through a pathway of intracellular amplification, whereas retroviral expression and persistence depend on a mechanism of genomic integration.

The Development of a ‘Large liner’ For the Processing and Solidification Of Intermediate Level Wastes

Magnox Electric have developed a ‘Large Liner’ for the processing and solidification of the majority of the slurry form intermediate level wastes which arise on its power station sites (operational or undergoing decommissioning). The Liner comprises a cylindrical vessel of approximate dimensions 1.7 m diameter by 1.2 m high and it has been developed to comply with the requirements of the current Nirex Package Specifications. It is manufactured from stainless steel, being of all welded construction, with a bolted lid, incorporating an ‘O’ ring elastomer seal. The Liner can be fitted with a paddle for the ‘in drum’ solidification of slurry from wastes. This paper describes the development of the Liner, outlining the main stages from initial concepts to the final testing and receipt of approval from Nirex, who have adopted the Liner as one of its ‘Standard’ waste containers.

Synthesis of a Full-Length Infectious cDNA Clone of Cucurbit Aphid-Borne Yellows Virus and Its Use in Gene Exchange Experiments with Structural Proteins from Other Luteoviruses

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as anin vitrotranscript, under control of a bacteriophage T7 RNA polymerase promoter, orin vivo,under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of itsin vitrotranscript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants could be transmitted by the aphid vectorsMyzus persicaeandAphis gossypii.The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

Nucleocapsid protein effects on the specificity of retrovirus RNA encapsidation

We have analyzed the roles of Gag protein nucleocapsid (NC) domains in the packaging or encapsidation of retroviral RNAs into virus particles. We found that mutation of both zinc finger motifs of the human immunodeficiency virus (HIV) NC domain reduced but did not eliminate encapsidation of the HIV viral RNA. However, the NC mutations also resulted in a three- to fourfold reduction in the specificity of RNA encapsidation, as determined by comparison of virus-associated genomic and spliced RNA levels. As a complementary approach, we replaced the NC domain of Moloney murine leukemia virus (M-MuLV) with that of HIV. Chimeric virus particles assembled efficiently, were of wild-type M-MuLV density, and cross-linked at NC cysteines. In encapsidation studies, wild-type M-MuLV precursor Gag (PrGag) proteins packaged M-MuLV transcripts more efficiently than HIV RNAs. In contrast, chimeric PrGag proteins possessing the HIV-1 NC domain in the context of the M-MuLV MA (matrix), p12, and CA (capsid) domains encapsidated HIV transcripts to a greater extent than M-MuLV transcripts. Our results support the notion that retroviral NC domains contribute toward both the efficiency and specificity of viral genomic RNA packaging.

The in Vitro Translocase Activity of λ Terminase and Its Subunits: KINETIC AND BIOCHEMICAL ANALYSIS

The terminase holoenzyme of bacteriophage lambda is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1.

RNA Binding, Packaging and Polymerase Activities of the Different Incomplete Polymerase Complex Particles of dsRNA Bacteriophage φ6

φ6 is an enveloped dsRNA bacterial virus. Its segmented genome resides inside the virion associated polymerase complex which is formed by four proteins (P1, P2, P4 and P7) encoded by the viral L segment. Complete and incomplete polymerase complex particles can be produced using cDNA copies of this largest genome segment. We have analysed the capacity of the different purified particles to (1) package φ6 (+) sense genomic precursors and unspecific RNA, (2) synthesize (−) and (+) strands and (3) bind φ6 specific and unspecific RNAs. Both (−) and (+ strand synthesis polymerase activities were found to be associated with protein P2. In addition to complete particles, particles lacking protein P2 were found to package and protect genomic precursor ssRNAs. Protein P7 was needed for efficient packaging. Regulation and specificity of the packaging were found to be independent of P2. Particles composed of proteins P1 and P4 did not package or protect RNA but did bind φ6 genomic (+) strand RNAs. The three φ6 (+) strands bound in equal amounts to the particles when tested alone in a filter binding assay. In competition experiments they competed each other for binding, indicating that individual binding sites for the three genomic (+) strands do not exist. Differences in RNA binding competition among the four particles were observed, suggesting that packaging specificity is achieved by complex interactions of proteins and genomic (+) strand RNAs during the advancement of the packaging process after the initial binding events.

Packaging in a yeast double-stranded RNA virus.

The yeast virus ScV-L1 has only two genes, cap and pol, which encode the capsid polypeptide and the viral polymerase, respectively. The second gene is translated only as a cap-pol fusion protein. This fusion protein is responsible for recognition of a specific small stem and loop region of the viral plus strands, of 19 to 31 bases in length, ensuring packaging specificity. We have used a related virus, ScV-La, which has about 29% codon identity with ScV-L1 in the most conserved region of the pol gene, to map the region in pol that is responsible for packaging L1. Characterization of a number of chimeric viral proteins that recognize L1 but have the La capsid region delimits the region necessary for recognition of L1 to a 76- to 82-codon portion of pol. In addition, we show that overproduction of the La capsid polypeptide results in curing of the ScV-La virus, analogous to the production of plants resistant to RNA viruses by virtue of systemic production of viral coat protein.

Modified Atmosphere Packaging Under Varying Surrounding Temperature

A mathematical model was developed to predict temperature and gas concentrations in modified atmosphere packages exposed to variable surrounding temperatures. Experiments were conducted with strawberries under constant and variable surrounding temperatures to validate the model for both conditions. For a constant surrounding temperature condition of 15.2 C, the mathematical model closely predicted the CO2 concentrations in the packages, whereas the O2 concentrations were underpredicted. For a variable surrounding temperature condition of 12 h at 18.6 C, followed by 48 h at 4.2 C, then 12 to 20 h at 18.6 C, temperatures and O2 concentrations predicted by the model closely agreed with the experimental data. The CO2 concentrations in some of the packages were slightly underpredicted. Natural variation in respiration rate between batches of the product caused the slight disagreement between experimental and model-predicted results. The mathematical model can be used not only to determine the package specifications for a given product and given surrounding temperatures, but also to predict the amount of time required for the packaging system to attain the designed optimum conditions.

Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.

Supporting distribution and dynamic reconfiguration in AdaPT

It is widely accepted that Ada83 provides inadequate support for the programming of distributed systems. Ada9X has introduced a unit of distribution called a partition. Partitions comprise aggregations of library units that collectively may execute in a distributed target execution environment. Each partition corresponds to a single execution site where all its library units occupy the same logical address space. The principal interface between partitions is one or more package specifications. Although Ada9X provides basic support for partitioning applications, it falls short of providing the full expressive power that would be expected of a language specially designed to support distributed systems. In particular, the unit of distribution is not a first class language object and methods for dynamic configuration are primarily left to the implementor. We briefly review the main requirements for programming distributed systems and illustrate where the current Ada9X proposals lack expressive power. We then introduce a new variant of Ada called AdaPT which has been designed to address explicitly partitioning and dynamic reconfiguration of distributed Ada programs. We illustrate how AdaPT programs can be written and detail their translation into Ada83 and Ada9X.