Mutational analysis of the gene start sequences of pneumonia virus of mice

The transcriptional start sequence of pneumonia virus of mice is more variable than that of the other pneumoviruses, with five different nine-base gene start (GS) sequences found in the PVM genome. The sequence requirements of the PVM gene start signal, and the efficiency of transcriptional initiation of the different virus genes, was investigated using a reverse genetics approach with a minigenome construct containing two reporter genes. A series of GS mutants were created, where each of the nine bases of the gene start consensus sequence of a reporter gene was changed to every other possible base, and the resulting effect on initiation of transcription was assayed. Nucleotide positions 1, 2 and 7 were found to be most sensitive to mutation whilst positions 4, 5 and 9 were relatively insensitive. The L gene GS sequence was found to have only 20% of the activity of the consensus sequence whilst the published M2 gene start sequence was found to be non-functional. A minigenome construct in which the two reporter genes were separated by the F-M2 gene junction of PVM was used to confirm the presence of two alternative, functional, GS sequences that could both drive the transcription of the PVM M2 gene

Epidemiology of parainfluenza virus type 3 in England and Wales over a ten-year period

We have analysed data on respiratory syneytial (RS) and parainfiuenza type 3 (PF3) viruses reported to the Communicable Disease Surveillance Centre. London, over the period 1978–87. These confirm the annual winter epidemic of RS virus and show that, in England and Wales, PF3 is a summer infection with regular yearly epidemics

The host-range tdCE phenotype of Chandipura virus is determined by mutations in the polymerase gene

The emerging arbovirus Chandipura virus (CV) has been implicated in epidemics of acute encephalitis in India with high mortality rates. The isolation of temperature-dependent host-range (tdCE) mutants, which are impaired in growth at 39 °C in chick embryo (CE) cells but not in monkey cells, highlights a dependence on undetermined host factors. We have characterized three tdCE mutants, each containing one or more coding mutations in the RNA polymerase gene and two containing additional mutations in the attachment protein gene. Using reverse genetics, we showed that a single amino acid change in the virus polymerase of each mutant was responsible for the host-range specificity. In CE cells at the non-permissive temperature, the discrete cytoplasmic replication complexes seen in mammalian cells or at the permissive temperature in CE cells were absent with the tdCE mutants, consistent with the tdCE lesions causing disruption of the replication complexes in a host-dependent manner

Cyclodextrin-based catalysts and molecular reactors

We suggest two nonparametric approaches, based on kernel methods and orthogonal series to estimating regression functions in the presence of instrumental variables. For the first time in this class of problems, we derive optimal convergence rates, and show that they are attained by particular estimators. In the presence of instrumental variables the relation that identifies the regression function also defines an ill-posed inverse problem, the “difficulty” of which depends on eigenvalues of a certain integral operator which is determined by the joint density of endogenous and instrumental variables. We delineate the role played by problem difficulty in determining both the optimal convergence rate and the appropriate choice of smoothing parameter.Supported in part by NSF Grants SES-99-10925 and SES-03-52675

Thirty Years of Turnstiles and Transport

To characterize transport in a deterministic dynamical system is to compute exit time distributions from regions or transition time distributions between regions in phase space. This paper surveys the considerable progress on this problem over the past thirty years. Primary measures of transport for volume-preserving maps include the exiting and incoming fluxes to a region. For area-preserving maps, transport is impeded by curves formed from invariant manifolds that form partial barriers, e.g., stable and unstable manifolds bounding a resonance zone or cantori, the remnants of destroyed invariant tori. When the map is exact volume preserving, a Lagrangian differential form can be used to reduce the computation of fluxes to finding a difference between the action of certain key orbits, such as homoclinic orbits to a saddle or to a cantorus. Given a partition of phase space into regions bounded by partial barriers, a Markov tree model of transport explains key observations, such as the algebraic decay of exit and recurrence distributions.Comment: Updated and corrected versio

Sequence variation in the haemagglutinin-neuraminidase gene of human parainfluenza virus type 3 isolates in the UK

The sequence variation in a 934 base-pair region of the gene encoding the haemagglutinin-neuraminidase of five human parainfluenza virus type 3 (HPIV3) isolates was determined together with that of a prototype UK strain. All of the clinical isolates were from the Manchester area of the UK and were obtained in 1990. 1991 and 1993. The gene segment was amplified by the polymerase chain reaction using HPIVB-specific oligonucleotide primers. The nucleotide homology of the strains was high, around 99% and specific differences in the UK sequences when compared with that of the US prototype strain were identified. In addition, a number of isolate-specific differences were seen. No correlation was detected between the observed nucleotide mutations and the year of isolation, which supports the hypothesis that HPIV3 shows cocirculation of a heterogeneous population of viruses rather than varying with time in a linear fashion. However, the data suggested that geographically-defined genetic lineages of HPIV3 may exist

Improving Instructional Practice: The Value of Classroom Goal Teams as Measured by Elementary Teachers\u27 Perceptions

Student achievement is in the forefront of education as never before. Educators, parents, business leaders, community members, and politicians are all actively watching reports of student achievement. Wong (2003) found in more than 200 studies, the only way to improve student achievement is with a knowledgeable and skillful teacher. The expertise of a teacher is a critical variable in effecting student achievement (Marzano, 2003). In this study, Classroom Goals Team Project (CGTP) was utilized as a professional development program to bring about improvements in teaching and learning in an effort to positively impact student achievement. The CGTP, implemented in a suburban school district in Nebraska, is a process where classroom teachers were asked to identify an area of concern within their classroom based upon student performance assessment data. The major finding of the CGTP indicates the teachers of this district view the CGTP as an effective professional development model and classroom goals team meetings were perceived as productive by 89% of the teachers. Other findings of this study focus on the impact of five constructs identified in the research as critical to effective professional development programs. These constructs are: learning community/ collaborative teams, quality teaching/ instructional practices, leadership, data driven decision making, and equity. A benefit of the CGTP was the foundation for fundamental change in attitudes and perceptions of what professional development looks like and sounds like in this district. Professional development has gone beyond a one day, shot in the dark event to a much higher level of active engagement and monitoring of successful implementation with consistent and frequent feedback from peers. Students had an increased opportunity to learn through the CGTP, which according to Berlinger & Biddle (1997) is the single most powerful predictor of student achievement. The results of the review of literature and the data from this study support the need to have a professional development program, which is .student achievement driven, and teacher focused in learning communities

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level

Retrograde transport pathways utilised by viruses and protein toxins

A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised

Seasonal Movements, Migratory Behavior, and Site Fidelity of West Indian Manatees along the Atlantic Coast of the United States as Determined by Radio-telemetry

The study area encompassed the eastern coasts of Florida, Georgia, and South Carolina, including inland waterways such as the St. Johns River (Fig. 1). Manatees inhabited the relatively narrow band of water that lies between the barrier beaches and the mainland, occasionally venturing into the ocean close to shore. Between Miami and Fernandina Beach, Florida, 19 inlets provided manatees with corridors between the intracoastal waters and the Atlantic Ocean; the distance between adjacent inlets averaged 32 km(SD = 24 km) and varied from 3 to 88 km. Habitats used by manatees along this 900-km stretch ofcoastline varied widely and included estuaries, lagoons, rivers and creeks, shallow bays and sounds, and ocean inlets. Salinities in most areas were brackish, but ranged from completely fresh to completely marine. The predominant communities of aquatic vegetation also varied geographically and with salinity: seagrass meadows and mangrove swamps in brackish and marine waters along the southern half of peninsular Florida; salt marshes in northeastern Florida and Georgia; benthic macroalgae in estuarine and marine habitats; and a variety of submerged, floating, and emergent vegetation in freshwater rivers, canals, and streams throughout the region. Radio-telemetry has been used successfully to track manatees in other regions ofFlorida (Bengtson 1981, Powell and Rathbun 1984, Lefebvre and Frohlich 1986, Rathbun et al. 1990) and Georgia (Zoodsma 1991), but these early studies relied primarily on conventional VHF (very high frequency) transmitters and were limited in their spatial and temporal scope (see O'Shea and Kochman 1990 for overview). Typically, manatees were tagged at a thermal refuge in the winter and then tracked until the tag detached, usually sometime between the spring and fall of the same year. Our study differs from previous research on manatee movements in several important respects. First, we relied heavily on data from satellite-monitored transmitters using the Argos system, which yielded a substantially greater number of locations and more systematic collection of data compared to previous VHF tracking studies (Deutsch et al. 1998). Second, our tagging and tracking efforts encompassed the entire range of manatees along the Atlantic coast, from the Florida Keys to South Carolina, so inferences were not limited to a small geographic area. Third, we often used freshwater to lure manatees to capture sites, which allowed tagging in all months of the year; this provided more information about summer movement patterns than had previous studies which emphasized capture and tracking at winter aggregations. Finally, the study spanned a decade, and success in retagging animals and in replacing transmitters allowed long-term tracking ofmany individuals. This provided the opportunity to investigate variation in seasonal movements, migratory behavior, and site fidelity across years for individual manatees. (254 page document.