No abstract

Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

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Composites of piezoelectric and magnetosirictive materials show a product property called magnetoelectricity which is absent in its constituent phases. The electric and magnetic fields are linked in the composite through the elastic stress-strain fields of the piezoelectric and magnetostrictive phases. Thus an applied magnetic field causes electric polarization or an electric field applied across the composite causes magnetization in the composite material. Such materials are potentially useful as magnetoelectric transducers. In this work, we report the results of a theoretical and experimental investigation of ME composites of PZT-CoFe2O4 and BaTiO3-CoFe2O4 with various connectivities. The magnetically induced ME effect was measured by applying an ac magnetic field of 1 kHz frequency with a variable dc magnetic bias field. The maximum values of the ME voltage coefficient, were 92.8 (V/m)/(kA/m) for the 2-2 CoFe2O4-PZT4 composites and 9.55 (V/m)/(kA/m) for CoFe2O4-BaTiO3 (20:80 mole %) ball mill mixed composites.Theoretical models were developed to calculate the ME voltage coefficient of 2-2 composites with different boundary conditions. Composites with 3-0 and 0-3 connectivities were modeled using a cubes model. The results show that the connectivities have a great effect on the magnetoelectric properties. An improvement of more than two orders of magnitude is possible by proper selection of materials and better process control to tailor the connectivity.

Studies in B cell-deficient mice generated by continuous injection of anti-mu antibodies (muSM) showed that T cell priming in lymph nodes was dependent on antigen presentation by B cells. This concept has recently become controversial since a wide range, from complete deficiency to near normal T cell responses, was reported in studies carried out with B cell-deficient mice generated by gene disruption (muMT). In this study we show that in the absence of B cells, T cell responses are greatly reduced in all the available muMT mouse strains although responses in muMT of the C57BL/6 background (which were used for most studies with muMT) were much more variable and could reach up to 42% of control. In contrast, T cell responses in muMT --> F(1) bone marrow chimeras which have the same phenotype as muMT were totally impaired, suggesting a principle difference between mice developing without B cells (muMT mice) and muSM which are made B cell deficient only after birth. Normal T cell priming was completely restored by reconstitution of muMT and muMT --> F(1) mice with syngeneic B cells. Interestingly, only B cell populations containing antigen-specific B cells were capable of reconstituting T cell responses. Monoclonal B cells taken from Ig transgenic mice could not reconstitute responses to an irrelevant antigen. We also found that B cells were also required for systemic T cell priming when antigen concentrations were limiting but were not required for priming (for T cell help) when mice were immunized with a high antigen dose.

A sintering, microstructural development and dielectric property study of BaTiO3–LiF ceramics was performed to assess the potential application of low-fired multilayer capacitors. Not only does LiF allow for sintering below 1000 °C, it also allows for the manipulation of dielectric properties and interfaces within BaTiO3–LiF ceramics. Using mixing laws, a model of the dielectric properties of the core-shell microstructures is presented that agrees well with the observed experimental data.

Spleen necrosis virus (SNV) is an avian retrovirus that efficiently infects some mammalian cells (e.g., dog and rat cells). We constructed an SNV-based vector, which contains less than 1 kilobase (kb) of the retrovirus sequence, and a number of derivatives containing selectable markers. We obtained high-titer virus stocks, over 106 transforming units per ml, with a vector whose genomic RNA consists of 1,850 bases (full-length SNV RNA is 7.7 kb). We also studied two vectors that both carry two genes which should be expressed from a single promoter, one gene from unspliced mRNA and the other gene from spliced mRNA. In one vector, both genes were efficiently expressed as expected. However, in the other vector, expression of the gene 3' to the splice acceptor was inhibited. When we selected for expression of the 3' gene is this latter case, we found that the resistant cells contained mutant proviruses in which the 3' gene could be expressed. Furthermore, we found that mutations were generated during a single round of virus replication (provirus to provirus) at a rate of approximately 0.5% mutations per cycle.Retroviruses are RNA viruses that replicate through a DNA intermediate. The DNA intermediate, the provirus, stably integrates into the chromosomal DNA of the host cell. Moreover, retroviruses can effectively infect most or all cells in a culture (37). Because of these properties, retroviral vectors represent an attractive system for stable introduction of exogenous genes into cells.Retroviral vectors that contain two expressed genes have been of particular interest because they can carry both a gene of interest and a selectable gene, enabling easy selection of cells that have been infected by vector-derived virus. There are two major ways such a vector can be constructed. One is to make a vector with an internal promoter so that one gene is expressed from the retrovirus long terminal repeat (LTR) promoter while the other gene is expressed from an internal promoter. The other method is to contruct a vector so that both genes are expressed from a single LTR promoter, one from unspliced retroviral RNA and the other from spliced retroviral RNA. Both types of vectors have been made (2, 4-6, 9, 10, 12, 15, 16, 20, 22, 26, 28-30, 32, 36, 40).We are interested in using spleen necrosis virus (SNV)-based retroviral splicing vectors to introduce different genes into avian cells, but we previously found that inserting different genes into reticuloendotheliosis virus (Rev) strain T (Rev-T) suppresses transformation by reducing the amount of spliced . Consequently, we thought that it would be prudent to study in detail the biological properties of retroviral vectors with spliced mRNA. We term these constructions splicing vectors. Moreover, because of the interest in using splicing vectors as vehicles in somatic therapy of human genetic diseases, a deeper understanding of their properties should be helpful in developing successful protocols for their use in this therapy. We constructed two SNV-based splicing vectors that each...

Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1⌬ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1⌬ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1⌬ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3 untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.Gene expression in eukaryotes is a highly diverse process, involving regulation at both transcriptional and posttranscriptional levels (47, 70). The process of mRNA turnover is an important posttranscriptional control point that helps to modulate the cellular abundance of a transcript. A large number of clinically relevant transcripts exhibit regulated decay in response to cellular signals, and the deregulation of their decay rates directly correlates with disease states (58,66,70). In the yeast Saccharomyces cerevisiae, the principal mRNA degradation pathway initiates with the removal of the poly(A) tail, followed by either decapping and 5Ј33Ј exonucleolytic decay or 3Ј35Ј exosome-mediated degradation (10, 64). In addition to this pathway, aberrant mRNAs harboring premature termination codons are degraded by an alternate nonsense-mediated decay (NMD) pathway that functions to ensure quality control of gene expression. NMD is initiated when a premature termination codon is recognized during translation termination and stimulates rapid deadenylation-independent decapping, followed by 5Ј33Јdegradation of the mRNA (4). Messages undergoing leaky scanning (69) or harboring upstream open reading frames (50, 59) represent some naturally occurring substrates that decay through this pathway.The turnover of mRNAs is mediated by the interplay between...