|
| ||
|
|
||
Binary Vector (binary + vector)
Selected AbstractsMolecular Characterization of a Strain of Squash Leaf Curl China Virus from the PhilippinesJOURNAL OF PHYTOPATHOLOGY, Issue 10 2003T. Kon Abstract The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA-A and DNA-B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA-A and two in DNA-B. Partial dimeric clones containing DNA-A and DNA-B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro-inoculated pumpkin plants (Cucurbita moschata). The total DNA-A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus -[Philippines] (SLCCNV-[PH]). [source] Genome Organization of an Infectious Clone of Tomato Leaf Curl Virus (Philippines), a New Monopartite Begomovirus*JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2002Tatsuya Kon Abstract Complete nucleotide sequence of infectious cloned DNA of Tomato leaf curl virus from Philippines (ToLCV-Ph) was determined. The single circular DNA molecule comprises 2755 nucleotides. ToLCV-Ph DNA contains six open reading frames (ORFs) each capable of encoding proteins with a molecular weight greater than 10 kDa. A partial dimeric ToLCV-Ph DNA clone was constructed in a binary vector and used to agroinoculate tomato plants (Lycopersicon esculentum Mill. cv. Zuikou 102). Typical leaf curl symptoms were observed, showing that the single DNA component is sufficient for infectivity. In total nucleotide sequence comparisons with other geminiviruses, ToLCV-Ph was most closely related to Ageratum yellow vein virus (AYVV) (79% identity), ToLCV-Laos (78%), Soyabean crinkle leaf virus -Thailand (78%) and ToLCV-Taiwan (77%). The significant but relatively low sequence identity in the genomic DNA between ToLCV-Ph and other geminiviruses suggests that it is a distinct geminivirus in the genus Begomovirus. [source] Optimization of the bioprocessing conditions for scale-up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression systemBIOTECHNOLOGY PROGRESS, Issue 3 2009Michael A. Plesha Abstract Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD600 of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] High-Level Transient Production of a Heterologous Protein in Plants by Optimizing Induction of a Chemically Inducible Viral Amplicon Expression SystemBIOTECHNOLOGY PROGRESS, Issue 6 2007Michael A. Plesha We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, ,-1-antitrypsin (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8-fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3-fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post-induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution. [source] Development of Auxotrophic Agrobacterium tumefaciens for Gene Transfer in Plant Tissue CultureBIOTECHNOLOGY PROGRESS, Issue 3 2004Jason I. Collens Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefacienswas achieved. [source] Modeling multiple-response categorical data from complex surveysTHE CANADIAN JOURNAL OF STATISTICS, Issue 4 2009Christopher R. Bilder Abstract Although "choose all that apply" questions are common in modern surveys, methods for analyzing associations among responses to such questions have only recently been developed. These methods are generally valid only for simple random sampling, but these types of questions often appear in surveys conducted under more complex sampling plans. The purpose of this article is to provide statistical analysis methods that can be applied to "choose all that apply" questions in complex survey sampling situations. Loglinear models are developed to incorporate the multiple responses inherent in these types of questions. Statistics to compare models and to measure association are proposed and their asymptotic distributions are derived. Monte Carlo simulations show that tests based on adjusted Pearson statistics generally hold their correct size when comparing models. These simulations also show that confidence intervals for odds ratios estimated from loglinear models have good coverage properties, while being shorter than those constructed using empirical estimates. Furthermore, the methods are shown to be applicable to more general problems of modeling associations between elements of two or more binary vectors. The proposed analysis methods are applied to data from the National Health and Nutrition Examination Survey. The Canadian Journal of Statistics © 2009 Statistical Society of Canada Quoique les questions du type « Sélectionner une ou plusieurs réponses » sont courantes dans les enquêtes modernes, les méthodes pour analyser les associations entre les réponses à de telles questions viennent seulement d'être développées. Ces méthodes sont habituellement valides uni-quement pour des échantillons aléatoires simples, mais ce genre de questions apparaissent souvent dans les enquêtes conduites sous des plans de sondage beaucoup plus complexes. Le but de cet article est de donner des méthodes d'analyse statistique pouvant être appliquées aux questions de type « Sélectionner une ou plusieurs réponses » dans des enquêtes utilisant des plans de sondage complexes. Des modèles loglinéaires sont développés permettant d'incorporer les réponses multiples inhérentes à ce type de questions. Des statistiques permettant de comparer les modèles et de mesu-rer l'association sont proposées et leurs distributions asymptotiques sont obtenues. Des simulations de Monte-Carlo montrent que les tests basés sur les statistiques de Pearson ajustées maintiennent généralement leur niveau lorsqu'ils sont utilisés pour comparer des modèles. Ces études montrent également que les niveaux des intervalles de confiance pour les rapports de cotes estimés à par-tir des modèles loglinéaires ont de bonnes propriétés de couverture tout en étant plus courts que ceux utilisant les estimations empiriques. De plus, il est montré que ces méthodes peuvent aussi êtres utilisées dans un contexte plus général de modélisation de l'association entre les éléments de deux ou plusieurs vecteurs binaires. Les méthodes d'analyse proposées sont appliquées à des données provenant de l'étude américaine « National Health and Nutrition Examination Survey » (NHANES). La revue canadienne de statistique © 2009 Société statistique du Canada [source] Variable Selection in High-Dimensional Multivariate Binary Data with Application to the Analysis of Microbial Community DNA FingerprintsBIOMETRICS, Issue 2 2002J. D. Wilbur Summary. In order to understand the relevance of microbial communities on crop productivity, the identification and characterization of the rhieosphere soil microbial community is necessary. Characteristic profiles of the microbial communities are obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16s rDNA from soil extracted DNA. These characteristic profiles, commonly called community DNA fingerprints, can be represented in the form of high-dimensional binary vectors. We address the problem of modeling and variable selection in high-dimensional multivariate binary data and present an application of our methodology in the context of a controlled agricultural experiment. [source] | |||||||||||||