RDP4 is the latest version of recombination detection program (RDP), a Windows computer program that implements an extensive array of methods for detecting and visualising recombination in, and stripping evidence of recombination from, virus genome sequence alignments. RDP4 is capable of analysing twice as many sequences (up to 2,500) that are up to three times longer (up to 10 Mb) than those that could be analysed by older versions of the program. RDP4 is therefore also applicable to the analysis of bacterial full-genome sequence datasets. Other novelties in RDP4 include (1) the capacity to differentiate between recombination and genome segment reassortment, (2) the estimation of recombination breakpoint confidence intervals, (3) a variety of ‘recombination aware’ phylogenetic tree construction and comparison tools, (4) new matrix-based visualisation tools for examining both individual recombination events and the overall phylogenetic impacts of multiple recombination events and (5) new tests to detect the influences of gene arrangements, encoded protein structure, nucleic acid secondary structure, nucleotide composition, and nucleotide diversity on recombination breakpoint patterns. The key feature of RDP4 that differentiates it from other recombination detection tools is its flexibility. It can be run either in fully automated mode from the command line interface or with a graphically rich user interface that enables detailed exploration of both individual recombination events and overall recombination patterns.

/pdf/rdp4-detection-and-analysis-of-recombination-patterns-in-598kf8ufr0.pdf

RDP4: Detection and analysis of recombination patterns in virus genomes.

rpd3 is a computer program for statistical identification and characterization of historical recombination events. Given a set of aligned nucleotide sequences, rpd3 will rapidly analyze these with a range of powerful non-parametric recombination detection methods (including bootscan, maxchi, chimaera, 3seq, geneconv, siscan, phylpro and visrd; Boni et al., 2007; Gibbs et al., 2000; Lemey et al., 2009; Padidam et al., 1999, Posada and Crandall, 2001; Weiller, 1998). It will provide a detailed breakdown of recombination breakpoint locations, and the identities of recombinant and parental sequences. For further downstream analyses, the program enables users to save edited sequence alignments with (i) recombinant sequences removed; (ii) recombinationally derived tracts of sequence removed; or (iii) recombinant sequences split into their constituent parts.

An important strength of rdp3 that makes it applicable to a variety of recombination analysis problems is that, unlike many other recombination detection programs such as simplot (Lole et al., 1999), dual brothers (Minin et al., 2005), jphmm (Schultz et al., 2006) or scueal (Kosakovsky et al., 2009), it does not screen predefined sets of potentially recombinant (or query) sequences against other predefined sets of non-recombinant (or reference) sequences. rdp3 instead treats every sequence within an input alignment as a potential recombinant and systematically screens large numbers of sequence triplets and/or quartets to identify sets of three or four sequences that contain a recombinant and two sequences resembling its parents. Such an approach means that rdp3 can simultaneously detect the entire scope of recombination evident within a dataset (i.e. not just that occurring between the reference strains or species) enabling its use in the characterization of complex recombinants such as those derived through recombination between parental sequences that were themselves recombinant. The drawback of such a flexible, exploratory framework is that it can often be difficult to assess the uncertainty associated with inferred recombination patterns. However, with its wide range of cross-checking tools, rpd3 is complementary to probabilistic recombination analysis approaches.

/pdf/rdp3-a-flexible-and-fast-computer-program-for-analyzing-dr8mg44rit.pdf

RDP3: a flexible and fast computer program for analyzing recombination

Summary: RDP2 is a Windows 95/XP program that examines nucleotide sequence alignments and attempts to identify recombinant sequences and recombination breakpoints using 10 published recombination detection methods, including GENECONV, BOOTSCAN, MAXIMUM χ2, CHIMAERA and SISTER SCANNING. The program enables fast automated analysis of large alignments (up to 300 sequences containing 13 000 sites), and interactive exploration, management and verification of results with different recombination detection and tree drawing methods.

Availability: RDP2 is available free from the RDP2 website (http://darwin.uvigo.es/rdp/rdp.html)

Contact: darren@science.uct.ac.za

Supplementary information: Detailed descriptions of RDP2 and the methods it implements are included in the program manual, which can be downloaded from the RDP2 website.

RDP2: recombination detection and analysis from sequence alignments

Imagine a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight. This could one day become reality as we learn to harness the power of plants for the production of recombinant proteins on an agricultural scale. Molecular farming in plants has already proven to be a successful way of producing a range of technical proteins. The first plant-derived recombinant pharmaceutical proteins are now approaching commercial approval, and many more are expected to follow.

/pdf/the-production-of-recombinant-pharmaceutical-proteins-in-h5b4asi42c.pdf

The production of recombinant pharmaceutical proteins in plants.

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated in Uganda in 1947. Although entomological and virologic surveillance have reported ZIKV enzootic activity in diverse countries of Africa and Asia, few human cases were reported until 2007, when a Zika fever epidemic took place in Micronesia. In the context of West Africa, the WHO Collaborating Centre for Arboviruses and Hemorrhagic Fever at Institut Pasteur of Dakar (http://www.pasteur.fr/recherche/banques/CRORA/) reports the periodic circulation of ZIKV since 1968. Despite several reports on ZIKV, the genetic relationships among viral strains from West Africa remain poorly understood. To evaluate the viral spread and its molecular epidemiology, we investigated 37 ZIKV isolates collected from 1968 to 2002 in six localities in Senegal and Cote d'Ivoire. In addition, we included strains from six other countries. Our results suggested that these two countries in West Africa experienced at least two independent introductions of ZIKV during the 20th century, and that apparently these viral lineages were not restricted by mosquito vector species. Moreover, we present evidence that ZIKV has possibly undergone recombination in nature and that a loss of the N154 glycosylation site in the envelope protein was a possible adaptive response to the Aedes dalzieli vector.

/pdf/molecular-evolution-of-zika-virus-during-its-emergence-in-3hvulu8zbk.pdf

Molecular Evolution of Zika Virus during Its Emergence in the 20th Century

Possible emergence of new geminiviruses by frequent recombination.

We have developed an inducible gene expression system with potential for field application using the ecdysone receptor (EcR) from the spruce budworm and the non-steroidal EcR agonist, methoxyfenozide. Chimeric transcription activators were constructed with EcR ligand binding domain, GAL4 and LexA DNA binding domains, and VP16 activation domain. In the presence of methoxyfenozide, the transcription activators induced expression of the luciferase reporter gene cloned downstream of a promoter containing GAL4- or LexA-response element and a minimal 35S promoter. Low basal and high induced luciferase expression was optimized by cloning the activator and the reporter genes in different tandem orientations. Many transgenic Arabidopsis and tobacco plants were obtained with little or no basal expression in the absence of methoxyfenozide and inducible expression that was several fold higher than that observed with the constitutive 35S promoter. Moreover, gene expression was controlled over a wide range of methoxyfenozide concentration. Our results demonstrate that the inducible gene expression system based on the spruce budworm EcR ligand binding domain with methoxyfenozide as a ligand is very effective in regulating transgenes in plants. It is suitable for field applications because methoxyfenozide is commercially available and has an exceptional health and environmental safety profile.

Chemical-inducible, ecdysone receptor-based gene expression system for plants.

The ecdysone receptor (EcR)-based gene regulation system is a tool for controlling gene expression. To improve the sensitivity of this system, we evaluated many two-hybrid format synthetic gene constructs in which the GAL4 DNA binding domain was fused to the ligand binding domain of the Choristoneura fumiferana EcR mutant V390I/Y410E (GEvy), and various activation domains--VP16, p53, p65, or E2F-i--were fused to the EF domains of chimeric human RXR. These gene switches were assayed in NIH3T3 cells, HEK293 cells, and in mouse quadriceps in the presence of the nonsteroidal inducer RG-115819 or GS-E. All of the two-hybrid format constructs had no or very low background in the "off" condition and high luciferase reporter gene expression levels in "on" conditions. Extremely high sensitivity was achieved, with EC50 values in the subnanomolar range and with maximal induction at 10 nM RG-115819. Co-expression of both receptor genes with encephalomyocarditis virus (EMCV) or eIF4G internal ribosome entry site (IRES) sequences gave robust induction levels. To reduce the size of the switch construct, we tested single receptor formats, in which any of 14 different activation domains were fused to GEvy. We identified several switches with acceptable levels of basal and maximal induction levels. The gene switches described here provide receptor configuration options suitable for gene function studies, therapeutic protein production in cell culture, transgenic mouse models, and gene/cell therapy.

https://www.future-science.com/doi/pdf/10.2144/05392ST01

Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size

Plant cells are commonly transformed with two or more tandemly arranged genes, but how orientation affects their expression is not well understood. We investigated the amount of transcriptional interference occurring between two adjacent genes by cloning luciferase and green fluorescent protein (GFP) genes (promoter--coding sequence--terminator) in all possible orientations and expressing the genes in tobacco protoplasts. When two genes are oriented head-to-tail (-->-->), the expression of the downstream gene was reduced 80% by the upstream gene. When two genes are oriented tail-to-tail (--> ). Using a chemically inducible gene expression system, we showed that the downstream gene expression was reduced 71% by the induction of an upstream gene. Inserting a mammalian transcription blocker sequence eliminated the interference between the genes in tail-to-tail orientation. The interference in the head-to-tail orientation was eliminated by inserting a 2322-bp lambda phage DNA fragment. The terminators in gene constructs did not prevent the transcriptional interference, and the interference was eliminated by designing the orientation of genes and by placing a transcription blocker or a lambda phage sequence between genes.

https://www.future-science.com/doi/pdf/10.2144/01312st04

Elimination of transcriptional interference between tandem genes in plant cells

Mammalian Markup Recombination techniques utilizing the Cre and Flp systems are now accepted as standard procedures in genome engineering, particularly in manipulating embryonic stem cells, replaci...

Malla Padidam

Papers

Possible emergence of new geminiviruses by frequent recombination.

Chemical-inducible, ecdysone receptor-based gene expression system for plants.

Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size

Elimination of transcriptional interference between tandem genes in plant cells

Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells