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5 results on '"Koohapitagtam M"'

1. The genomes of several plant species contain endogenous geminiviral sequences

Author

Filloux, Denis, Murrell, Sasha, Koohapitagtam, M., Golden, Michael, Julian, Jacqueline, Galzi, Serge, Uzest, Marilyne, Rodier-Goud, Marguerite, D'Hont, Angélique, Vernerey, Marie-Stéphanie, Wilkin, P., Peterschmitt, Michel, Winter, Stephan, Murrell, Ben, Martin, Darren Patrick, Roumagnac, Philippe, Filloux, Denis, Murrell, Sasha, Koohapitagtam, M., Golden, Michael, Julian, Jacqueline, Galzi, Serge, Uzest, Marilyne, Rodier-Goud, Marguerite, D'Hont, Angélique, Vernerey, Marie-Stéphanie, Wilkin, P., Peterschmitt, Michel, Winter, Stephan, Murrell, Ben, Martin, Darren Patrick, and Roumagnac, Philippe

Abstract

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements (GRD) and the geminivirus-like elements (EGV) found respectively within the nuclear genomes of several Nicotiana species and various Dioscorea spp. of the Enantiophyllum clade. Those two new classes of endogenous plant virus sequence are apparently derived from ancient geminiviruses in the genus Begomovirus. GRD and EGV sequences likely became integrated millions years ago. Interestingly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. We recently found using in silico searches that other ssDNA virus-like sequences are included within complete or draft genomes of various plant species, including apple tree (Malus domestica), black cottonwood (Populus trichocarpa), various Coffea spp, eggplant (Solanum melongena), lettuce (lactuca sativa), and Tepary bean (Phaseolus acutifolius), which suggests that endogenous geminiviruses may be more common in plant genomes than has previously been appreciated. (Texte intégral)

Published
2016

2. The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed

Author

Filloux, Denis, Murrell, Sasha, Koohapitagtam, M., Golden, Michael, Julian, Charlotte, Galzi, Serge, Uzest, Marilyne, Rodier-Goud, Marguerite, D'Hont, Angélique, Vernerey, Marie-Stéphanie, Wilkin, P., Peterschmitt, Michel, Winter, Stephan, Murrell, Ben, Martin, Darren Patrick, Roumagnac, Philippe, Filloux, Denis, Murrell, Sasha, Koohapitagtam, M., Golden, Michael, Julian, Charlotte, Galzi, Serge, Uzest, Marilyne, Rodier-Goud, Marguerite, D'Hont, Angélique, Vernerey, Marie-Stéphanie, Wilkin, P., Peterschmitt, Michel, Winter, Stephan, Murrell, Ben, Martin, Darren Patrick, and Roumagnac, Philippe

Abstract

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum c

Published
2015

3. Characterization of new endogenous geminiviral elements in yam (Dioscorea spp.) genomes

Author

Filloux, Denis, Koohapitagtam, M., Golden, Michael, Julian, Charlotte, Galzi, Serge, Rodier-Goud, Marguerite, D'Hont, Angélique, Vernerey, Marie-Stéphanie, Wilkin, P., Peterschmitt, Michel, Martin, Darren Patrick, and Roumagnac, Philippe

Subjects
H20 - Maladies des plantes, F30 - Génétique et amélioration des plantes
Abstract

Geminivirus sequences integrated into the genomes of various plant species (Nicotiana species, Lactuca sativa, Malus domestica and Populus trichocarpa) have been reported. The best studied of these integrated geminivirus sequences are the so-called "geminivirus-related DNA" or GRD elements within the genomes of various Nicotiana species. Our study aimed at assessing whether such integrated geminiviral sequences are present within the genomes of yam species. We have used a polyphasic approach combining a first step of plant genome screening (in silico EST analysis and viral metagenomics) followed by a second step of molecular characterization of the integrated sequences (Long-PCR and Inverse-PCR). We have found two new phylogenetically related endogeneous geminivirus (EGV) elements within the genome of the majority of Asian Dioscorea spp. section Enantiophyllum. Based on multiple lines of evidence, such as (i) seed transmission of the two EGV sequences, (ii) no amplification by rolling circle amplification, (iii) worldwide presence of the two EGV sequences in a range of asympomatic Asian Dioscorea species and (iv) lack of sequences of coat protein genes (i.e. they are not likely to be insect transmissible), we hypothesized that those two EGV elements were likely to have become integrated within an ancestor of the Asian Dioscorea species. We then confirmed the presence of the two EGV elements within D. alata and D. nummularia genomes by fluorescence in situ hybridization. The predicted proteins expressed by the two EGV sequences had detectable homology to begomovirus replication enhancer and replication associated proteins but only one out of the two EGV sequences contains a GC rich sequence resembling the conserved hairpin structures found at geminivirus virion strand origins of replication (including the TAATATTAC sequence in the likely loop region) and is organized as 2.6 Kb tandem repeats. The discovery and analysis of such fossilized geminivirus sequences within plant genomes would certainly help to more accurately date events deep in the evolutionary history of the Geminiviridae family such as the most recent geminivirus common ancestor, and the origins of the various geminivirus genera. (Texte intégral)

Published
2013

4. The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Author

Filloux D, Murrell S, Koohapitagtam M, Golden M, Julian C, Galzi S, Uzest M, Rodier-Goud M, D'Hont A, Vernerey MS, Wilkin P, Peterschmitt M, Winter S, Murrell B, Martin DP, and Roumagnac P

Abstract

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus . These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21-24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

Published
2015
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5. Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library.

Author

Koohapitagtam M, Rungpragayphan S, Hongprayoon R, Kositratana W, and Sirinarumitr T

Subjects
Animals, Cloning, Molecular, DNA Primers metabolism, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Genetic Variation, Mice, Transformation, Genetic, Immunoglobulin Heavy Chains genetics, Immunoglobulin Light Chains genetics, Immunoglobulin Variable Region genetics, Peptide Library, Polymerase Chain Reaction methods
Abstract

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V(kappa) and V(H) genes with newly designed primer sets. Initially, we amplified V(kappa) and V(H) genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V(kappa) and V(H) genes from the framework region 1 to the joining region. The V(kappa) and V(H) genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.

Published
2010
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