Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report

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Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have ... continued below

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3 pages

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Grant, S. R. February 5, 1999.

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Description

Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

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3 pages

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OSTI as DE00825155

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  • Other Information: PBD: 5 Feb 1999

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  • Report No.: None
  • Grant Number: FG02-95ER20186
  • DOI: 10.2172/825155 | External Link
  • Office of Scientific & Technical Information Report Number: 825155
  • Archival Resource Key: ark:/67531/metadc787798

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  • February 5, 1999

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  • Dec. 3, 2015, 9:30 a.m.

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  • Aug. 4, 2016, 7:41 p.m.

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Grant, S. R. Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report, report, February 5, 1999; United States. (digital.library.unt.edu/ark:/67531/metadc787798/: accessed September 21, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.