Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis
| Autor: | Joel J. Milner, Simon N. Covey, Edi Cecchini, Bernard J. Mulligan |
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| Rok vydání: | 1998 |
| Předmět: |
Health
Toxicology and Mutagenesis Transgene Restriction Mapping Mutant Arabidopsis Locus (genetics) Biology Amidohydrolases Gene product chemistry.chemical_compound Caulimovirus Genetics Promoter Regions Genetic Molecular Biology Sequence Deletion Southern blot Dose-Response Relationship Radiation Plants Genetically Modified Molecular biology Recombinant Proteins chemistry Agrobacterium tumefaciens Cesium Radioisotopes Gamma Rays Mutagenesis Suppression subtractive hybridization Restriction fragment length polymorphism DNA |
| Zdroj: | Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 401:199-206 |
| ISSN: | 0027-5107 |
| Popis: | Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35S∷tms2 construct introduced as a transgene, were γ-irradiated at a range of doses from 20–120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures. |
| Databáze: | OpenAIRE |
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