%0 Journal Article %J Plant Physiol %D 2011 %T Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis. %A Bollmann, Stephanie R %A Tominey, Colin M %A Peter D Hoffman %A Hoffman, Taylor M C %A John B Hays %K Alleles %K Amino Acid Substitution %K Arabidopsis %K Base Sequence %K Gene Dosage %K Genes, Reporter %K Genetic Engineering %K Glucuronidase %K Immunohistochemistry %K Ions %K Metals %K Molecular Sequence Data %K Mutagenesis %K Mutant Proteins %K Mutation %K Plants, Genetically Modified %K Sequence Analysis, DNA %K Transgenes %K Ultraviolet Rays %X

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS- proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.

%B Plant Physiol %V 155 %P 1286-300 %8 2011 Mar %G eng %N 3 %R 10.1104/pp.110.167726 %0 Journal Article %J Planta %D 2009 %T Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines. %A Curtis, Marc J %A Belcram, Katia %A Bollmann, Stephanie R %A Tominey, Colin M %A Peter D Hoffman %A Mercier, Raphael %A John B Hays %K Arabidopsis %K Arabidopsis Proteins %K Chromosomes, Plant %K DNA, Bacterial %K DNA-Directed DNA Polymerase %K Flowers %K Gene Frequency %K Genotype %K Heterozygote %K In Situ Hybridization, Fluorescence %K Models, Genetic %K Mutagenesis, Insertional %K Mutation %K Pollen %K Translocation, Genetic %X

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3 (+/2 ) single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV3 ( 2/2 ) homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I-V translocation. Spreads of meiosis I chromosomes in selfing AtREV3 (+/2 ) heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered.

%B Planta %V 229 %P 731-45 %8 2009 Mar %G eng %N 4 %R 10.1007/s00425-008-0868-0 %0 Journal Article %J Genes Dev %D 2004 %T Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis. %A Peter D Hoffman %A Leonard, Jeffrey M %A Lindberg, Gerrick E %A Bollmann, Stephanie R %A John B Hays %K Arabidopsis %K Arabidopsis Proteins %K Base Pair Mismatch %K DNA Repair %K Genomic Instability %K Microsatellite Repeats %K Mutation %K MutS Homolog 2 Protein %K Plants, Genetically Modified %K Reproduction, Asexual %K Seeds %X

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

%B Genes Dev %V 18 %P 2676-85 %8 2004 Nov 01 %G eng %N 21 %R 10.1101/gad.1217204