%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 Plant J %D 2004 %T Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues. %A García-Ortiz, Maria Victoria %A Ariza, Rafael R %A Peter D Hoffman %A John B Hays %A Roldán-Arjona, Teresa %K Alternative Splicing %K Amino Acid Sequence %K Arabidopsis %K Arabidopsis Proteins %K DNA-Directed DNA Polymerase %K Gene Expression %K Gene Library %K Molecular Sequence Data %K Phylogeny %K Plants, Genetically Modified %K Recombinant Fusion Proteins %K Sequence Homology, Amino Acid %X

Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily. We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs. The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.

%B Plant J %V 39 %P 84-97 %8 2004 Jul %G eng %N 1 %R 10.1111/j.1365-313X.2004.02112.x %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 %0 Journal Article %J Mol Gen Genet %D 1996 %T PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases. %A Peter D Hoffman %A Batschauer, A %A John B Hays %K Amino Acid Sequence %K Apoenzymes %K Arabidopsis %K Arabidopsis Proteins %K Cryptochromes %K Deoxyribodipyrimidine Photo-Lyase %K DNA Repair %K DNA, Complementary %K Drosophila Proteins %K Escherichia coli %K Eye Proteins %K Flavoproteins %K Fungal Proteins %K Genes, Plant %K Genomic Library %K Introns %K Membrane Glycoproteins %K Molecular Sequence Data %K Photoreceptor Cells, Invertebrate %K Plant Proteins %K Plasmids %K Receptors, G-Protein-Coupled %K Saccharomyces cerevisiae %K Transformation, Genetic %K Ultraviolet Rays %X

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.

%B Mol Gen Genet %V 253 %P 259-65 %8 1996 Nov 27 %G eng %N 1-2