To investigate UV light response mechanisms in higher plants, we isolated a UV light-sensitive mutant, rev3-1 , in Arabidopsis. The root growth of rev3-1 was inhibited after UV-B irradiation under both light and dark conditions. We found that chromosome 1 of rev3-1 was broken at a minimum of three points, causing chromosome inversion and translocation. A gene disrupted by this rearrangement encoded the catalytic subunit of DNA polymerase ( AtREV3 ), which is thought to be involved in translesion synthesis. The rev3-1 seedlings also were sensitive to ␥ -rays and mitomycin C, which are known to inhibit DNA replication. Incorporation of bromodeoxyuridine after UV-B irradiation was less in rev3-1 than in the wild type. These results indicate that UV light-damaged DNA interrupted DNA replication in the rev3-1 mutant, leading to the inhibition of cell division and root elongation.

SUMMARYTo investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to c-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast twohybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after c-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP.

The significant differences in DNA methylation that are considered to be a biomarker for the diagnosis of cancer are a barrier to the application of biomarkers in the clinical field. In the present study, new primers were designed and further standard controls were set up to validate the accuracy of the methylation‑specific PCR (MSP), a method widely used to analyze DNA methylation. By analyzing the methylation pattern of breast cancer 1 (BRCA1) and estrogen receptor (ER) in 60 patients with breast cancer, the number of cases of methylated BRCA1 and ER detected by the primer was 7/60 and 21/60, respectively, whereas that detected by the previous widely used primers was 25/60 and 47/60, respectively. Sequencing of the MSP products indicated that the 18 and 26 false-positive methylations of BRCA1 and ER, respectively, were due to insufficient validation of the previously used primers. Thus, the present study proposes that all studies based on the MSP approach should incorporate more controls to validate the precision of the MSP primers.

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.