ABSTRACT
It is generally and widely accepted that the biological effects of a given dose of ionizing radiation, especially those of low linear energy transfer radiations like X-ray and gamma ray, become smaller as the dose rate becomes lower. This phenomenon, known as 'dose-rate effect (DRE),' is considered due to the repair of sublethal damage during irradiation but the precise mechanisms for DRE have remained to be clarified. We recently showed that DRE in terms of clonogenic cell survival is diminished or even inversed in rodent cells lacking Ku, which is one of the essential factors in the repair of DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Here we review and discuss the involvement of NHEJ in DRE, which has potential implications in radiological protection and cancer therapeutics.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA , DNA Repair , Linear Energy TransferABSTRACT
The biological effects of ionizing radiation, especially those of sparsely ionizing radiations like X-ray and γ-ray, are generally reduced as the dose rate is reduced. This phenomenon is known as 'the dose-rate effect'. The dose-rate effect is considered to be due to the repair of DNA damage during irradiation but the precise mechanisms for the dose-rate effect remain to be clarified. Ku70, Ku86 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are thought to comprise the sensor for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). In this study, we measured the clonogenic ability of Ku70-, Ku86- or DNA-PKcs-deficient rodent cells, in parallel with respective control cells, in response to high dose-rate (HDR) and low dose-rate (LDR) γ-ray radiation (~0.9 and ~1 mGy/min, respectively). Control cells and murine embryonic fibroblasts (MEF) from a severe combined immunodeficiency (scid) mouse, which is DNA-PKcs-deficient, showed higher cell survival after LDR irradiation than after HDR irradiation at the same dose. On the other hand, MEF from Ku70-/- mice exhibited lower clonogenic cell survival after LDR irradiation than after HDR irradiation. XR-V15B and xrs-5 cells, which are Ku86-deficient, exhibited mostly identical clonogenic cell survival after LDR and HDR irradiation. Thus, the dose-rate effect in terms of clonogenic cell survival is diminished or even inversed in Ku-deficient rodent cells. These observations indicate the involvement of Ku in the dose-rate effect.
Subject(s)
Clone Cells/radiation effects , Ku Autoantigen/metabolism , Animals , Cell Line , Cell Survival/radiation effects , Cesium Radioisotopes , Cobalt Radioisotopes , DNA End-Joining Repair/radiation effects , DNA-Activated Protein Kinase/metabolism , Dose-Response Relationship, Radiation , Gamma Rays , Mice, SCIDABSTRACT
Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.
Subject(s)
Carica , DNA, Plant/isolation & purification , Food Analysis/methods , Food, Genetically Modified , Genome, Plant/genetics , Plants, Genetically Modified , Carica/genetics , Polymerase Chain Reaction/methods , Silica Gel , Silicon DioxideABSTRACT
The yield of genomic DNA extracted from corn-processed foods, such as corn flake and one of the corn snack what is called "Jumbo corn", using an ion-exchange resin type kit (Gtip) has been reported to be very low, and it is thought to be difficult to detect the intrinsic corn gene "Zein" in the foods. Therefore, we developed a new method using Gtip, which we called the "KNG-Gtip method," by modification of the Ministry of Health, Labour and Welfare (MHLW) method using Gtip (MHLW-Gtip method). We compared the KNG-Gtip method, MHLW-Gtip method, the Gtip method for detection of allergen (ALG-Gtip method), and the Gtip method according to the Ministry of Agriculture, Forestry and Fisheries (MAFF) (JAS-Gtip method) in terms of the yield and quality of genomic DNA and the detection probabilities of the PCR-amplified Zein gene. The concentrations of DNA and the detection probabilities of the PCR-amplified Zein gene of genomic DNA extracted from 4 g corn flake and 4 g Jumbo corn by the KNG-Gtip method were larger than those by using the conventional methods. In addition, the PCR-amplified Zein gene from 4 g of corn starch could be detected by the KNG-Gtip method. We propose that the KNG-Gtip method, in which requires sample weight of four grams, is practical and useful to extract genomic DNA from corn flake and Jumbo corn.
