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DNA damage has been considered to be the universal critical lesion in cells after exposure to ionizing radiation. Measuring radiation-induced DNA damage is important to understand the mechanisms of radiation-induced toxicity and monitor DNA damage repairs. Currently the most widely used methods to measure DNA damage are pulsed-field gel electrophoresis (PFGF) and single-cell gel electrophoresis (also known as the comet assay), both of which are technically challenging and time consuming. Long range quantitative polymerase chain reaction (LR-QPCR) has been used successfully to measure nuclear and mitochondrial DNA damage in mammalian and several model organism cells. The principle of this assay is that DNA lesions will slow down or block the progression of DNA polymerase. Therefore, the amplification efficiency of DNA with fewer lesions will be higher than DNA with more lesions under the same reaction condition. Here, we developed the LR-QPCR assay primers and reaction conditions to quantify DNA damage in Cryptococcus neoformans (C. neoformans) and Saccharomyces cerevisiae (S. cerevisiae) after gamma ray exposure. Under these conditions, long DNA targets of C. neoformans H99 and S. cerevisiae BY4741 (17.6 and 16.4 kb for nuclear DNA and 15.3 and 14.6 kb for mitochondrial DNA) were quantitatively amplified using extracted DNA templates, respectively. Two short mitochondrial DNA targets of these two species (207 bp and 154 bp) were also quantitatively amplified and used to monitor the number of mitochondria. Using the LR-QPCR method, we showed that the frequency of radiation-induced mitochondrial and nuclear DNA lesions had a significant linear correlation with the radiation doses (from 500 Gy to 3000 Gy) in both species. Furthermore, the faster disappearance of DNA damage detected in C. neoformans H99S strain compared to H99 strain may help to explain the different radiation sensitivity of these two strains. In summary, we developed a simple, sensitive method to measure radiation-induced DNA damage, which can greatly facilitate the study of radiation-induced toxicity and can be widely used as a dosimetry in radiation-induced cell damage.
This article was published in the following journal.
Name: PloS one
Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monoval...
Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections an...
Cryptococcosis is a life-threatening mycosis primarily occurring in adult patients particularly those with immunosuppression such as HIV infection/AIDS. The number of reported cases of pediatric crypt...
Cryptococcus neoformans is a yeast fungus, which causes cryptococcosis, triggered by basidiospore inhalation and consequent dissemination to the central nervous system. In this study, we analyzed the ...
Saccharomyces cerevisiae survival fractions (SFs) in response to X-ray radiation were determined by two new methods: an OD600-based method and a 96-well method. For the OD600-based method, cells were ...
Evaluation of the Association of Polymorphisms in the Innate Immune System With the Risk for Cryptococcus Neoformans Infection in Patients Not Infected With HIV and Complications Associated With Cryptococcus Neoformans Infection
Innate immunity plays an important role for fungal recognition and initiation of fungicidal activity. We hypothesize that subtle differences in different molecules of innate immunity may ...
This study is designed to evaluate the impact of oral consumption of the probiotic yeast CNCM I-3856 on healthy vaginal microbiota
The goal of this randomized clinical trial is to compare early versus standard timing of initiation of antiretroviral therapy (ART) with respect to clearance of Cryptococcus neoformans fro...
In the radiotherapeutic treatment of lung cancer, the dose that can be safely applied to the tumour is limited by the risk of radiation induced lung damage. This damage is characterized by...
Rationale: Radiation induced cardiac toxicity after treatment for intrathoracic tumours is a clinically relevant problem. So far, no clinical prediction models exist for cardiac toxicity....
A species of the fungus CRYPTOCOCCUS. Its teleomorph is Filobasidiella neoformans.
A member of the Rho family of MONOMERIC GTP-BINDING PROTEINS from SACCHAROMYCES CEREVISIAE. It is involved in morphological events related to the cell cycle. This enzyme was formerly listed as EC 22.214.171.124.
Proteins obtained from the species SACCHAROMYCES CEREVISIAE. The function of specific proteins from this organism are the subject of intense scientific interest and have been used to derive basic understanding of the functioning similar proteins in higher eukaryotes.
A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.
A set of nuclear proteins in SACCHAROMYCES CEREVISIAE that are required for the transcriptional repression of the silent mating type loci. They mediate the formation of silenced CHROMATIN and repress both transcription and recombination at other loci as well. They are comprised of 4 non-homologous, interacting proteins, Sir1p, Sir2p, Sir3p, and Sir4p. Sir2p, an NAD-dependent HISTONE DEACETYLASE, is the founding member of the family of SIRTUINS.
Bioinformatics is the application of computer software and hardware to the management of biological data to create useful information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied...
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...