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1.
Nucleic acids circulating in blood plasma and other biological fluids are of interest as potential markers for the diagnosis of various pathologies and the monitoring of stresses. Mitochondrial DNA (mtDNA) is a more vulnerable target for many genotoxic agents than nuclear DNA, and mutations in the mitochondrial genome can serve as markers for many diseases. In the present study, extracellular mtDNA with mutations was assayed in the blood plasma of mice exposed to X radiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained by the hybridization of mtDNA PCR amplicons (ND3 gene and D loop region) from the blood plasma of irradiated and control mice were cleaved with CEL endonuclease, a mismatch-specific enzyme. The total amount of mtDNA (ND4 gene) copies vs. nuclear DNA (GAPDH gene) was measured by real-time PCR. The content of mtDNA with mutations in murine blood plasma remained high within one month after irradiation but varied with time. The measurements were performed on days 1, 4, 8, 14, and 28 after irradiation, and the maximum level was detected on day 14. The elevated content of extracellular mutant mtDNA in blood plasma of X-irradiated mice is a sensitive candidate biomarker for the assessment of radiation injury and effects of other genotoxic agents.  相似文献   

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Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of >0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 x 10(-5) mutations/ nucleotide, and for the controls it was 8.65 x 10(-5) mutations/ nucleotide, with a procedural error rate of 2.65 x 10(-5) mutations/nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P = 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-dose-rate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation.  相似文献   

4.
Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.  相似文献   

5.
Mitroshina  I. Yu.  Sirota  N. P.  Prokofiev  V. N.  Kuznetsova  E. A. 《Biophysics》2021,66(2):310-315

We studied the levels of extracellular nuclear and mitochondrial DNA of blood serum and DNA damage in leukocytes of healthy donors of different sex and age groups. The baseline levels of DNA damage in leukocytes and serum DNA levels were shown to vary greatly among different donors. The baseline level of DNA damage in leukocytes was not associated with the presence of chronic deceases or an occupational health risk for elderly donors. It was found that extracellular DNA concentrations were generally higher in men than in women. There is a tendency towards an increase in the relative mitochondrial DNA copy number determined by ΔCt in women but not in men: the relative mtDNA copy number in elderly individuals varies significantly in both sexes, possibly due to age-related physiological changes. It is necessary to consider the gender and age of patients when using an indicator such as the level of extracellular DNA of blood serum for diagnosis and monitoring.

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6.
Abdullaev  S. A.  Gulyaeva  N. A.  Bezlepkin  V. G.  Gaziev  A. I. 《Biophysics》2010,55(1):142-147
Mutations in brain mtDNA of mice X-irradiated to 5 Gy were determined using a mismatch-specific endonuclease (CEL I) and by temporal temperature gradient gel electrophoresis (TTGE). The CEL I nuclease method allows simultaneous analysis of multiple DNA samples and proves to yield better results than TTGE; it is more sensitive, quite reproducible, and economical, requiring no complicated equipment. The CEL I nuclease assay for mtDNA mutations in the brain of x-irradiated mice has shown that the amount of mutant mtDNA copies is markedly reduced (2–3 times) from the 8th to the 28th day after irradiation, while the overall amount of mtDNA copies in the brain tissue remains fairly constant over this period, though lower than in the control. It can be suggested that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the postirradiation period.  相似文献   

7.
Mitochondrial respiratory chain deteriorates with age, mostly in tissues with high energy requirements. Damage to mitochondrial DNA (mtDNA) by reactive oxygen species is thought to contribute primarily to this impairment. However, the overall extent of random mtDNA mutations has still not been evaluated. We carried out molecular and biochemical analyses in muscle biopsies from healthy young and aged subjects. Deleted mtDNA accumulation was followed by both quantitative PCR analysis to quantify total mtDNA, and Southern-blotting, to determine deleted to full length mtDNA ratio. Enzymatic activities of the mitochondrial respiratory chain were measured in all subjects. Randomly deleted mtDNA appeared mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules. The activities of complexes III and IV of the respiratory chain, complexes with mtDNA encoded subunits, are lower in the aged subjects. Physical activity could be one major parameter modulating the mitochondrial respiratory chain activity in aged muscle.  相似文献   

8.
Some properties of the cell-free DNA (cfDNA) of peripheral blood plasma were assessed in 153 employees of atomic industry enterprises. The contents of ribosomal repeat (rDNA) and its concentration in plasma increased in cfDNA of the group of persons in comparison with non-irradiated individuals. The contents of satellite III in cfDNA of donors and of irradiated persons do not differ and less than in DNA nucleus. The correlation between cumulative dose of radiation, contents of rDNA in cfDNA and the frequency of lymphocytes bearing mutations at T-cell receptor (TCR) locus was obtained. The definition of three indications in irradiated persons: the contents of ribosomal genes in cfDNA, TCR-mutant cell frequency and concentration of ribosomal genes in blood plasma--may be useful for revealing individuals in organism of which an intensive cell apoptosis takes place and there is an increased probability of carcinogenesis and of progress of disease of immune system.  相似文献   

9.
Quantitative and qualitative changes in circulating extracellular DNA (ec-DNA) of blood plasma are considered as markers for diagnosis and prognosis of tumor pathology. We have investigated the content of mutant copies of the circulating mitochondrial DNA (ec-mtDNA) in blood plasma in 8 patients with lung cancer before and after radiotherapy as well as in healthy young and elderly donors. It was found that in plasma of healthy elderly donors the proportion of ec-mtDNA with mutations (in the total circulating DNA) is much higher than in young donors. Before radiotherapy the proportion of ec-mtDNA with mutations was higher in plasma of lung cancer patients (aged 70–76 years) than that of healthy elderly donors. After radiotherapy of lung cancer patients a twofold increase in the proportion of ec-mtDNA with mutations was observed in total circulating plasma DNA. This may be attributed to release of mutant DNA copies from dying tumor cells and also from normal cells injured by the radiation treatment.  相似文献   

10.
We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.  相似文献   

11.
For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.  相似文献   

12.
The cell-mediated immune responses of 12 healthy, coccidioidin skin-test positive subjects (Group I) were compared with those of 15 healthy, coccidioidin skin-test positive persons who had primary asymptomatic coccidiodomycosis, (Group II), 12 patients with active, pulmonary coccidioidomycosis (Group III), four patients with disseminated disease (Group IV), and five patients who had been in clinical remission for 1 year or longer (Group V). Lymphocytes from healthy subjects in Groups I and II responded in vitro to Coccidioides immitis antigen by undergoing an increased DNA synthesis (lymphocyte transformation) and/or by producing macrophage migration inhibitory factor (MIF). In contrast, patients in Groups III and IV failed to respond to Coccidioides antigens in vivo (skin tests) or in vitro (lymphocyte transformation and production of MIF). The responses of subjects in Group V with inactive disease fell in between those of healthy donors in Groups I and II and patients in Groups III and IV. The cellular immune defect, in terms of antigen recognition, appeared to be specific for C. immitis in all but one patient.  相似文献   

13.
Highly diverse results have been reported for mitochondrial DNA (mtDNA) hetero-plasmy in nuclear-transferred farm animals. In this study, we cloned genetically defined mice and investigated donor mtDNA inheritance following somatic cell cloning. Polymerase chain reaction (PCR) analysis with primers that were specific for either the recipient oocytes or donor cells revealed that the donor mtDNA coexisted with the recipient mtDNA in the brain, liver, kidney, and tail tissues of 96% (24/25) of the adult clones. When the proportion of donor mtDNA in each tissue was measured by allele-specific quantitative PCR and subjected to ANOVA analysis, a tissue-specific mtDNA segregation pattern (P < 0.05) was observed, with the liver containing the highest proportion of donor mtDNA. Therefore, the donor mtDNA was inherited consistently by the cloned offspring, whereas donor mtDNA segregation was not neutral, which is in accordance with previous notions about tissue-specific nuclear control of mtDNA segregation.  相似文献   

14.
Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.  相似文献   

15.
The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.  相似文献   

16.
We have developed a technique called "LSSP-PCR" (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique "gene signature." We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing.  相似文献   

17.
In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.  相似文献   

18.
Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.  相似文献   

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Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.  相似文献   

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