Study of genome instability using DNA fingerprinting of the offspring of male mice subjected to chronic low dose gamma irradiation

By a polymerase chain reaction with an arbitrary primer (AP-PCR), the possibility of transmission of genome instability to somatic cells of the offspring (F1 generation) from male parents of mice exposed to chronic low-level gamma-radiation was studied. Male BALB/c mice 15 days after exposure to 10-50 cGy were mated with unirradiated females. Biopsies were taken from tale tips of two month-old offspring mice and DNA was isolated. The primer in the AP-PCR was a 20-mer oligonucleotide flanking the microsatellite locus Atp1b2 on chromosome 11 of the mouse. A comparative analysis of individual fingerprints of AP-PCR products on DNA-templates from the offspring of irradiated and unirradiated male mice revealed an increased variability of microsatellite-associated sequences in the genome of the offspring of the males exposed to 25 and 50 cGy. The DNA-fingerprints of the offspring of male mice exposed to chronic irradiation with the doses 10 and 25 cGy 15 days before fertilization (at the post-meiotic stage of spermatogenesis) showed an increased frequency of "non-parent bands". The results of the study point to the possibility of transmission to the offspring somatic cells of changes increasing genome instability from male parents exposed to chronic low-level radiation prior to fertilization.     

The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.     

Detection of genomic instability in offspring of male mice chronically exposed to gamma-radiation by the "adaptive response" test

The genomic instability (GI) in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-dose gamma-radiation was studied by comparative analysis of chromosome damage. BALB/C male mice exposed to 0.1 Gy (0.01 Gy/day) and 0.5 Gy (0.01 and 0.05 Gy/day) were mated with unirradiated females 15 days after irradiation. For comparison of radiosensitivity, two-month-old males, the descendants of irradiated and unirradiated animals, were subjected to irradiation with a dose of 1.5 Gy (0.47 Gy/min) from a 60Co source. GI was revealed by the standard scheme of adaptive response. The experiments indicated that, by using the test "adaptive response", it is possible to detect the transition of gamma-radiation-induced genomic instability in sex cells of male parent into somatic cells of mice (F1 generation) either from changes in radiosensitivity or by the absence of the adaptive response induced by a standard scheme.     

Increased genomic instability in somatic cells of the progeny of female mice exposed to acute X-radiation in the preconceptional period

The level of genome instability (GI) was studied in the progeny of female mice exposed in the preconceptional period to radiation doses of 0.5, 1, and 2 Gy in comparison to that in the progeny of the same parent pairs born before irradiation of the females. To assess the level of genome instability, we analyzed polymorphism of DNA fragments from postmitotic (blood and brain) and proliferating (spleen and tail tip) tissues amplified by AP-PCR (PCR amplification with an arbitrary primer). It was found that polymorphism of the spectrum of AP-PCR products, which is a multilocus genetic marker (MGM), in the genome of somatic cells in the progeny of female mice exposed to 2 Gy was higher than in the progeny of male mice exposed to the same doses. In the progenies of female mice born before and after irradiation, tissue-specific variations in the level of DNA polymorphism were detected. The maximum value of this polymorphism (with respect to the frequency of “nonparental bands”) was determined for peripheral blood DNA in comparison with the other tissues. Estimations of the MGM polymorphism with the AP-PCR method demonstrate an increased level of genome instability in somatic cells of offsprings from female mice exposed to a single acute dose of X-rays (0.5, 1, and 2 Gy) in the preconceptional period. Radiation-induced transgenerational genome instability with an increase in the dose of preconceptional irradiation of female mice was more pronounced in DNA of the postmitotic tissues (blood and brain DNA) than in DNA of the proliferating tissues (spleen and tail tip epithelium).     

Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.     

Transmission of genome damage from irradiated male rats to their progeny

The effect of gamma-radiation (3Gy) on slowly proliferating liver tissue of male rats and their progeny was investigated with respect to induction and duration of latent damage. The irradiation caused latent cytogenetic damage in the liver in irradiated males of the F(0) generation, which manifested itself in different ways during proliferation of hepatocytes induced by partial hepatectomy: a reduced proliferating activity, a higher frequency of chromosomal aberrations and a higher proportion of cells with apoptotic DNA fragments were observed, compared with non-irradiated rats. In the progeny of irradiated males (F(1) and F(2) generation), the latent genome damage manifested itself during regeneration of the liver after partial hepatectomy by similar, but less pronounced changes compared with those seen in irradiated males of the parental generation. This finding gave evidence of the transfer of part of the radiation-induced genome damage from parents to their offspring. Irradiation of F(1) and F(2) progeny of irradiated males (their total radiation load being 3 + 3 and 3 + 0 + 3 Gy, respectively) caused less change as irradiation of progeny of non-irradiated control males (their total radiation load being 0 + 3 and 0 + 0 + 3 Gy, respectively).     

The phenomenon of the single-strand DNA breaks level decrease in blood system cells in first generation chronically irradiated mice

For the first time has been shown experimentally that chronic low dose-rate gamma-radiation (0.04 mGy/hr) exposure leads to decrease of the single-strand DNA breaks level in spleen cells in 2.3 times (p < 0.001) and blood leukocytes in 6.1 times (p < 0.01), a decrease of the apoptotic cells frequency in 1.3 times (p < 0.05) and an increase in the spleen relative mass ratio B 1.2 times (p < 0.001) in CBA mice offspings (F1) from the chronically irradiated parents and exposed to chronic irradiation during the embryonic and postembryonic periods. A hypothesis about the more compact chromatin structure of blood system cells in the individuals of the first generation from chronically irradiated mice is proposed.     

Detection of gamma-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka (Oryzias latipes) using AP-PCR fingerprinting.

Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of gamma-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which, to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F1 hybrids were the sum of those of their parents. Based on these findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy gamma-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy gamma-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of gamma-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.     

Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbitrarily primed PCR (AP-PCR) fingerprinting.

DNA repair mechanisms are important to maintain the stability of the genome. In Drosophila melanogaster, the mus-201 gene is required in the excision repair process. To study the contribution of the mus-201 gene in the stability of the Drosophila genome, we have used the arbitrarily primed PCR fingerprinting method (AP-PCR). We have analysed the changes in the genomic DNA fingerprints from the progeny of wild-type males crossed with mus-201 repair-deficient or repair-proficient females. After induction of DNA damage with 2-acetylaminofluorene (2-AAF) in the wild-type parental males, quantitative and qualitative differences in the AP-PCR fingerprints were detected between the two crosses, and the estimate of the genomic damage detected by AP-PCR has clearly shown that the mus-201 repair deficiency is associated with an increase of genomic damage. The predominant type of alterations detected by AP-PCR under the mus-201 repair-deficient conditions agree with the results obtained in microsatellite PCR analysis, suggesting that the role of the mus-201 gene, necessary in excision repair, is not associated to the mismatch repair process. The work reported here demonstrates that the AP-PCR is a suitable technique to analyse genetic alterations in D. melanogaster and, consequently, can be used to compare the susceptibility to genomic damage of different DNA repair mutants.     

The estimation of molecular and cytogenetic effects in mice exposed to chronic low dose gamma-radiation

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate gamma-radiation (62 cGy/year) for 40, 80, 120, 210, and 365 days. After 40 days of exposure (6.7 cGy), spleen lymphocyte susceptibility to hydrogen peroxide was shown to increase. However, beginning from the day 120 of the treatment (20.4 cGy), the opposite effect was observed. An increase in number of the DNA-protein crosslinks was recorded in spleen lymphocytes only on day 40 of the experiment. The number of DNA breaks increased significantly beginning from day 120 of the experiment, as shown by the DNA-comet method. On the day 210 of irradiation, the frequency of abnormal sperm heads in the mice significantly increased. The number of normochromatic micronucleated erythrocytes of the peripheral blood remained unchanged.     

Elevated Micronucleus Frequency in Bone Marrow Erythrocytes in the Progeny of Male Mice Exposed to Chronic Low-Dose Gamma Irradiation

The micronucleus frequency in bone marrow erythrocytes from the F1 progeny of male mice exposed to chronic low-dose -irradiation was determined. Male BALB/c mice were irradiated with 10, 25 and 50 cGy at dose rates of 1, 5, and 15 cGy/day and mated with unirradiated females on day 15 after irradiation. The obtained offspring had an elevated micronucleus frequency in bone marrow erythrocytes at the age of 2 months. This suggests the transmission of genome instability from damaged germ-line cells of irradiated male parents to somatic cells of the progeny.     

Mitochondrial Genome Fingerprinting in Hybrid Rice and Its Associated Lines Using the Primed Polymerase Chain Reaction

A new economic and efficient DNA polymorphism assay was developed in 1990 that is based on the amplification by polymerase chain reaction (PCR) of random DNA segments using primers of arbitrary nucleotide sequence. Authors have now adapted this type of amplification to rice mitochondrial genome. Using 6 rice varieties in conjunction with 7 of 20–27 mer oligonucleotide primers, the AP-PCR products revealed that the amplified DNA bands fell into two categories, the evolutively conserved the cytoplasmic-specific. It is suggested that AP-PCR assay of mtDNA may help to classify or identify the cytoplasms in rice. By comparing "fingerprints" among the WA type cytoplasmic male sterility (CMS) rice, its Maintainer and Restorer lines, as well as its hybrid, one CMS cytoplasm-specific band (primer R2/630 bp) and one normal cytoplasm-specific segment (primer V5/707 bp) could be directly identified among the set of amplified DNA fragments. Further, some difference in the amplification patterns of mtDNA between CMS line and its hybrid, which infers that rearrangement of mitochondrial genome in hybrid rice probably happened.     

Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between slightly divergent strains of any organism. Single oligodeoxyribonucleotide (oligo) primers have been used to generate such fingerprints, with the same primer being present at the 5' end of both strands for every PCR product. We used three arbitrary oligos, individually and in pairs, to generate six different genomic fingerprints of the same mouse genomic DNAs. Fewer than half of the products in genomic fingerprints generated using the oligos in pairs were the same as those produced by AP-PCR using one of the three oligos alone. Thus, a few oligos could be used in a very large number of single and pairwise combinations, each producing a distinct AP-PCR fingerprint with the potential to identify new polymorphisms. For example, 50 oligos can be used in a matrix of pairwise combinations to produce 2,500 fingerprints, in which at least half the data can be expected to be unique to each pair. We demonstrate this principle by using two oligos, alone and together, to generate three sets of fingerprints and map thirteen polymorphisms in the C57BL/6J x DBA/2J set of recombinant inbred mice.     

Isolation and characterization of microsatellites from the canine genome

Microsatellite sequences comprising (dC-dA)_n.(dG-dT)_n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the micro-satellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.     

Ethylene dibromide: effects of paternal exposure on the neurotransmitter enzymes in the developing brain of F1 progeny

The effects of ethylene dibromide (EDB) exposure to male rats on several neurotransmitter enzymes have been examined in various brain regions of the F1 progeny, from 7 to 90 days of age. The choline acetyltransferase activity was significantly increased at 21 days old, in most brain regions studied in the F1 progeny of the EDB-treated males, but not at 7, 14 or 90 days old. The acetylcholinesterase activity was altered in different brain regions of the F1 progeny of the EDB-exposed males at both 14 and 21 days old but not at 7 or 90 days old. Glutamic acid decarboxylase activity was increased in corpus striatum but decreased in frontal cortex only at 21 days of age. These neurochemical changes in the developing brain of F1 progeny of EDB-treated males at low doses may be associated with behavioral abnormalities observed early in their development.     

The radiationally induced change of level of double-stranded breaks DNA in neuroblasts of larvae and frequency of lethal mutations in sex cells of males Drosophila melanogaster

The level of damage DNA in neyroblastes of larvae and frequency of recessive sex-linked lethal mutations of males from chronically irradiated populations Drosophila melanogaster, differing on mobile P-elements patterns, was estimated. Received results testify, that exposition in conditions a chronic gamma-radiation (absorbed radiation dose at one generation is compounds 10 mGy) result to increase of significance of parameters and change of sensitivity of cells to following of an acute irradiation in a dose of 3 Gy.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Dose response of hepatic and renal DNA synthetic rates to continuous exposure of bromodeoxyuridine (BrdU) via slow-release pellets or osmotic minipumps in male B6C3F1 mice

We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with the 4-day time point. In some cases, the labeling indices at 7 days were significantly decreased compared with those at 4 days. In contrast, a time-related increase in nuclear labeling was seen in hepatocytes and Kupffer cells of mice exposed to BrdU minipumps. Therefore, the method used to administer BrdU chronically to the animal appears to play an important role in presenting the true proliferative scenario in cell kinetic studies. Our findings also provide evidence for an effect of BrdU on normal proliferation rates in these tissues.     

植物不同种属间共用微卫星引物的研究

微卫星标记是近年来发展起来的建立在PCR基础卜的第二代分子标记,具有分布均匀、多态性高、共显性、选择中性等优点,已用于品种鉴定、构建连锁图谱、构建物理图谱、遗传多样性、居群和进化等方面的研究。由于来源于数据库的微卫星序列不能代表整个基因组微卫星的分布情况,而且数据库中的DNA序列仅局限于一些模式植物及经济作物。同时,通过基因组文库筛选获得微卫星序列既耗时又耗资。最近有很多研究表明,从某种植物来的微卫星引物可用于其近缘物种的扩增。本文概括介绍了植物不同种属间共用微卫星引物的研究情况及意义。     

Inhibition of clastogenic effect of radiation by Liv. 52 in the bone marrow of mice

The frequency of micronuclei in bone marrow cells of mice treated or not with Liv. 52 and then exposed to 4.5 Gy of gamma-radiation was evaluated from 6 h to 14 days post irradiation. The frequency of micronuclei increased from 6 h to 24 h post irradiated in both irradiated groups and declined thereafter, the frequency of micronuclei remaining significantly lower in the Liv. 52-treated group. These data demonstrate that Liv. 52 protects the bone marrow of mice against radiation injury.