RESUMO
In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.
Assuntos
Antígenos Nucleares , DNA Helicases , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Integrases/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Transferência Genética Horizontal , Autoantígeno Ku , Modelos Genéticos , Mutação , Plasmídeos , Recombinação Genética , Retroelementos , Especificidade por SubstratoRESUMO
Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.
Assuntos
Apoptose/efeitos da radiação , Proteínas Fúngicas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Recombinação Genética , Telômero/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Linhagem Celular Transformada , Proteínas de Ligação a DNA , Fibroblastos , Proteínas Fúngicas/genética , Raios gama , Teste de Complementação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/efeitos da radiação , Proteínas Supressoras de TumorRESUMO
A simple model of homogenous chromatin distribution in HeLa-cell nuclei suggests that the track of an energetic ion hits 30 nm chromatin fibers with a mean distance of 0.55 mum. To test this assumption, living HeLa-cells were irradiated at the irradiation setup of the ion microprobe SNAKE using the ion beams provided by the Munich 14 MV tandem accelerator. After irradiation, the distribution of 53BP1 protein foci was studied by immunofluorescence. The observed 53BP1 distribution along the tracks of 29 MeV (7)Li ions and 24 MeV (12)C ions differed significantly from the expectations resulting from the simple chromatin model, suggesting that the biological track structure is determined by cell nuclear architecture with higher order organisation of chromatin.
Assuntos
Cromatina/química , Cromatina/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , DNA/química , DNA/efeitos da radiação , Simulação por Computador , Proteínas de Ligação a DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HeLa , Íons Pesados , Humanos , Transferência Linear de Energia , Modelos Químicos , Modelos Moleculares , Doses de RadiaçãoRESUMO
Inactivation of the Saccharomyces cerevisiae gene YKU70 (HDF1), which encodes one subunit of the Ku heterodimer, confers a DNA double-strand break repair defect, shortening of and structural alterations in the telomeres, and a severe growth defect at 37 degrees. To elucidate the basis of the temperature sensitivity, we analyzed subclones derived from rare yku70 mutant cells that formed a colony when plated at elevated temperature. In all these temperature-resistant subclones, but not in cell populations shifted to 37 degrees, we observed substantial amplification and redistribution of subtelomeric Y' element DNA. Amplification of Y' elements and adjacent telomeric sequences has been described as an alternative pathway for chromosome end stabilization that is used by postsenescence survivors of mutants deficient for the telomerase pathway. Our data suggest that the combination of Ku deficiency and elevated temperature induces a potentially lethal alteration of telomere structure or function. Both in yku70 mutants and in wild type, incubation at 37 degrees results in a slight reduction of the mean length of terminal restriction fragments, but not in a significant loss of telomeric (C(1-3)A/TG(1-3))(n) sequences. We propose that the absence of Ku, which is known to bind to telomeres, affects the telomeric chromatin so that its chromosome end-defining function is lost at 37 degrees.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Telômero , Cromossomos Fúngicos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Amplificação de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Mutagênese , Proteínas Serina-Treonina Quinases , Proteína Rad52 de Recombinação e Reparo de DNA , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos , TemperaturaRESUMO
Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.
Assuntos
Aberrações Cromossômicas , Cromossomos Fúngicos/efeitos da radiação , Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos da radiação , Cromossomos Fúngicos/genética , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Raios gama , Cariotipagem , Proteína Rad52 de Recombinação e Reparo de DNA , Saccharomyces cerevisiae/genéticaRESUMO
In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.
Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologia , Ciclo Celular/genética , Reparo do DNA , Diploide , Deleção de Genes , Genes Fúngicos , Haploidia , Autoantígeno Ku , Fenótipo , Tolerância a Radiação/genética , Saccharomyces cerevisiae/efeitos da radiaçãoRESUMO
Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.
Assuntos
Ácido Hipocloroso/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Esterilização , Soluções Tampão , Relação Dose-Resposta à Radiação , FiltraçãoRESUMO
The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to gamma-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb. The two assays gave similar results for the frequency of dsb ((1.07 +/- 0.06) x 10(-9) Gy-1 bp-1 and (0.93 +/- 0.09) x 10(-9) Gy-1 bp-1, respectively). The dsb frequency was found to be linearly dependent on dose.
Assuntos
Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA , DNA Fúngico/efeitos da radiação , Saccharomyces cerevisiae/genética , Radioisótopos de Cobalto , DNA/efeitos da radiação , Eletroforese em Gel de Campo Pulsado , Raios gama , RadiogenéticaRESUMO
The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.
Assuntos
Adenosina Trifosfatases , Dano ao DNA/genética , Genes Fúngicos , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Modelos Genéticos , Radiação Ionizante , Saccharomyces cerevisiae/efeitos da radiaçãoRESUMO
The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA-PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA , Mutação , Proteínas Serina-Treonina Quinases/genética , Tolerância a Radiação/genética , Recombinação Genética/genética , Animais , Células CHO , Cricetinae , Proteína Quinase Ativada por DNA , Relação Dose-Resposta à Radiação , Teste de Complementação Genética , Mutagênicos/farmacologia , Raios XRESUMO
Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.
Assuntos
Anormalidades Múltiplas/genética , Transformação Celular Viral , Quebra Cromossômica , Fibroblastos/virologia , Anormalidades Múltiplas/patologia , Antineoplásicos/farmacologia , Bleomicina/farmacologia , Camptotecina/farmacologia , Linhagem Celular , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Pré-Escolar , DNA/efeitos dos fármacos , DNA/genética , DNA/efeitos da radiação , Dano ao DNA , Reparo do DNA , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Células HeLa , Humanos , Mitomicina/farmacologia , Mutação , Síndrome , Raios XRESUMO
CONTEXT: Radio-sensitivity in normal tissue is characterized by heterogeneity throughout the population and the absence of pre-diagnostic biomarkers. OBJECTIVE: We conducted a proteomic approach to search for radiation characteristic protein regulation. MATERIALS AND METHODS: Cell lines were 10 Gy irradiated and analysed by 2D-DIGE after 24 h. RESULTS were analysed intra- and inter-individually. The principal component analysis and hierarchical clustering was applied to all datasets. RESULTS: Differences in intra-individual spot abundance prior and post irradiation exactly show the separation of sample classes in two groups: sham-irradiated and irradiated. The inter-individual datasets clustered according to the cell line. The intra-individual differences on protein level after gamma-irradiation are very low, compared with the inter-individual differences among cell lines derived from the same tissue. CONCLUSION: The application of 2-D DIGE may offer a realistic chance for a better molecular characterization of radio-sensitivity and for the discovery of candidate biomarkers.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Proteínas/metabolismo , Proteômica , Tolerância a Radiação/efeitos da radiação , Linhagem Celular , Raios gama , Humanos , Fatores de Tempo , Eletroforese em Gel Diferencial BidimensionalRESUMO
The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.
Assuntos
Cromatina/química , Dano ao DNA/genética , Reparo do DNA/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transativadores/química , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Difusão , Humanos , Modelos Genéticos , Modelos Estatísticos , Osteossarcoma/química , Osteossarcoma/genética , Ligação ProteicaRESUMO
An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.
Assuntos
Bioensaio/métodos , Sobrevivência Celular/efeitos da radiação , Técnicas Citológicas/métodos , Programas de Rastreamento/métodos , Mitocôndrias/efeitos da radiação , Monitoramento de Radiação/métodos , Tolerância a Radiação , Adulto , Humanos , Masculino , Doses de RadiaçãoRESUMO
Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a "pulsed mode" that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, A(L) cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately (P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.
Assuntos
Aberrações Cromossômicas , Prótons , Animais , Linhagem Celular , Cricetinae , Relação Dose-Resposta à Radiação , Humanos , Células Híbridas , Micronúcleos com Defeito Cromossômico , Neoplasias/radioterapia , Eficiência Biológica Relativa , Raios XRESUMO
To obtain greater insight into the future potential of tumor radiotherapy using proton beams generated from high-intensity lasers, it is important to characterize the ionization quality of the new beams by measuring the relative biological effectiveness (RBE) under conditions where the full dose at one irradiation site will be deposited by a few proton pulses less than 1 ns in duration. HeLa cells attached to a Mylar foil were irradiated with 70 kV X rays to obtain a reference dose-response curve or with 3 Gy of 20 MeV protons at the Munich tandem accelerator (Garching), either using a continuous mode where a cell sample was irradiated within a 100-ms time span or using a pulsed mode where radiation was given in a single proton pulse of about 1 ns. After irradiation cytochalasin B was added; 24 h later cells were fixed and stained with acridine orange and micronuclei were counted. The X-ray dose-response curve for the production of micronuclei in HeLa cells followed a linear-quadratic model. The corresponding RBE values for 20 MeV protons in pulsed and continuous irradiation modes were 1.07 +/- 0.08 and 1.06 +/- 0.10 in the first proton experiment and 1.09 +/- 0.08 and 1.05 +/- 0.11 in the second, respectively. There was no evidence for a difference in the RBE for pulsed and continuous irradiation of HeLa cells with 20 MeV protons.
Assuntos
Prótons , Relação Dose-Resposta à Radiação , Células HeLa , Humanos , CariotipagemRESUMO
A procedure for the quantification of double-strand breaks in yeast is presented that utilizes pulsed field gel electrophoresis (PFGE) and a comparison of the observed DNA mass distribution in the gel lanes with calculated distributions. Calculation of profiles is performed as follows. If double-strand breaks are produced by sparsely ionizing radiation, one can assume that they are distributed randomly in the genome, and the resulting DNA mass distribution in molecular length can be predicted by means of a random breakage model. The input data for the computation of molecular length profiles are the breakage frequency per unit length, alpha, as adjustable parameter, and the molecular lengths of the intact chromosomes. The obtained DNA mass distributions in molecular length must then be transformed into distributions of DNA mass in migration distance. This requires a calibration of molecular length vs. migration distance that is specific for the gel lane in question. The computed profiles are then folded with a Lorentz distribution with adjusted spread parameter gamma to account for band broadening. The DNA profiles are calculated for different breakage frequencies alpha and for different values of gamma, and the parameters resulting in the best fit of the calculated to the observed profile are determined.
Assuntos
Simulação por Computador , Dano ao DNA , DNA Fúngico/análise , Eletroforese em Gel de Campo Pulsado , Modelos Estatísticos , Saccharomyces cerevisiae/genética , DNA Fúngico/efeitos da radiação , Raios gama , Processamento de Imagem Assistida por ComputadorRESUMO
In the hypusine-containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4-amino-2-hydroxybutyllysine). The HP has been designated as an eucaryotic translation initiation factor--eIF-5A--because of its stimulating effect in the methionyl-puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1-gene is identical to the ANB1-gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2-gene has not been elucidated. By using pulsed-field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2-gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Diploide , Eletroforese em Gel de Campo Pulsado , Haploidia , Recombinação GenéticaRESUMO
An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells irradiated with gamma-rays or restriction enzyme-treated human DNA was resolved according to size using pulsed field gel electrophoresis, and the resulting DNA mass distributions were measured in ethidium bromide-stained gels. The DNA profiles thus obtained were compared to the predictions on DNA fragment size distribution which follow from a so-called random breakage model to test whether the experimental outcome is compatible with the assumption of a random localization of breaks. Comparisons of fragment distributions may be performed utilizing two equivalent representations that are linked by an adequate transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in units of molecular length, we show for gamma-irradiated samples that the predictions derived from the employed models agree well with the observed data, thus allowing an immediate quantification of double-strand breaks (DSB). Using restriction enzyme-treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability of our approach to the detection of a non-random distribution of DSB. Therefore, we suppose that our approach may also be useful to reveal a clustering of DSB, which is postulated to occur after damage induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.